key: cord-007305-pkjfnhro authors: iosub, silvia; gromisch, donald s. title: leukonychia partialis in kawasaki disease date: 1984-10-17 journal: j infect dis doi: 10.1093/infdis/150.4.617-a sha: doc_id: 7305 cord_uid: pkjfnhro nan legend. different treatment courses for travellers highly suspected of being infected with borrelia recurrentis. no t some of the travellers in groups iii and iv had minor signs of tbrf (headache and low grade fever). colleagues -some areas in the northern part of the israel desert (negev) are known to be endemic for borrelia recurrentis infection [1] (tick-borne relapsing fever). four groups of subjects, aged 18-21 years, had an overnight stay in a few caves along a small valley in this area. the groups camped separately overnight in the same caves within an interval of four weeks. after an overnight stay in the caves, 50% of the individuals in each group found ticks or had signs of tick bites on their bodies. most actually noted the ticks biting them. fifteen (52%) of 29 subjects in groups i and ii had signs of bites. four to five days later, 12 of them developed fever (>39 c), rigors, headache, anorexia, nausea, vomiting, and generalized muscular pain. the clinical diagnosis of tick-borne relapsing fever was confirmed by dark-field microscopy of fresh blood samples (12 patients) or serologically (proteus oxk agglutination test) in the three patients in whom spirochete was not identified in the blood. tetracycline (tevacycline; teva ltd, jerusalem) [2, 3] was administered in a dose of 2.0 g/day for seven days and all of the patients recovered. following this experience, 13(46%) of 28 individuals (groups iii and iv) were given tetracycline (1.0 g/day) please address requests for reprints to dr. eithan galun, department of medicine a, hadassah university hospital, ein kerem, jerusalem 91120, israel for three to five days, 24-48 hr after evidence of tick bites. only two of these 13 subjects developed fever « 38c) for one day and three had headache for about two days. none of them developed overt symptoms and signs of tick-borne relapsing fever as observed in the subjects from groups i and ii. it would appear that a 'short-term, low-dose course of tetracycline may have prevented the appearance of the disease following a tick bite. ) . examination of the patients' nails showed that most fingernails and some toenails were abnormally white proximally, with a distinct sharp 2-4 mm transverse band of normal pink nail distally. the free edge of the nail was of normal white color and the nails were of normal texture. the color changes persisted for four to seven days. leukonychia (white nails) has been divided into four types: total, partial, striate, and punctate. all but the punctate type may be hereditary (autosomal dominant) or acquired. our patients had nail abnormalities typical for leukonychia partialis of the acquired type, since color changes had not been noted before the present illness and were transient. this type of leukonychia has been associated with tuberculosis, arteriosclerosis, nephritis, hodgkin's disease, chilblains, metastatic carcinoma, anemia, and hepatic cirrhosis [1, 2] . most researchers agree that it is caused by abnormal keratinization [2] [3] [4] . some [2] postu-correspondence late that altered vascular patterns of the nailbed or intravascular changes (anemia and hypoproteinemia) may also lead to increased whiteness of the nail. since our patients were not anemic or hypoproteinemic, we suggest that vasculitis was the cause of leukonychia partialis. although the heart and coronary arteries are mainly involved in kawasaki disease, other small and medium-sized arteries may exhibit intimal thickening, round cell infiltration, and, rarely, fibrinoid necrosis [5] . according to kawasaki et al. [6] an arteritis similar to that seen in infantile periarteritis nodosa and accompanied by coronary thrombosis and aneurysm was found in 13 autopsy cases. we would welcome correspondence from others who have seen nail color abnormalities in kawasaki disease. relapsingfever in children in the northern negev borrelia recurrentis infection: singledose antibiotic regimens and management of the jarisch-herxheimer reaction single-dose doxycyclinetreatment of louse-borne relapsingfever and epidemictyphus plorde 11. penicillin vs. tetracycline in the treatment of louse-borne relapsing fever phagocytosis of borreliarecurrentis by blood polymorphonculear leukocytesis enhanced by antibiotic treatment diseases of the skin acute febrile mucocutaneous lymph node syndrome a new infantile acute mucocutaneous lymph node syndrome (mlns) prevailing in japan no.4· october 1984 © 1984 by the university of chicago. all rights reserved colleagues -we examined paired sera from 62 infants with acute non bacterial gastroenteritis and from 50 age-matched controls (admitted to the hospital for nondiarrheal diseases) for antibody to human coronavirus (hcv) oc43 and neonatal-calf diarrhea coronavirus (ncdcy). antibody response was observed with greater frequency in patients (27.4010) than in controls (2.0%). it was characterized by the presence of neutralizing and often hal antibody to hcv oc43, but not to the antigenically related ncdcv [1] . these serological data suggested indirectly the existence of a human enteric coronavirus (hecy) antigenically related to hcv oc43, and prompted us to examine both infants and young children with acute gas-this work was supported by grant grt c6/i81/124 from the world health organization.please address requests for reprints to professor goiuseppe gerna, virus laboratory, institute of infectious diseases, university of pavia, 27100 pavia, italy. troenteritis and age-matched controls for detection of antibody to coronavirus in sera and coronavirus-like particles in stools. coronavirus-like particles were detected by electron microscopy in 34 (16.3%) of 208 patients and in 3 (1.6%) of 182 controls tested (p < .01), subsequently, we purified hecvs from stools of two patients (va24 and va 35) by sucrose density gradient centrifugation. antisera from mice and guinea pigs immunized with purified virus were examined by conventional immune electron microscopy (lem) [2] for reactivity to hcv oc43, ncdcv, and the two hecv strains. results showed a two-way crossreactivity between hcv oc43 and the two hecv strains. the antigenic relatedness to hcv oc43, as well as the typical morphology (figure), suggest that the coronavirus-like particles detected were actually hecvs. furthermore, the buoyant density of hecv-24 and hecv-35 in sucrose was approximately 1.20 glml, a value in agreement with those reported for human and animal coronavirus. convalescent-phase sera from all the patients positive for excretion of coronavirus-like particles in stools, and seronegative for previous hcv oc43 infections, reacted by iem with hecy-24 and hecv-35 and, to a lesser extent, with hcy oc43. acute-phase sera from the same patients were poorly reactive or nonreactive by iem. conversely, key: cord-267458-uofy7jyx authors: jiang, xiao-lin; zhang, xiao-li; zhao, xiang-na; li, cun-bao; lei, jie; kou, zeng-qiang; sun, wen-kui; hang, yang; gao, feng; ji, sheng-xiang; lin, can-fang; pang, bo; yao, ming-xiao; anderson, benjamin d; wang, guo-lin; yao, lin; duan, li-jun; kang, dian-ming; ma, mai-juan title: transmission potential of asymptomatic and paucisymptomatic sars-cov-2 infections: a three-family cluster study in china date: 2020-04-22 journal: j infect dis doi: 10.1093/infdis/jiaa206 sha: doc_id: 267458 cord_uid: uofy7jyx data concerning the transmission of sars-cov-2 in asymptomatic and paucisymptomatic patients are lacking. we report a three-family cluster of infections involving asymptomatic and paucisymptomatic transmission. eight (53%) of 15 members from three families were confirmed with sars-cov-2 infection. of eight patients, three were asymptomatic and one was paucisymptomatic. an asymptomatic mother transmitted the virus to her son, and a paucisymptomatic father transmitted the virus to his three-month-old daughter. sars-cov-2 was detected in the environment of one household. the complete genomes of sars-cov-2 from the patients were >99.9% identical and were clustered with other sars-cov-2 sequences reported from china and other countries. m a n u s c r i p t severe acute respiratory syndrome coronavirus 2 (sars-cov-2), that causes coronavirus disease 2019 (covid-19), emerged in december 2019 in wuhan, china [1] . it has since been declared a global pandemic with over 1,000,000 cases reported as of april 3, 2020 [2] . person-to-person transmission has been established [3] [4] [5] [6] [7] [8] , and asymptomatic transmission of sars-cov-2 has been reported [9] . however, studies on the potential transmission of sars-cov-2 by asymptomatic persons and those with mild illness have been limited [10] . herein, we report a 3-family cluster study of eight patients associated with asymptomatic and pauciasymptomatic (one mild symptom only) sars-cov-2 transmission in shandong province, china. the first positive sars-cov-2 patients in this cluster were identified on january 21, 2020 triggering an epidemiological investigation by the local center for disease control and prevention. to identify the possible infective source, the epidemiological investigation focused on exposure history before the onset of illness, such as travel history to wuhan or hubei province, visiting live animal markets, and contact history with febrile persons. medical records were also closely reviewed to verify the timelines of events and clarify clinical progressions. to examine possible environmental contamination of sars-cov-2 in households, select surfaces that may be frequently touched by family members were sampled in the bedroom (door handle, bedside light switch, and sliding of wardrobe door), kitchen (door handle, faucet switch, light switch, rice cooker plug), and bathroom (door handle, handrail, the surface of the toilet bowl, sink). one swab per site (room) with multiple surfaces was collected. all close contacts of sars-cov-2 positive patients were traced, including family members who lived with the patients and individuals who had contact with the patients within 1 meter without wearing proper personal protection. close contacts were quarantined at home and monitored for fever (≥38°c) and symptoms. in addition, nasopharyngeal swabs of close contacts were collected every 24 a c c e p t e d m a n u s c r i p t 5 hours from day 1 to 14 to detect sars-cov-2 by molecular assay. if any close contact had positive detection of sars-cov-2, they were sent to a hospital for isolation and treatment. all collected environmental and patient samples were stored at −80°c before being transported using cold chain to a biosafety level 2 enhanced laboratory to perform molecular detection of sars-cov-2. a real-time reverse transcriptase pcr (rrt-pcr) test kit (gz-d2rm, shanghai geneodx biotech co., ltd) targeting the orf1ab and n genes of sars-cov-2 was used. a cycle threshold (ct) value less than 37 was interpreted as positive for sars-cov-2 rna and a ct value of 40 or more was defined as a negative test. a medium load (weakly positive), defined as a ct value of 37 to less than 40, required confirmation by retesting. positive samples were sequenced directly from the original specimens as previously described [11] . the maximum likelihood phylogenetic tree of the complete genomes was conducted by using raxml software (version 8.2.9) [12] with 1000 bootstrap replicates, employing the general time-reversible nucleotide substitution model. patients 1 (62-year-old woman) and 2 (65-year-old man) were a couple who lived with their son (patient 3), daughter-in-law (patient 4), and two grandchildren. patient 1 presented with cough, rhinorrhea, and sputum on january 12, 2020 ( figure 1 ). on january 15, she visited a health clinic and was diagnosed with a common cold. she was prescribed intravenous infusions of ampicillin and sulbactam, ribavirin, and traditional medicine for five days. on january 16, she developed a fever (38°c). on january 17, patient 2 reported symptoms of fever (37.8°c), cough, sputum, earache, and upset stomach. he was also diagnosed with a common cold at the health clinic and received the same prescription as patient 1 for three days. however, their symptoms did not resolve at the conclusion of the treatment regimen leading both to seek care at a local hospital on january 21. nasopharyngeal swabs were collected from both patients at the hospital and confirmed positive for the infant had no clinical symptoms before, during, or after hospitalization. the chest ct images on admission or hospitalization showed that patients 1-6 had ground-glass opacities. however, no significant abnormalities were observed for patients 7 and 8 (supplemental a c c e p t e d m a n u s c r i p t 9 we report a unique three-family cluster of infection with sars-cov-2, in which eight of 15 members were confirmed with sars-cov-2 infection. particularly interesting is that of 6 secondary patients, two were asymptomatic, one was paucisymptomatic, and three were symptomatic. our findings show that the transmission of sars-cov-2 by individuals with asymptomatic or paucisymptomatic infections is possible. patients 1 and 2 were likely first exposed to sars-cov-2 after visiting their hometown in xiaogan hubei province, china. their son (patient 3) and daughter-in-law (patient 4, asymptomatic), whom they live with, were later found to be infected with sars-cov-2. patient 5 (asymptomatic) was identified to be infected with sars-cov-2 after frequent contact with patients 3 and 4 during work and home visits. she transmitted the virus to her son (symptomatic) whom she lives with. patient 7 (paucisymptomatic) was found to be infected with sars-cov-2 after frequent contact with patient 3 during work. he likely transmitted the virus to his daughter (patient 8, asymptomatic). in addition, consistent with previous studies [5] [6] [7] [8] , the transmission of sars-cov-2 during the incubation period of patient 3 likely occurred. patients 5 and 7 were infected after their exposures to a presymptomatic patient 3 during working or home visits. these findings may help explain the rapid spread of sars-cov-2 between person-to-person. the currently available evidence shows that sars-cov-2 is transmitted between people through droplets and close contact [13] . a recent study showed extensive environmental contamination by a sars-cov-2 patient [14] , suggesting the contaminated environment as a potential medium of transmission. in this study, we detected sars-cov-2 in two environmental swabs from the household of patient 3. such detection of sars-cov-2 in contaminated environments of the household may provide an additional contribution to virus transmission among family members as the virus can remain viable and infectious on the surface up to seven days [15] . however, the direct researchbased evidence describing exactly how sars-cov-2 is transmitted is limited, and further studies are required. we cannot rule out the possibility of unknown covid-19 patients (e.g., asymptomatic carriers) transmitting the virus. however, according to screening protocols implemented by the provincial, municipal, and county-level center for disease control and prevention, all close contacts were a c c e p t e d m a n u s c r i p t 10 traced, and all patients with positive rrt-pcr results in this study were confirmed by whole-genome sequencing, including those who were asymptomatic or pauciasymptomatic (patients 4, 5, 7, and 8). m a n u s c r i p t 11 we thank all patients involved in the study. we also thank dr. hong-guang ren from beijing institute of biotechnology for generating phylogenetic tree. a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster novel coronavirus pneumonia emergency response epidemiology t transmission of 2019-ncov infection from an asymptomatic contact in germany a familial cluster of infection associated with the 2019 novel coronavirus indicating potential person-to-person transmission during the incubation period potential presymptomatic transmission of sars-cov-2 presymptomatic transmission of sars-cov-2 -singapore presumed asymptomatic carrier transmission of covid-19 covert coronavirus infections could be seeding new outbreaks genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies advice on the use of masks in the community, during home care and in healthcare settings in the context of the novel coronavirus (covid-19) outbreak surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 a c c e p t e d m a n u s c r i p t key: cord-277673-kvh60zd5 authors: kanzawa, mia; spindler, hilary; anglemyer, andrew; rutherford, george w title: will coronavirus disease 2019 become seasonal? date: 2020-06-21 journal: j infect dis doi: 10.1093/infdis/jiaa345 sha: doc_id: 277673 cord_uid: kvh60zd5 this manuscript explores the question of the seasonality of severe acute respiratory syndrome coronavirus 2 by reviewing 4 lines of evidence related to viral viability, transmission, ecological patterns, and observed epidemiology of coronavirus disease 2019 in the southern hemispheres’ summer and early fall. although the object of much speculation, few data exist that bear on the question of the seasonality of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] or, more succinctly, the question of whether the virus will "disappear magically by summer. " there are 4 lines of evidence that bear on this question: (1) seasonality of other human coronaviruses and influenza a, (2) in vivo experiments with influenza transmission, (3) ecological data, and (4) the observed epidemiology of coronavirus disease 2019 in the southern hemispheres' summer and early fall. human alpha and beta coronaviruses and influenza peak in winter months whereas many other respiratory viral pathogens do not [2] [3] [4] [5] . surges in incidence of these infections are thought to be due in part to environmental effects on viral stability and transmission as well as host behavior (eg, clustering indoors) and changes in immunity level over time [5, 6] . more specifically, winter months are generally associated with decreased temperatures, decreased absolute humidity, and decreased indoor relative humidity (where we spend most of our time interacting). cool, dry environments have been associated with increased influenza and coronavirus stability and transmissivity [5] [6] [7] [8] [9] . this is thought to be due to changes in essential viral outer proteins and lipids [10] as well as droplet matrices during droplet, and fomite transmission [7] . for example, environments with lower relative humidity may lead to droplet evaporation and smaller droplet sizes. this affects how far virus-containing droplets travel through the air and where they deposit in the airways. associations between environmental factors and viral propagation are typically made through controlled laboratory experiments and natural history observations. laboratory experiments have demonstrated that controlling temperature and humidity affects the viability of coronavirus and influenza. severe acute respiratory syndrome coronavirus 1 was found to have longer viability at temperatures typical to air-conditioned environments (22-25°c) with relative humidity of 0-50% compared with higher temperatures (>38°c) and higher relative humidity (>95%) [9] . severe acute respiratory syndrome coronavirus 2 has been found to have similar stability under experimental conditions [11] . one study found that the viability of sars-cov-2 decreased at higher temperatures. in this study, investigators incubated sars-cov-2 in virus transport medium with a final concentration of approximately 6.8 log units of 50% tissue culture infectious dose per milliliter and found only a reduction of 0.7 log units on day 14 at 4°c compared with complete inactivation of the virus at 14 days in 22°c and at 2 days in 37°c [12] . beyond the impact temperature and humidity have on viral stability, researchers have also investigated the impact they have on transmission. lowen and steel [8] exposed individually housed guinea pigs to nebulized influenza virus at controlled temperatures and humidity levels. transmission was found to be highly efficient at ambient temperatures of 5°c, variable at 20°c, and inefficient at 30°c [8] . lower relative humidity (20%-35%) was also found to be more favorable for spread of the virus [8] . data may be emerging regarding sars-cov-2 viability in different environments within engineered aerosols and simulated body fluids [13] . laboratory experiments offer the possibility of strict control over environments with limited variables; however, these studies typically rely on animal models and/or engineered culture mediums, aerosols, or droplets and therefore may not generalize to clinical conditions. because sars-cov-2 is an emerging virus, we do not have the longitudinal data to determine whether covid-19, the disease caused by sars-cov-2, cycles seasonally. however, recent research, still preprint and not yet peer-reviewed, has examined ecological associations between transmission patterns and climate. in one study, researchers found that before march 22, 2020, 90% of sars-cov-2 global transmissions occurred within areas with temperatures ranging from 3°c to 17°c and absolute humidity ranges between 4 and 9 g/m 3 daily [13] . similarly, sajadi et al [14] found that areas across the globe with significant community spread (defined as ≥10 reported deaths by march 10, 2020) remained in a distribution approximately in the range of 30-50° north latitude with average temperatures of 5-11°c and low absolute humidity (4-7g/m 3 ). in addition, notari [15] estimated that among countries with at least 30 covid-19 cases and 12 days of data before april 1, 2020, the maximal transmission peak occurred at 7.7°c with decreased comparative doubling times at higher and lower temperatures. supporting the ecological theory that temperature and humidity may impact the transmission of covid-19, some investigators have found that as little as a 1°c increase in temperature and a 1% increase in relative humidity can lower the daily effective reproductive number (r e ) by 0.0383 and 0.0224, respectively [16] . similarly, a study investigating 30 chinese provinces found a 1°c increase in average temperature was associated with a decrease in daily confirmed cases by 36%-57% when average relative humidity ranged from 6% to 85.5% [17] . furthermore, a 1% increase in average relative humidity led to a decrease in daily confirmed cases by 11%-22% when average temperature was between 5.04-8.2°c [17] . athough these findings are not consistent throughout china, their conclusions that higher viral spread is observed in areas with moderate temperatures and lower humidity are similar. however, the evidence is still developing and is somewhat inconsistent [18] . in fact, recent research from a public bath center in huai'an, jiangsu province, china found that a bathhouse with high temperature and humidity was the source of a cluster of covid-19 cases [19] . it is important to note that causal inference is limited in these ecological studies exploring the relationship between environment and covid-19 transmission rates. moreover, these studies are limited by data quality, strong modeling assumptions, and reveal that temperature and humidity are only 2 of the many factors involved in viral transmission [13] . two primary factors that potentially confound the relationship between environment and transmission include travel and behavioral patterns, which are also driven by public health policy, testing capacity, quality of healthcare, and per capita income. furthermore, rapid viral spread is not solely limited to areas within ranges of 3-17°c temperature and 4-9 g/m 3 humidity. for example, covid-19 has already spread very rapidly throughout iran and louisiana. finally, we should note that sars-cov-2 has occurred in 14 countries and 3 overseas french dependencies lying entirely in the southern hemisphere since january 25, 2020. on february 1, 12 cases had been reported from australia with evidence of community transmission, but by the vernal equinox (march 19), 1055 cases had been reported from countries lying entirely south of the equator, including argentina, australia, bolivia, chile, eswatini, french polynesia, mayotte, new zealand, namibia, paraguay, réunion, rwanda, seychelles, south africa, tanzania, uruguay, and zambia [20] . an additional 598 were from countries that crossed the equator, including major foci in brazil, ecuador, and peru. all but 70 of these were from countries that the world health organization had classified as having local transmission. so, although data suggest temperature and humidity may affect viral viability and transmission, given our limited data on the emerging pandemic, the seasonality of covid-19 cannot yet be definitively determined. in addition, human factors, such as lack of sustainable social distancing and low immunity to a novel virus with high transmissibility, will likely outweigh the climate effects on transmission. therefore, it seems unlikely that the coming northern hemisphere summer will have a significant effect on sars-cov-2 transmission reduction. as to whether covid-19 will enter into regular circulation like other human coronaviruses and influenza, this will depend largely on the duration of immunity to the virus, which remains unknown. one study predicts that if duration of immunity to sars-cov-2 mimics other related coronaviruses, recurrent outbreaks are likely to occur [21] . potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. rapid expert consultation on sars-cov-2 survival in relation to temperature and humidity and potential for seasonality for the covid-19 pandemic accessed 1 when is flu season? available at coronavirus occurrence and transmission over 8 years in the hive cohort of households in michigan epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method seasonality of respiratory viral infections low ambient humidity impairs barrier function and innate resistance against influenza infection mechanistic insights into the effect of humidity on airborne influenza virus survival, transmission and incidence roles of humidity and temperature in shaping influenza seasonality the effects of temperature and relative humidity on the viability of the sars coronavirus coronavirus envelope protein: current knowledge aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 stability of sars-cov-2 in different environmental conditions will coronavirus pandemic diminish by summer? ssrn temperature, humidity and latitude analysis to predict potential spread and seasonality for covid-19 temperature dependence of covid-19 transmission. medrxiv high temperature and high humidity reduce the transmission of covid-19 accessed 4 covid-19 transmission in mainland china is associated with temperature and humidity: a time-series analysis the role of absolute humidity on transmission rates of the covid-19 outbreak. medrxiv possible transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in a public bath center in huai'an accessed 1 projecting the transmission dynamics of sars-cov-2 through the postpandemic period key: cord-007201-m87jid5l authors: tzipori, saul; angus, kenneth w.; campbell, iris; sherwood, david title: diarrhea in young red deer associated with infection with cryptosporidium date: 1981-08-17 journal: j infect dis doi: 10.1093/infdis/144.2.170 sha: doc_id: 7201 cord_uid: m87jid5l in an outbreak of diarrhea among 82 artificially reared red deer calves, 56 developed the disease and 20 subsequently died. during the outbreak 80% of diarrheal and 50% of apparently healthy calves excreted cryptosporidial oocysts in feces. the coincidence of infection with cryptosporidium and clinical diarrhea suggested a causal relationship. histologic examination of intestinal sections from a necropsied deer calf showed lesions consistent with field and experimental cryptosporidiosis in other species. the deer cryptosporidium subclinically infected newborn mice; in indirect immunofluorescence tests, it could not be distinguished from a calf cryptosporidium. diarrhea has been noted among captive deer, which appear to be susceptible to enteropathogens, such as escherichia coli [1] and rotavirus [2] , that commonly infect other domestic species. infection with cryptosporidium has been reported among several species of mammals, birds, and reptiles [3] but has not previously been observed in deer. although cryptosporidium is classified with other enteric coccidia, it has some distinctive characteristics. it is an extracellular parasite that infects the brush borders of enterocytes [4] , has a rapid life cycle and lacks host specificity [5] , and is pathogenic for much younger animals than the age group generally affected by other coccidia. features of cryptosporidial infections that make recognition difficult are that the oocysts are small (diameter, 4 urn) and they are excreted in small numbers. cryptosporidium has been associated with diarrhea in several mammalian species. outbreaks have been most commonly observed among calves [4] [5] [6] [7] [8] , but there have been reports of similar occurrences among lambs [9, 9a] , and humans [10] [11] [12] . experimental cryptosporidiosis in newborn lambs in-received for publication february 3, 1981 [13] with severe histopathologic lesions [13a] . in contrast, experimental exposure of laboratory animals to cryptosporidium induces only subclinical infection [13] . in the present communication, an association between severe diarrhea in artificially reared red deer calves and infection with cryptosporidium is described. in addition, the serologic relationship between the cryptosporidium isolated from the deer to a bovine cryptosporidium is reported. case history. during december 1979 an outbreak of diarrhea in suckled beef calves occurred on a research station in scotland, which, after investigation, was attributed to cryptosporidiosis [14] . during june-july 1980, 82 red deer calves, up to one week in age, were caught and brought to the research station to be reared artificially. they were divided into small groups, housed in pens indoors, and fed ewes' milk substitute (nutrilamb"; scottish agricultural industries, edinburgh, scotland) three to four times a day with access to dried grass and concentrate pellets. within two weeks, a few of the calves developed diarrhea, and over the next four weeks 56 (68070) had diarrhea that lasted two to 14 days. twenty animals (25070) died during this period. concurrently two suckled deer calves aged eight to 10 days and reared naturally outdoors were found dead. ileal and cecal contents from both calves were obtained for microbiologic examination. one three-week-old live deer calf which had been scouring intermittently for two weeks was necropsied. the animal appeared to have recovered after an initial seven days of illness but relapsed two days later for two days, appeared to recover for two days, and then became bloated and constipated. on the 14th day it began to scour again and refused milk. on arrival at the laboratory a day later, it appeared alert and was not scouring. during the two-week period of illness, three fecal samples had been collected, and the calf had been treated with ampicillin, neomycin, chloramphenicol, and sulfamethazine. samples for histopathologic and electron microscopic examination and for cryostat sectioning were taken from seven intestinal sites from the calf while it was under anesthesia [13] . microbiologic examination. fifty-six fecal swabs obtained during the outbreak (34 from scouring and 22 from nonscouring calves), 58 fecal swabs collected at the end of the outbreak, and ileal and cecal contents of two dead, naturally reared calves were examined for microbes. all samples were examined for enteric viruses by direct electron microscopy with use of negative staining [15] , for rotaviruses by enzyme-linked immunosorbent assay, for cryptosporidial oocysts in direct smears treated with giemsa stain by light microscopy using an oil-immersion lens [8] , and for the adherence antigen (k99) of enterotoxigenic e. coli by a slide agglutination method [16] . because the intestinal contents of the dead suckled deer contained large amounts of blood, they were also checked for salmonellae and clostridia. inoculation of mice. two litters of newborn, specific pathogen-free c57 mice were inoculated orally with 0.1 ml (20010 [vol/vol] in 0.5% phosphate-buffered saline) of feces containing cryptosporidial oocysts from a diarrheal red deer calf. fecal smears from the mice were examined daily for crypt ospori dial oocysts. between four and 14 days after inoculation, randomly selected mice from each litter were killed, smears prepared from the large bowel were treated with giemsa stain and examined, and sections from the upper, middle, and lower intestine taken for histologic and electron microscopic examination. to confirm that the mouse colony was free from naturally acquired cryptosporidial infection, age-matched, conventionally reared control mice were killed before and after the experimental period. serology. the indirect immunofluorescence test 171 was used for serologic examinations. cryostat-cut sections obtained from a specific pathogen-free lamb that was heavily infected with cryptosporidium derived originally from calves [14] were reacted with 12 convalescent-phase sera from red deer that were recovering from diarrhea. fluorescein-conjugated pig antiserum to sheep y-globulin, known to cross-react with deer y-globulin (h. w. reid, personal communication), was used as an indicator. cryostat-cut sections of intestine from uninfected, specific pathogen-free lambs were used as negative controls. the 12 red deer sera were diluted 1:10 and the conjugate 1:40 before the test. this system was tested previously with a large number of positive sera obtained from specific pathogen-free lambs experimentally infected with calf cryptosporidium and with negative sera from uninfected, specific pathogen-free lambs. it had also been tested against k99-specific hyperimmune rabbit serum and did not cross-react (s.t., unpublished observation). cryptosporidial oocysts measuring about 4 lim in diameter were observed in fecal smears from 27 (80%) of the 34 deer calves with diarrhea and from 11 (50%) of the remaining 22 apparently healthy animals during the outbreak. however, by the end of the outbreak only one of 58 fecal swabs was positive for oocysts. five of the 56 fecal smears from diarrheal deer calves during the outbreak contained astroviruslike particles [17] . these particles were observed in the feces of two scouring calves, one of which was also excreting cryptosporidial oocysts, and three normal calves, one of which was shedding oocysts. no other viral or bacterial enteropathogen was observed. the ileal and cecal contents of the two suckled deer calves which contained large amounts of blood were negative for salmonellae and clostridia, but all samples contained large numbers of cryptosporidial oocysts. pathology. in the necropsied deer calf, the small intestine was slightly congested and the mesenteric lymph nodes were enlarged. both the small and large intestines were infected with cryptosporidia; typically, organisms were attached to the microvillar borders of enterocytes, as demonstrated by light and electron microscopy (figure 1). cryptosporidia were most numerous in the cecum and colon and were also present, although in smaller numbers, in the jejunum and upper ileum. very few organisms were seen in the terminal ileum, with none in the two most proximal intestinal sites. atrophy was almost total in the villi of the ileum (figure 2, top), which showed fusion of villi and striking elongation of crypts. changes were much less severe in the jejunum, and some infected areas had a normal morphology. focal areas of villous atrophy were present in the villi of the upper jejunum; however, these areas were associated with adherence of bacteria but not with the presence of cryptosporidia. the lamina propria of the small intestine contained moderate numbers of mononuclear cells. by contrast, the large intestine was heavily infiltrated by macrophages and other mononuclear cells; the crypts were dilated and contained few mucus-secreting cells. cryptosporidia were numerous, both at the mucous surface and in the crypts. vasculitis involving submucosal venous branches was also present in the cecum (figure 2, bottom); the walls of affected vessels were heavily infiltrated with neutrophils. cryostat-cut sections of intestine failed to react in indirect immunofluorescence tests with serum containing antibody to lamb rotavirus and with k99-specific hyperimmune rabbit serum. the only enteric pathogens identified in the three fecal samples from this calf during the two-week period of illness were cryptosporidial oocysts. inoculation of mice. mice orally inoculated with feces containing cryptosporidia from the deer excreted oocysts between seven and 10 days later, but they remained clinically healthy. histologic and electron microscopic examination of mouse intestinal sections revealed widespread infection of the lower small intestine ( figure 3) . serology. the convalescent-phase sera collected from the deer surviving two weeks after the outbreak of diarrhea contained antibody to the bovine cryptosporidium, as demonstrated by the indirect immunofluorescence test. fluorescence was observed on the surface of villi in sections from infected but not from control deer. the results of the present communication demonstrate, on the basis of the excretion of typical oocysts in the feces and the attachment of typical stages of the organism to the brush borders of enterocytes, that these artificially reared red deer were infected with cryptosporidium, there is thus strong circumstantial evidence to incriminate cryptosporidium as the cause of diarrhea and mortality in the outbreak. the diarrhea coincided with the period in which 80070 of the deer tested were shedding oocysts. if sampling had not been confined to a single collection, the infection rate may have been higher. it should also be emphasized that the method of detection of infection is not very sensitive -there are generally few oocysts in fecal smears treated with giemsa stain, and often a positive diagnosis is based on detection of very small numbers. the clinical disease was similar to that observed in field outbreaks of diarrhea associated with cryptosporidium in calves [14] , lambs [9a] , and experimentally infected, specific pathogen-free lambs [13] . intestinal lesions in an affected deer were also similar to those observed in experimentally infected lambs [13a] . not all of the infected deer developed diarrhea, a result which suggests that in cryptosporidiosisas in infections with enterotoxigenic e. coli, rotavirus, and coronavirus -contributing factors (for example, inadequate passive immunity) may play a significant role in precipitating diarrhea. initially the disease was thought to be confined to artificially reared red deer, as is the case in lambs [9a] , but the involvement of a few suckled deer suggests that in the latter group colostral protection was insufficient to prevent infection. a possible explanation for this lack of protection is that the organisms were introduced recently to the deer population. in past years and under the same system of management, diarrhea in young red deer had occurred only sporadically and less severely (w. corrigall and g. a. m. sharman, personal communication). furthermore, the occurrence of an outbreak of diarrhea in suckled beef calves on the same premises six months earlier may have had important epidemiologic significance. cryptosporidia isolated from those calves and from the deer both infected laboratory animals and were serologically indistinguishable by the indirect immunofluorescence method used. the role of astrovirus-like particles in this outbreak of diarrhea is uncertain. on the basis of the evidence presented here and its doubtful role as an enteropathogen in other species [18, 19] , it was probably only of minor importance. in conclusion, the present communication describes another possible enteropathogen, cryptosporidium, to be considered in future investigations of outbreaks of diarrhea in deer. diarrhea in captive mule deer fawns attributed to escherichia coli caple, 1. w. isolation of a rotavirus from deer protozoan parasites of domestic animals and of man cryptosporidiosis as a probable factor in neonatal diarrhea of calves cryptosporidium: evidence for a single-species genus pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhoea neonatal calf diarrhoea: pathology and microbiology of spontaneous cases in dairy herds and incidence of the enteropathogens implicated as etiological agents cryptosporidia associated with rota virus and an escherichia coli in an outbreak of calf scour ovine cryptosporidiosis diarrhea in artificially reared lambs due to cryptosporidium infection overwhelming watery diarrhea associated with a cryp-cryptosporidium diarrhea in red deer tosporidium in an immunosuppressed patient acute enterocolitis in a human being infected with the protozoan cryptosporidium campbell, i. vomiting and diarrhea associated with cryptosporidial infection diarrhea in lambs experimentally infected with cryptosporidium isolated from calves intestinal pathology in specific pathogen-free lambs associated with a cryptosporidium from calves with diarrhea an outbreak of calf diarrhoea attributed to cryptosporidial infection detection and transmission of 30 nm virus particles (astroviruses) in faeces of lambs with diarrhoea improved minca medium for the detection of k99 antigen in calf enterotoxigenic strains of escherichia coli detection of astrovirus in the faeces of red deer pathogenesis of diarrhoea caused by astrovirus infections in lambs isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves key: cord-011708-naezfola authors: frank, gregory m.; adalja, amesh; barbour, alan; casadevall, arturo; dormitzer, philip r.; duchin, jeff; hayden, frederick g.; hirsch, martin s.; hynes, noreen a.; lipsitch, marc; pavia, andrew t.; relman, david a. title: infectious diseases society of america and gain-of-function experiments with pathogens having pandemic potential date: 2016-05-01 journal: j infect dis doi: 10.1093/infdis/jiv474 sha: doc_id: 11708 cord_uid: naezfola nan the rapid pace of technological advances in the life sciences has provided powerful tools for understanding and managing human-microbe interactions. these new capabilities can aid the development of vaccines, diagnostic tools, and therapeutic interventions. these same capabilities, especially the ability to manipulate genomes and, therefore, the properties of bacteria, viruses, and other infectious agents, could also pose important risks. efforts to study and/or predict the natural evolution and emergence of pathogenic microbes by deliberately creating, in the laboratory, pathogens with enhanced disease-causing or transmission-promoting properties pose the greatest concern. examples of this type of gain-of-function (gof) research include the recent creation of highly pathogenic avian influenza viruses with altered host range, enhanced transmissibility, and/or the ability to evade certain forms of human immunity. as infectious diseases society of america (idsa) members, most of whom are involved in direct patient care, research, and public health responses, we recognize that some of the same laboratories, technologies, and types of research that have given rise to concern are also essential to protect public health. just as we have an ethical responsibility to first do no harm to our patients, we are also responsible for ensuring that we do no harm to the public, either through unnecessarily dangerous scientific experimentation or, conversely, by unduly burdening and delaying scientific work that serves essential clinical and public health purposes. this responsibility means we must identify those experiments whose potential benefits to scientific knowledge and public health clearly outweigh the risks they pose to the public and could not be achieved by safer means. in fall 2014, the us government issued a pause of gof research projects of concern and tasked the national science advisory board for biosecurity (nsabb), a federal advisory committee formed at the request of the us government to address issues related to biosecurity and dual use research of concern, to establish recommendations on how gof research of concern should be assessed for its risk and benefit to public health. the nsabb recently developed a framework to guide its efforts and is working with a contractor to undertake a risk-benefit assessment (rba) of the paused gof projects. given the robust public discussion about this topic, the nsabb plans to engage with stakeholders as it continues to develop its final recommendations [1] [2] [3] [4] . in their review in this issue of the journal of infectious diseases, kilianski et al remind us of the importance of including the clinician perspective in any conversation about how best to assess the risk and benefit of gof research [5] . infectious diseases specialists will be among the physicians who will respond to any microbial disease outbreak and care for affected individuals, be it of natural origin or laboratory engineered through accidental or deliberate means. infectious diseases specialists are also among those leading research efforts to counter these disease threats. accordingly, they are especially well positioned to understand the risks and benefits posed by potentially dangerous experiments involving pathogenic microbes and can be valuable advisors for those who will need to undertake the complex rba for gof research. the idsa appreciates the nsabb's efforts on the gof debate and its willingness to engage with the public as it develops its final recommendations. we agree with kilianski et al that this effort represents a key opportunity for clinicians to join the discussion about gof research. to that end, we highlight below 6 key recommendations that the idsa recently shared with the nsabb as we discuss how best to assess the benefits and risk of gof research of concern. the idsa remains concerned that the nsabb framework's broad definition of gof research of concern may inadvertently capture areas of research that pose a lower risk to the public. for example, while the nsabb recognizes the benefit of research aiding the development or selection of new or more-effective vaccines, its framework still targets influenza vaccine production methods that rely on adaptation of vaccine candidate viruses for improved growth in culture as gof research. the adaptation and manipulation of wild-type influenza virus for growth in eggs or mammalian cell lines are critical to vaccine manufacturing. this approach to producing high-growth vaccine candidates has been practiced since the 1940s [6, 7] and is essential to protect the public from both seasonal and pandemic influenza. the idsa strongly urges the nsabb to narrow its definition of gof research to be considered for risk-benefit assessment (rba), to avoid this inadvertent capture of low-risk research, which is not mentioned in the white house office of science and technology policy's original description of the types of research that should be included in the deliberative process. we recommend that the rba process focus on research that is reasonably anticipated to result in a pathogen that combines a high risk of transmissibility with severe pathogenicity in humans, as this combination poses the greatest risk to public health. such research may involve enhancing one of these properties in a pathogen already possessing the other or simultaneously enhancing both properties. whereas other types of gof research are also of concern, notably those involving increased resistance to known medical countermeasures, they are secondary to the two properties described above. the idsa believes that this definition strikes a balance between impeding experiments with a lower public health risk that society has accepted for many years while ensuring that experiments of special concern are assessed appropriately. the risk assessment process to be used by the nsabb will use estimated data in the models, as it will have to make assumptions on both risks and benefits. although the idsa understands that assumptions are necessary to assess risk and benefit, we urge the nsabb to hold robust discussions with experts about the uncertainty of its estimates of risk. we also recommend that the nsabb ensure that any analysis of uncertainty not only include uncertainties in the outcome of the research, such as the pathogenicity changes in a gof organism, but also the uncertainties in the assessments of the likelihood of misuse of the science, as well as the consequences of accidents, misuse, and regulations on the conduct of the science. whereas the nsabb will use a qualitative assessment of the benefits of gof research, we urge that the uncertainties about the benefits of research be explicitly considered. finally, the idsa recommends that the nsabb consider communicating specific assumptions used in its modeling as well as error due to uncertainty to assist other policy makers in better understanding the risk/ benefit estimates. the nsabb has indicated that it will interview subject-matter experts to obtain additional input to aid its rba efforts. the idsa strongly supports these actions and also urges the nsabb to consider seeking additional perspectives to inform the rba process, including those of a range of experts in vaccine development, microbial risk assessment, and public health response; physicians whose work is primarily clinical; and the public. in addition, the moral and ethical implications surrounding gof research have not been adequately addressed in the nsabb framework [8] . several experts in this field are actively engaged in the gof research debate, and their unique viewpoints can be valuable to the rba process. some stakeholders have expressed concern that the experts best positioned to evaluate the risk and benefits of gof research are in some cases the ones who are actively conducting the research. the idsa agrees that this potential conflict of interest is an issue that should be considered and strongly believes that, while this rba evaluation needs as many expert perspectives as possible, those perspectives must be transparent, with all relevant interests disclosed. one important type of risk that is not included in the nsabb framework is the ethical, reputational, and credibility risk for science with the public. the recent laboratory mishaps at some of the nation's most prestigious laboratories have placed a strain on the public's trust for scientific research. should a us government-funded gof study result in an accident or a deliberate act that places the public at risk, the credibility of science as a whole may suffer. this occurrence, in turn, could lead the public to question the quality of public stewardship of biomedical funding and the reliability of scientific and medical advice on risk. such a loss of public trust could significantly impair science's ability to inform evidence-based policy decisions. the idsa recommends that the nsabb consider recruiting additional perspectives, such as those from individuals with expertise in sociology and ethics, to assess this risk, as the nsabb develops its final recommendations. the ability of humanity to protect itself against pathogens with pandemic potential rests on a vigorous and healthy scientific enterprise. some, including idsa members, have raised the concern that as controversy swirls around gof experiments, investigators might abandon certain types of scientific approaches that are powerful tools of scientific inquiry [9] . furthermore, the concern has been raised that the best and brightest researchers will avoid these areas of inquiry simply because of the weight of regulation, the uncertainty in planning careers in areas subject to moratoriums and increased scrutiny, and the controversial nature of the work [10] . if this happens, humanity will be more vulnerable to future threats. the idsa recommends that the possible risk of regulation to the scientific enterprise and, in particular, to certain fields of inquiry be factored in the overall rba. in the nsabb's assessment approach for gof research benefit, it states that it will evaluate "other gof experiment types" in addition to alternative approaches. the idsa believes these efforts will yield valuable information that may be useful in developing constructive recommendations on how gof research may be conducted more safely. for example, during the december 2014 national academies of science discussion on the pause in gof research, one researcher presented data on how to engineer highrisk influenza virus strains to only generate productive infection in experimental animals, posing minimal risk to public health [11] . this search for pragmatic solutions that lower the risk of gof has not been widely discussed, and the idsa urges that such solutions be a more prominent component in the nsabb's final recommendations. the idsa is committed to ensuring that the broader scientific and science policy community participates in efforts to guide gof research appropriately. to complement the nsabb's efforts, the idsa calls for a continued series of transparent, broad discussions on gof research and dual use research of concern among stakeholders, including scientists, healthcare workers, policy makers, ethicists, and representatives from the public. these discussions should include a consideration of rba methods, governance models, classified research, and social responsibilities of scientists and journal editors; increased vigilance of biosafety and security concerns; and societal values. the discussions should also solicit international input. we look forward to working with the public, as well as with the us government, to ensure that gof research of concern is conducted appropriately and only when the risk is outweighed by the benefit to public health. potential conflicts of interest. p. r. d. was an employee and shareholder in novartis influenza vaccines during the drafting of this manuscript, is a pfizer employee and shareholder, and is listed as an inventor on multiple influenza and vaccinerelated patent applications and patents. m. l. has received grants from pfizer and path vaccine solutions on vaccine modelling. a. t. p. has received royalties from antimicrobial therapy, honoraria from webmd for cme materials preparation, and compensation from biofire diagnostics for advisory board participation. all other authors report no potential conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. risks and benefits of gain-of-function experiments with pathogens of pandemic potential, such as influenza virus: a call for a science-based discussion gain-of-function experiments: time for a real debate moratorium on research intended to create novel potential pandemic pathogens the irrationality of gof avian influenza virus research gainof-function research and the relevance to clinical practice growth of influenza virus in the allantoic cavity of the chick embryo office of the surgeon general, united states army. a clinical evaluation of vaccination against influenza: preliminary report the increasingly compelling moral responsibilities of life scientists an epistemological perspective on the value of gain-offunction experiments involving pathogens with pandemic potential is the debate and "pause" on experiments that alter pathogens with pandemic potential influencing future plans of graduate students and postdoctoral fellows? micro-rna-based strategy to mitigate the risk of gain-offunction influenza studies key: cord-002921-i5jxn1vj authors: morens, david m; fauci, anthony s title: pandemic zika: a formidable challenge to medicine and public health date: 2017-12-15 journal: j infect dis doi: 10.1093/infdis/jix383 sha: doc_id: 2921 cord_uid: i5jxn1vj nan it has been 150 years since the noted german physician-scientist and public health advocate rudolf virchow made his prescient observation about new diseases. since then, the world has seen a dizzying succession of newly emerging and reemerging infectious diseases that have changed the landscape of medical practice and of biomedical research [2] . the zika pandemic was first recognized in 2013 and is still unfolding in alarming and unprecedented ways. because of the pandemic's uniqueness and the insidious ability of zika virus to harm unborn children, the pandemic has captured the attention of infectious disease researchers and practitioners of clinical and public health medicine around the world, as well as the attention of allied colleagues working in entomology, vector control, informatics, teratology, immunology, and a host of other disciplines [3] [4] [5] . zika virus has now spread to >80 countries or territories [5] , including 50 in the americas and the caribbean, causing millions of infections. an enormous body of information about zika has rapidly accumulated and the scientific community is now energetically organizing and translating new data into control and prevention approaches. in this issue of the journal, we review what we have learned so far and how we may best apply it to blunt zika's alarming progress. eighteen expert groups discuss multiple aspects of the zika pandemic, including its history, epidemiology, virology, vector characteristics, immunology, teratology, and vaccinology. our collective goal is to stimulate thought and discussion so we can more effectively work together in pursuit of preventing zika infections, developing treatments for those affected, and ending the pandemic. among the many important aspects of the zika pandemic covered in these reviews, its history, epidemiology, and vector biology are of fundamental interest [4] [5] [6] [7] [8] . zika virus was first thought of as an almost completely inconsequential virus that had smoldered at a low level in remote african/asian sylvatic cycles for decades, if not centuries. zika virus then suddenly and unexpectedly surged, and the virus was spread around the world by a nonsylvatic-vector mosquito, the globally ubiquitous aedes aegypti. this is the same mosquito that also has transmitted yellow fever, dengue, and chikungunya viruses around the world. a. aegypti is intensely anthropophilic (preferring blood meals from human beings as opposed to other animal hosts). while human travel and population growth and crowding clearly have played an important role in zika's emergence, these factors alone seem insufficient to explain its sudden pandemicity, since the virus has apparently long persisted in populous and mobile urbanized asian/southeast asian countries with hyperabundant a. aegypti mosquitoes [4] . it is conceivable that one or more viral mutation occurred to facilitate viral spread. this remains a critically important research question. preliminary data show no evidence for phenotypic changes related to human pathogenic or transmissibility properties of the virus [9] and fortunately no evidence of viral evolution that would hinder diagnostic detection [10] . however, surprising preliminary evidence is consistent with the new (asian lineage-derived) pandemic strain causing earlier and more-frequent viral infection of mosquito salivary glands [11, 12] . most pandemic zika cases appear to have been acquired from a. aegypti, a tenacious mosquito vector intimately associated with human dwellings and extremely difficult to eliminate or even control effectively. some evidence also suggests that at least 3 other aedes species have also vectored zika outbreaks. this is of great concern not only because of the global abundance of 2 of these mosquitoes (aedes albopictus and aedes polynesiensis), but because of the possibility that zika virus may be able to adapt to become more efficiently transmitted by new vectors, as has been seen with the chikungunya alphavirus [13] . this would have alarming implications for enhanced zika virus spread and more broadly for the possibility of emergence of other diseases via enhanced vector capacity. in addition to vector transmission, zika virus also is transmitted from mothers to fetuses and infants, via sexual transmission, via laboratory accident, and possibly through blood transfusion and organ transplantation [7] . these realities greatly complicate public health control efforts. understanding the pathogenesis and natural history of zika virus infection has been facilitated by decades of research with flaviviruses [14] . however, despite its genetic similarity to the other 58 flaviviruses (including its close cousins, the 4 dengue viruses), zika virus has some unique or at least uncommon phenotypic properties. these include the ability to cause congenital infections and marked teratogenicity; to be simultaneously viscerotropic and neurotropic, causing not only dengue-like febrile illnesses, but also neurologic conditions, such as guillain-barré syndrome and encephalitis [15] ; to transmit sexually; and to persist for long periods in multiple sites of the body. furthermore, some studies have suggested that preexisting flavivirus immunity (eg, from prior dengue virus infection) might potentiate zika [16] via antibody-dependent infection enhancement in some circumstances [17] , while other research has countered this view [18] . regardless, the serologic and immune responses to zika virus in the background of high population immunity to related flaviviruses is complex and raises difficult questions about diagnosis and pathogenesis [10, 19] . whether this proves to be the case, the serologic and immune responses to zika virus in the background of high population immunity to related flaviviruses is complex and raises difficult questions about diagnosis and pathogenesis [10, 19] . the emergence of zika virus has shown it to be among the most unique and least understood of flaviviruses, necessitating intensive experimental study to determine how it causes disease. as with most flaviviruses, small-animal models of zika virus infection and disease have been problematic, but considerable progress has nonetheless been made, including important new information bearing on teratogenicity and vaccine design strategy [20] . moreover, exciting nonhuman primate work is yielding insights into the natural history not only of typical infection, but also of placentally acquired infection of fetuses [21] . unquestionably, the most disturbing aspect of the zika pandemic is the secondary pandemic of zika-associated microcephaly and many other birth defects (congenital zika syndrome), as well as fetal losses that have affected a large but still unknown percentage of women infected with zika virus during pregnancy [22] . a large body of experimental work, including epidemiological and clinical studies being conducted in south and central america, represents the first time that an epidemic of a human teratogenic/fetal infectious disease has been intensively investigated in real time. these efforts are critically important because of the unfolding tragedy of the loss of thousands of babies and the birth of thousands more who will be incapacitated, sometimes severely, throughout their lifetimes. to make matters worse, it is suspected that many infected babies born in apparent health will manifest delayed effects of intrauterine zika virus infection as they grow into infancy, toddler age, and early school age. the epidemic of congenital zika syndrome represents not only a profound medical tragedy, but a societal challenge, as well: thousands of parents and caregivers will need financial, medical, and social support for decades to come. in this regard, it is worth reflecting upon and studying the rubella epidemic of the mid 1960s, when tens of thousands of babies were born with congenital rubella syndrome in the united states [23] , the deadliest epidemic of infectious birth defects ever documented. study of this rubella epidemic and the ongoing occurrences of other congenital infections, such as cytomegalovirus infection, have made clear the importance of understanding zika through large-scale prospective epidemiologic studies, such as those now underway in the americas. effective antiviral therapy for zika virus infection is still an unrealized goal, but numerous compounds, including repurposed drugs that are already in use, as well as antiviral antibodies, are being examined in preclinical studies [23] . because the most important clinical application of an anti-zika drug would be treatment of infected pregnant women and infected fetuses, unprecedented challenges are now being considered [24] . another critically important research goal is the development of a preventive zika vaccine [25, 26] . almost a century of work on flavivirus vaccines (going back to the 1920s) [27] has provided us with several viable vaccine approaches and platforms, including those using other flavivirus proteins, and could readily be switched to zika virus proteins. at least 7 different zika vaccines are currently (in late 2017) in clinical trials, with ≥40 more in preclinical development [25, 26] . zika vaccine trials face significant challenges, including the fact that the zika pandemic is waning in many areas and that flavivirus outbreaks normally appear sporadically and unexpectedly. furthermore, it is unknown which of several possible vaccination strategies is optimal, eg, inducing sterilizing immunity in vulnerable individuals, protecting pregnant women by vaccine induction of population herd immunity, or vaccinating children to induce partial population immunity [25] . in addition, as noted above, serious questions about vaccine-associated immunologically enhanced disease remain unanswered. finally, it is important that we not assume that pandemic zika is a one-time crisis that can be met, controlled, and then forgotten or relegated to historical review. over the past 4 decades, we have seen the dawn of a new infectious disease era in which mostly quiescent arboviruses have begun to aggressively emerge and reemerge around the globe, including dengue virus, west nile virus, and chikungunya virus. all the tropical world and much of the temperate world is now at risk and is likely to remain at risk for the foreseeable future. how we deal with the zika pandemic is likely to become a roadmap for future challenges. this issue of the journal outlines some of the important thoroughfares on that roadmap and will hopefully contribute to the difficult journey ahead. notes supplement sponsorship. this work is part of a supplement sponsored by the national institute of allergy and infectious diseases (niaid), part of the national institutes of health (nih). potential conflicts of interest. all authors: no reported conflicts. ueber die neueren fortschritte in der pathologie, mit besonderer beziehung auf öffentliche gesundsheitspflege und aetiologie. (rede, gehalten in der zweiten allgemeinen sitzung der deutsches naturforscher-versammlung zu frankfurt a m am 20 september 1867) in: virchow r, ed. gesammelte abhandlungen aus dem gebiete der öffentlichen medicin und der seuchen-lehre. erster band. xi. berlin: august hirschwald the perpetual challenge of infectious diseases zika virus in the americas-yet another arbovirus threat history and emergence of zika virus epidemiology of zika virus infection quantifying zika: advancing the epidemiology of zika with quantitative models modes of transmission of zika virus zika virus mosquito vectors: competence, biology and vector control zika virus evolution and spread in the americas diagnosis of zika virus infections: challenges and opportunities a zika virus from america is more efficiently transmitted than an asian virus by aedes aegypti mosquitoes from evolutionary enhancement of zika virus infectivity in aedes aegypti mosquitoes meeting the challenge of epidemic chikungunya zika virus structure, maturation and receptors neurological implications of zika virus in the adult population enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity antibody-dependent enhancement of infection and the pathogenesis of viral disease zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus humoral immune responses against zikv infection and the importance of pre-existing flavivirus immunity small animal models of zika virus non-human primate models of zika virus infection, immunity and therapeutic development zika virus (zikv) infection in pregnancy: maternal, fetal and neonatal considerations pathogenesis of other congenital viral infections small molecules and antibodies for zika therapy zika virus vaccine development zika vaccines: role for controlled human infection clinical development strategies and considerations for zika vaccine licensure all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-255927-0tp4ig4o authors: hayman, david t s title: african primates: likely victims, not reservoirs, of ebolaviruses date: 2019-11-15 journal: j infect dis doi: 10.1093/infdis/jiz007 sha: doc_id: 255927 cord_uid: 0tp4ig4o nan ebola virus disease (evd) kills almost half those people infected. four different viruses from 4 ebolavirus species have caused evd in africa: zaire ebolavirus (ebov), sudan ebolavirus (sudv), bundibugyo ebolavirus (bdbv), and tai forest ebolavirus (tafv), with all but tafv causing fatal human disease. outbreaks have been sporadic and unpredictable, but the frequency and size of outbreaks appear to be increasing [1, 2] . the ongoing outbreak in the democratic republic of the congo (drc) is the second largest on record, with >500 cases and >290 deaths reported from 15 health zones [3] . the largest evd outbreak, originating in guinea, west africa, in 2013 [4] , ended in 2016 after 28 616 cases and 11 310 deaths [5] . preventing evd outbreaks is challenging because the "reservoir hosts" of the viruses that cause the disease are not known. the weight of evidence suggests that fruit bats are the natural hosts, but this is uncertain [6] . the uncertainty is partially because most outbreaks in people are not directly linked to bats. circumstantial evidence linked bats to the 2013 west african outbreak [7] , but index case exposure to bats has only once been reported with any confidence [8] . in contrast, hunting or butchering primates has been linked to several evd index cases. in particular, africa's great apes, gorillas and chimpanzees, have been sources of human infection, and human evd outbreaks have occurred concurrently with outbreaks in apes in central and west africa [9, 10] . high case fatality rates among apes [11] [12] [13] , however, suggest they are not maintenance reservoir hosts [14] . wildlife mortality events during evd outbreaks have involved other mammals, including monkeys, pigs, and antelope [15] . contact with monkeys has been reported in human outbreaks in central africa [16, 17] and chimpanzees in ivory coast [10] . monkeys themselves appear to be susceptible to ebov infection, at least experimentally [18] . outside of africa, reston ebolavirus (restv) has been linked to monkeys, with macaques imported to the united states from the philippines infected [19] , but the mammals linked to restv in asia are similar to africa, with pigs, monkeys, and bats all implicated as hosts [20] [21] [22] . serological data may be well suited for surveillance studies, because antibodies are longer lasting than viral infection and provide evidence of survival. experimental evidence suggests that ebov infection in bats may be acute, nonfatal, and short-lived, but induces antibodies [23] . this experimental work is supported by field data from related marburg viruses, first identified after african monkeys infected people in europe [24] , which apparently persist within large colonies of cave-dwelling egyptian fruit bats, and restv in asian bats. in both cases, viruses or viral rna and antibodies were detected in apparently healthy bats [22, 25] . just 1 study has detected ebov rna in bats, but anti-ebov antibodies are widespread in african bats and the rna-positive bats were, again, apparently healthy [11, [26] [27] [28] [29] . in contrast, while anti-ebov antibodies have been observed in african apes and monkeys [30, 31] , suggesting that nonlethal infections might occur, the prevalence of antibodies is low (similar to those reported for restv in asian macaques [21] ), and ebov rna has been isolated from dead apes [32] . thus, together the evidence for bats being the true reservoir host for evd causing viruses is convincing, but relies on serological evidence of infection rather than virus detection, and the role of nonhuman primates as reservoirs remains uncertain. the role of primates in evd epidemiology has been unclear largely because study sample sizes have been small. serology is further complicated by different methodologies and antibody-positive sera cross-reacting among different evd-causing viruses. a report by ayouba et al, in this issue of the journal of infectious diseases, has taken a significant step toward addressing these problems [33] . the team utilized a large sample (n = 4649) of tissues from multiple species of african primates, collected from 1999 to 2016 from ivory coast in west africa and drc and cameroon in central africa. the study more than triples the number of all previous primate samples reported and is similarly powered to some studies showing high seroprevalence of anti-ebov antibodies in certain african fruit bat species [26, 27] . a single luminex-based serological assay that included antigens from 4 viruses (ebov, sudv, bdbv, and restv) was used, and the team discovered that none of 2327 ape samples and only 1 of 2322 monkey samples met their seropositive criteria. the data strongly suggest that the primates sampled are unlikely reservoir hosts. the work highlights the importance of multiyear, multisite empirical studies and archiving samples. specimen collection in general has created some controversy in areas such as conservation biology [34] , but for epidemiologists tissue archives may enable us to better understand the epidemiology of infectious diseases. here, the primate samples were collected for lentivirus research (eg, human immunodeficiency virus [hiv] and its relatives), then repurposed for evd research. in other systems, archived sample banks have helped identify middle east respiratory syndrome coronavirus-seropositive camels in east africa over 11-year (kenya) and 30-year (sudan and somalia) periods, suggesting extensive virus circulation in camels prior to the first human outbreaks [35] [36] [37] [38] . some impressive examples of using archaeological samples have led to the sequencing of yersinia pestis genomes from black death victims in london, england, dated to 1348-1350 [39] , and bronze age hepatitis b viral dna [40] . the instability of rna viruses will prevent paleovirological studies on these timeframes, though gene sequencing from archived samples has helped identify hiv type 1 (hiv-1) sequences predating the first aids diagnosis, with hiv sequences from 1959 and 1960 in drc informing our understanding of pandemic hiv-1 origins and evolution [41, 42] . ideally, evd-causing viruses themselves will be isolated in space and time through wildlife surveillance to understand viral transmission dynamics. phylogenetic models that estimate the relationship between genetic sequences have been used with sample location data to place the first 1976 case from drc near the root of the ebov phylogenetic tree, suggesting that all other known outbreaks descended from a closely related virus [43] . although the analysis contained just a few viral fragments, it suggested that later outbreaks were epidemiologically linked and occurred in a wave-like pattern, spreading at approximately 50 km per year. once ebov rna fragments were discovered in bats, the same team used similar models to reconstruct the ancestry of ebov, including fragments of viral rna from bats [44] . their analyses suggested that all of the genetic variation present in ebov, including from fruit bats, was the product of mutations accumulated within a 30-year time period, supporting the ancestry of ebov in bat reservoirs and the role of bats in ebov epidemiology. the absence of robust data on ebola virus reservoirs makes forecasting when and where outbreaks may occur difficult, limiting preventive measures [45, 46] . the lack of data relating to bats themselves led researchers to characterize the traits of all filovirus-seropositive and virus-positive bat species to predict potential undetected bat species [47] . putative bat hosts have been included in models to predict the spatial risk of human outbreaks [48] . similar modeling approaches have been used to model the spatial and temporal risk of human and ape evd, finding the greatest risk during wet to dry season transitions in sparsely populated regions of tropical africa [49] , supporting previous work [50] . all of these studies are limited by data, but ayouba et al's comprehensive study supports the assumption that bats, not primates, are likely reservoir hosts and that nonhuman primates may be viewed as both sentinels for human infection and victims of evd [9, 15, 33, 51] . these are important findings because they can inform field and surveillance studies, which are costly and difficult in most areas where evd outbreaks occur and for the species linked to evd. to really manage and prevent evd, however, we also need to understand why outbreaks appear to be increasing in frequency. recent analyses of forest fragmentation and evd emergence suggest there may be links [52, 53] . if so, there may be management options that can be implemented alongside human and wildlife surveillance and public health interventions to reduce the risk of human and, potentially, primate evd emergence in the first place. financial support. the author was supported by the rutherford discovery fellowship (award number rdf-mau1701) and the marsden fund (award number mau1503). potential conflicts of interest. author certifies no potential conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. temporal and spatial analysis of the 2014-2015 ebola virus outbreak in west africa ebola (ebola virus disease) world health organization. ebola virus disease-democratic republic of the congo emergence of zaire ebola virus editorial commentary • jid 2019:220 (15 november) • 1549 disease in guinea situation report: ebola virus disease filoviruses in bats: current knowledge and future directions investigating the zoonotic origin of the west african ebola epidemic human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo wild animal mortality monitoring and human ebola outbreaks, gabon and republic of congo ebola virus outbreak among wild chimpanzees living in a rain forest of côte d'ivoire fruit bats as reservoirs of ebola virus catastrophic ape decline in western equatorial africa identifying reservoirs of infection: a conceptual and practical challenge morbidity and mortality of wild animals in relation to outbreaks of ebola haemorrhagic fever in gabon ebola virus disease in the democratic republic of congo clinical management of patients and deceased during the ebola outbreak from transmission of ebola virus (zaire strain) to uninfected control monkeys in a biocontainment laboratory preliminary report: isolation of ebola virus from monkeys imported to usa discovery of swine as a host for the reston ebolavirus epidemiology of ebola (subtype reston) virus in the philippines molecular evidence of ebola reston virus infection in philippine bats experimental inoculation of plants and animals with ebola virus a hitherto unknown infectious disease contracted from monkeys seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus spatial and temporal patterns of zaire ebolavirus antibody prevalence in the possible reservoir bat species long-term survival of an urban fruit bat seropositive for ebola and lagos bat viruses ebola virus antibodies in fruit bats a serological survey of ebola virus infection in central african nonhuman primates ebola and rift valley fever virus antibodies in east african primates isolates of zaire ebolavirus from wild apes reveal genetic lineage and recombinants extensive serological survey of multiple african nonhuman primate species reveals low prevalence of immunoglobulin g antibodies to 4 ebolavirus species specimen collection: an essential tool antibodies against mers coronavirus in dromedary camels isolation of a novel 1550 • jid 2019:220 (15 november) • editorial commentary coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus, in a patient transferred to the united kingdom from the middle east a draft genome of yersinia pestis from victims of the black death ancient hepatitis b viruses from the bronze age to the medieval period direct evidence of extensive diversity of hiv-1 in kinshasa by 1960 an african hiv-1 sequence from 1959 and implications for the origin of the epidemic wavelike spread of ebola zaire recent common ancestry of ebola zaire virus found in a bat reservoir field investigations of an outbreak of ebola hemorrhagic fever, kikwit, democratic republic of the congo, 1995: arthropod studies search for the ebola virus reservoir in kikwit, democratic republic of the congo: reflections on a vertebrate collection undiscovered bat hosts of filoviruses mapping the zoonotic niche of ebola virus disease in africa spatiotemporal fluctuations and triggers of ebola virus spillover trigger events: enviroclimatic coupling of ebola hemorrhagic fever outbreaks multiple ebola virus transmission events and rapid decline of central african wildlife habitat fragmentation, biodiversity loss and the risk of novel infectious disease emergence the nexus between forest fragmentation in africa and ebola virus disease outbreaks key: cord-257521-1amcsgmj authors: hirsilä, maija; kauppila, jaana; tuomaala, katri; grekula, birgitta; puhakka, tuomo; ruuskanen, olli; ziegler, thedi title: detection by reverse transcription–polymerase chain reaction of influenza c in nasopharyngeal secretions of adults with a common cold date: 2001-04-15 journal: j infect dis doi: 10.1086/319675 sha: doc_id: 257521 cord_uid: 1amcsgmj the lack of practical methods for a laboratory diagnosis of influenza c virus infections and the seemingly benign nature of the virus contribute to the fact that 50 years after its first isolation, relatively little is known about the epidemiology and the clinical impact of this virus. reverse transcription–polymerase chain reaction (rt-pcr) was used to amplify influenza c rna fragments from clinical specimens. two hundred otherwise healthy adults with recent onset of a common cold were studied. nasopharyngeal aspirates were collected at entry to the study and 1 week later. serum samples for antibody determinations were obtained at the first visit and after 3 weeks. influenza c was detected in 7 of the 200 patients by 2 different rt-pcr formats. all 7 patients had a significant increase in antibody titers between serum samples collected during the acute and convalescent phases of the illness. influenza c appears to be one of the many viruses that cause acute upper respiratory tract infections in adults antibodies in acute-and convalescent-phase serum samples from these individuals. fifty-nine men (mean age, 24.0 years) and 141 women (mean age, 24.1 years) were recruited from october 1994 through november 1995 for a study of the etiology of the common cold [6] . criteria for inclusion were the presence of acute rhinorrhea, nasal congestion, and/or sore throat of !48-h duration. a recent upper respiratory tract infection, tonsillitis, a history of allergic rhinitis, any chronic illness, or use of regular medication were reasons for exclusion. an npa was collected at entry to the study and at the first follow-up visit 1 week later. npas were kept frozen at ϫ70њc for р5 years before being analyzed in this study. for antibody testing, serum samples were obtained at study entry and 3 weeks later and were kept at ϫ20њc until use. virus propagation was done in madin-darby canine kidney cells (mdck-i-n; provided by h. nishimura, yamagata university school of medicine, yamagata, japan). cells were grown and maintained at 36њc in dulbecco's modified eagle medium (dmem) supplemented with 0.2% bovine serum albumin, 25 mm hepes, and antibiotics (dmem-bsa) containing 7% fetal bovine serum (fbs). influenza c/ann arbor/1/50 was grown in mdck-i-n cells, using dmem-bsa supplemented with 4 mg/ml of 1-tosylamide-2-phenylethyl chloromethyl ketone-trypsin (tpcktrypsin). five days after inoculation, medium was collected, was clarified by centrifugation, and was stored in aliquots at ϫ80њc. this virus stock served as positive control in the rt-pcr and was used in the antibody assay. viral rnas were extracted from npas by use of a commercial kit (nucleospin virus; macherey-nagel). influenza c virus rna was reverse transcribed and was amplified by 2 separate rt-pcrs, using 2 sets of primer pairs (table 1), one specific for the hemagglutinin-esterase fusion protein gene (hef) [4] and the other for the nonstructural protein gene (ns-1) [5] . the reaction mixture contained 3 mm mgcl 2 , each dntp (finnzymes) at 0.25 mm, 9.4 pmol of the forward primer, which also primed the cdna reaction and the reverse primer, 15 u of rnasin (promega), 2.5 u of dna polymerase (dynazyme; finnzymes), 6 u of the enzyme avian myeloblastosis virus reverse transcriptase (promega), and 10 ml of extracted rna. cdna was synthesized at 42њc for 45 min, followed by a 10-min incubation at 95њc. thereafter, cdna was amplified by 35 cycles consisting of denaturation at 95њc for 30 s, primer annealing at 50њc for 30 s, and primer extension at 72њc for 30 s. in the final cycle, primer extension was continued for 5 min. rna from c/ann arbor/1/50 served as positive control in each run. reaction tubes containing water instead of rna were included as negative controls. amplified products were detected by electrophoresis in 1% agarose gel after ethidium bromide staining. an immunoperoxidase staining assay was developed for the detection of influenza c-specific antibodies. ninety-six-well microtiter plates (nunc) were seeded with mdck-i-n cells. approximately cells in 200 ml of dmem-bsa containing 4% fbs were 4 2.5 ϫ 10 added to each well, and the plates were incubated at 36њc in a humidified 5% co 2 atmosphere. when the cells were confluent, 100 ml of the growth medium was replaced by 100 ml of dmem-bsa containing c/ann arbor/1/50 and 8 mg/ml of tpck-trypsin. the plates were centrifuged at 700 g at ambient temperature for 45 min and were incubated at 34њc for 2-3 days. the amount of virus was chosen so that ∼10%-20% of cells became infected. the cells then were washed with pbs, fixed with 80% acetone in pbs at room temperature for 10 min, and washed twice again with pbs, and the plates were kept at 4њc until use within 1 week. serum samples were diluted in pbs containing 5% nonfat dry milk powder (pbs-m) and were tested in serial 4-fold dilutions starting at 1:200. a known positive serum sample and fbs as a negative control were included on each plate. serum incubation was done at 37њc for 60 min. after the wells were washed with pbs, peroxidaselabeled rabbit antibodies to human igg (dako) diluted 1:10,000 in pbs-m were added to each well, and plates were incubated at 37њc for 60 min. the wells were washed again, and 40 ml of trueblue substrate (kirkegaard & perry) was added to each well for 30 min. wells were rinsed twice with pbs, and the plates were read with an inverted microscope at ϫ100 magnification. distinct blue cytoplasmic and nuclear staining of infected cells in the absence of background staining of uninfected cells was considered to be a positive result. a у4-fold increase in titer between acute and convalescent serum was considered to be a significant result. with c/ann arbor/1/50, the detection limit of the 2 rt-pcr protocols was ∼1 tcid 50 with the ns-1 primer pair and 10 tcid 50 with the hef primers, respectively. of the 200 npas collected during the first visit, 7 showed a distinct band of appropriate size with both primer pairs (table 2). only 1 of the 7 patients from whom these samples were obtained was still positive by pcr at the first control visit 1 week later, but a signal was detected only with the ns-1 primer pair. the remaining 193 patients had negative pcr results at the initial visit. the second npa of 107 randomly selected patients with initially negative pcr results also was tested by both pcrs, but none yielded positive signals. influenza c-specific antibodies were detected in all study participants. a у8-fold increase between acute-and convalescent-phase serum samples was found in all 7 individuals with pcr-positive results (table 2). in addition, 1 study participant with pcr-negative results had an 8-fold increase in titer. no significant fluctuation of antibody levels was detected in the remaining 192 individuals with pcr-negative results. all 8 influenza c cases occurred during winter and spring. the duration of illness in the 7 patients with pcr-positive results varied, 3 of whom had a fever of 1 or 2 day's duration, from 9 to 19 days (table 2). sinusitis was confirmed radiologically in 4 of the 8 individuals with laboratory evidence for a recent infection with influenza c (table 2) . to our knowledge, this is the first successful attempt to detect influenza c rna in clinical specimens by means of rt-pcr. of 200 adults with a common cold of !48-h duration, 7 were diagnosed with an influenza c infection. specimens of all 7 patients were found to be positive by both rt-pcrs, and all specimens were found to be positive on repeated examination. no other pathogen could be identified for any of these influenza c-positive patients (results not shown). all 7 individuals with pcr-positive results had a у8-fold increase in antibody titers between serum samples collected during the acute and convalescent phases of the illness. this further underlines the specificity of our pcr results. in addition, 1 study participant with pcr-negative results also had a significant increase in antibody titer (patient 160; table 2). the titer found in the first serum sample from this patient was as high as those in the convalescent-phase serum samples of the 7 patients with pcr-positive results; however, over the 3-week observation period, this patient showed an 8-fold increase in antibody titer to influenza c. this patient also had a significant titer increase to coronavirus (results not shown). this person possibly experienced an influenza c infection a few weeks earlier and entered the study because of an acute infection with a coronavirus. with laboratory-grown virus material, the sensitivity of both pcrs was relatively low. however, if a significant increase in antibody titers is accepted as definite proof of recent infection with a virus, the diagnostic sensitivity of our pcr assays is satisfactory. prolonged storage of npas at ϫ70њc and repeated freezing and thawing evidently did not affect the ability to detect virus-specific gene sequences in these specimens. npas obtained from the 7 patients with rt-pcr-positive results at the first control visit 1 week after study entry also were tested. only one of these patients still had pcr-positive results, which indicates that the virus is rapidly cleared from the upper respiratory tract. furthermore, this patient also had a longer duration of fever and illness than all other influenza c-positive patients (patient 162; table 1). although rt-pcr may be a rather sophisticated technique for diagnosing a relatively benign respiratory virus, the antibody assay described is easy to perform and allows for the simultaneous testing of numerous specimens. intense nuclear staining of infected cells indicates that this assay predominantly measures antibodies to the nucleoprotein and the matrix protein, which is in contrast to the strain-specific hemagglutination inhibition (hi) test, which measures antibodies to hef. as in influenza a and b, the nucleoprotein and matrix protein of influenza c virus are well preserved through evolution and thus offer a reliable source of antigen in the absence of more recent isolates, which could be used in hi tests [8] . in an earlier study, an eia was shown to be more sensitive than the hi test in detecting titer increases [9] . in that study, the antigen probably consisted mainly of nucleoprotein and matrix protein. none of our 200 patients was seronegative to influenza c, which is in accordance with another study in which high seroprevalence and antibody titers were found in adults by means of eia [9] . however, an epidemiologic study in france found a seropositivity rate of !80% in the age group reflecting our study population [10] . the authors of that study used a highly purified virus antigen in an eia. it remains unclear why a significant fraction of their study population lacked antibodies to influenza c. it is likely that a virus causing acute upper respiratory tract infection in adults would readily and efficiently infect susceptible younger individuals; therefore, antibodies should be detectable in virtually all adults. evidently all our influenza c patients experienced re-infection with this virus. all 7 influenza c-positive cases occurred from december through may. however, the number of positive findings is too small to draw conclusions on the epidemiology and seasonality of the virus. in a recent study from israel, 2 cases of influenza c were identified during outbreaks of influenza a and b during the winter of 1996-1997 [11] . the common cold is an illness of viral etiology. before this study, a viral cause was established for 148 of our 200 patients [6] , whereas evidence for a bacterial infection was found in only 7 patients. yet, a large proportion of patients with a common cold seeking medical attention receive antibiotic treatment [12] . sinusitis, a frequent complication of the common cold, is considered to be of bacterial origin. in a recent study, though, most patients with acute sinusitis in connection with a common cold lacked laboratory parameters that are indicative of bacterial infection [7] . a better understanding of the common cold's etiology and accumulation of information about the inflammatory processes that cause the characteristic symptoms may result in a more rational use of existing drugs and in accelerated research toward new therapeutic interventions. with an incidence of 3.5% in our study, influenza c caused as many cases of common cold as did parainfluenza viruses; as many episodes as adenoviruses, respiratory syncytial virus, and enteroviruses combined; and an equal number of cases as the 4 bacterial pathogens chlamydia pneumoniae, mycoplasma pneumoniae, streptococcus pneumoniae, and haemophilus influenzae [6] . our findings indicate that influenza c is a relevant pathogen that causes acute respiratory infections. influenza viruses community-acquired influenza c virus infection in children concepts and procedures for laboratorybased influenza surveillance detection and identification of human influenza viruses by the polymerase chain reaction type-specific identification of influenza viruses a, b, and c by the polymerase chain reaction viruses and bacteria in the etiology of the common cold sinusitis in the common cold epidemiology of influenza c virus in man: multiple evolutionary lineages and low rate of change comparison of enzyme-linked immunosorbent assay and hemagglutination inhibition in a seroepidemiological study of influenza type c infection influenza c virus infection in france isolation of influenza c virus during an outbreak of influenza a and b viruses antibiotics and upper respiratory infection: do some folks think there is a cure for the common cold? age a /sex key: cord-010638-xjtapifg authors: law, carmella l. h.; qassim, mohammed; cunningham, anthony l.; mulhall, brian; grierson, jean m. title: nonspecific proctitis: association with human immunodeficiency virus infection in homosexual men date: 1992-01-17 journal: j infect dis doi: 10.1093/infdis/165.1.150 sha: doc_id: 10638 cord_uid: xjtapifg in a cross-sectional study of 140 homosexual men attending a sexually transmissible diseases clinic, the association between the presence of antibody to the human immunodeficiency virus (hiv) and the presence of proctitis, as determined by histologic examination, as well as past or present exposure to other pathogens and details of sexual practices was analyzed. significant associations with hiv seropositivity were found with the number of lifetime partners, positive treponemal serology, and evidence of previous infection with herpes simplex virus. however the major and unique finding was the strong and independent association between proctitis diagnosed by histologic criteria and seropositivity for hiv. whether this is cause or effect awaits further elucidation. in a cross-sectional study of 140 homosexual men attending a sexually transmissible diseases clinic, the association between the presence of antibody to the human immunodeficiency virus (hiv) and the presence of proctitis, as determined by histologic examination, as well as past or present exposure to other pathogens and details of sexual practices was analyzed. significant associations with hiv seropositivity were found with the number of lifetime partners, positive treponemal serology, and evidence of previous infection with herpes simplex virus. however the major and unique finding was the strong and independent association between proctitis diagnosed by histologic criteria and seropositivity for hiv. whether this is cause or effect awaits further elucidation. among homosexual men, acquisition of human immunodeficiency virus (hiv) has been associated with multiple sexual partners and with receptive anal intercourse [1] [2] [3] . al-though proctins is a well-described phenomenon in homosexual men, and when it is associated with symptoms it is often associated with sexually transmissible agents [4] , no pathogenic agent is identified in as many as 26% of patients [5] . we investigated the association of rectal inflammation (proctitis), as determined by histologic examination, in homosexual men attending a sexually transmissible diseases (std) clinic and the presence of antibodies to hiv. patients. subjects were drawn from consecutive homosexual or bisexual males attending the std centre, sydney hospital, and seen by one of the authors (el.) in the period march 1985 march -1988 . they were mostly healthy, were attending for std and hiv screening, and were identified as either homosexual or bisexual on the basis of the sexual history obtained. a self-administered questionnaire was given to each subject to record past medical and std history and details of sexual practices. for this project, we analyzed the data on 140 ofthe original 200 enrolled in a study on std and enteric infections in homosexual men, who had completed questionnaires, undergone rectal biopsies, and turned in the full complement of fecal specimens. a comparison of the study group and the group that was excluded due to incomplete data indicated that there was no significant difference in age, sexual practices, or gastrointestinal symptoms. screening tests for std consisted ofcultures for neisseria gonorrhoeae and chlamydia trachomatis from the rectum, urethral meatus, and pharynx and culture for herpes simplex virus (hsv) from the rectum alone. three rectal biopsies were taken under direct vision from each individual at 15, 10, and 5 ern from the anal verge using flexible colonoscopy biopsy forceps. clinical features of proctitis such as contact bleeding with rectal mucosal congestion, presence of a mucopurulent exudate, or superficial ulceration were noted. blood was collected for routine laboratory investigations. each subject was also asked to deliver fecal samples on 3 separate days over a period of 1-2 weeks. laboratory tests. screening tests for treponemal infections were done using standard methodology: venereal diseases reference laboratory (vdrl) and treponema pal/idum hemagglutination (tpha) tests. swabs for culture of n. gonorrhoeae were directly innoculated onto modified new york media, and those for culture of c. trachomatis and hsv were placed into transport media and subsequently innoculated onto buffalo green monkey cells and human embryonic fibroblast cells, respectively. serologic tests for previous infection with hsv were done using a herpes simplex virus complement fixation test (hsv cft) and for hiv-i antibody by elisa (abbott laboratories, north chicago; wellcozyme, wellcome diagnostics, beckenham, uk). positive antibody tests were confirmed by western blot (dupont, carlingford, australia). lymphocyte subpopulations were quantified using fluorescein-conjugated monoclonal antibodies to leu-2a, leu-sa, and leu-4 (becton dickinson, mountain view, ca) and flow cytometry (facs 440; becton dickinson, sunnyvale, ca). total and differential white blood cell counts were done on edta-treated blood in a coulter counter. two rectal biopsies (from 5 and 10 em) were routinely handled after formalin fixation, paraffin embedding, sectioning, and hematoxylin-eosin staining. they were examined by an experienced histopathologist (1.m.g.) and graded as follows. grade i, normal biopsy as defined by the presence of an inflammatory cell infiltrate in the lamina propria within the usual subjective normal limits, that is, a scattered mixed cellular infiltration of plasma cells, lymphocytes, eosinophil leukocytes, mast cells, and macrophages, seen in small numbers and distributed predominantly within the superficial part of the lamina propria. in this grade, no associated inflammatory vascular, epithelial, or crypt injury was present. grade ii, chronic proctitis as defined by an increase in chronic inflammatory cells throughout the lamina propria with minimal vascular, epithelial, and crypt changes. grade iii, acute proctitis or active chronic proctitis as defined by a marked or intense infiltration of acute and chronic inflammatory cells throughout the lamina propria, with vascular, epithelial, and crypt damage as well as possible mucosal ulceration. this grading was influenced by previous studies of a similar nature [6] . the histologic assessment also allowed for reporting of other diagnostically important changes. these included traumatic changes, the presence of some organisms, foreign body and other granulomas, cases of classic ulcerative, pseudomembranous, and collagenous colitis, and the presence of tumors. the histopathologist was blinded to the clinical history and other laboratory data. specimens from each of the 3 days were screened for cysts, ova, and parasites, and specimens from the first 2 days were cultured for bacteria (salmonel/a, shigel/a, aeromonas, campylobacter, and yersinia, species and clostridium diffici/e); organisms were identified by standard methods. the first-day specimens were also examined for viruses by electron microscopy and culture [7] . statistical analysis. pearson's x 2 test with continuity correction and fisher's exact test, where appropriate, were used to examine the association between the variables collected and proctitis or a positive hiv antibody result. all variables considered statistically or biologically significant were entered into a logistic regression model to evaluate interactions while controlling for possible confounders. pearson's correlation coefficient (r) was used to examine the collinearity between the variables that were finally entered into the logistic regression model. therefore, if two variables were found to be highly correlated, one of them was removed from the analysis as both ofthem were predictive of the same outcome. all 140 subjects underwent rectal biopsies, but some information was not available for < 14% of the subjects. the mean age of the study group was 34.4 years (range, 21-68). the median number of partners over the subjects' lifetimes was 150 (range, 2-6400) and the mean number of years of homosexual activity was 15.4 (range, 2-40). sexual practices were divided into "never," "sometimes," and "usually" categories, with most individuals admitting to having ever engaged in oral-genital contact (95%), receptive anal intercourse (91 %), and insertive anal intercourse (90%). a lower percentage admitted to other sexual practices: oralanal contact (53%), use of enemas or dildos (34%), or fisting (8%). because the study group was recruited from an std center, it was not unexpected that the percentage seropositive for hiv was higher than that reported for homosexual or bisexual men by another primary care service in a general practice setting in sydney (30%) over a similar period of time [8] . of the 63 individuals (45%) who tested positive for hiv antibody, only 1 presented with centers for disease control class iv disease (overt aids), 15 had class iii disease (persistent generalized lymphadenopathy), and the rest were class ii (asymptomatic) [9, 10] . the t cell helper-to-suppressor (cd4+:cd8+) ratio was within the normal range (1.2-4.1) in 76 patients (54%), while in 46% the ratio was below the normal range for our laboratory. the absolute values oft helper (cd4+) cells were <500 and~500 x 10 9/1 in 24% (33) and 76% of patients (107), respectively. the majority (56%, 79/ 140) had absolute t suppressor values (cd8+) within the normal range (230-660 x 10 9/1), but 41 % and 2% of patients, respectively, had values above or below these limits. the mean absolute numbers of cd4+ and cd8+ cells were 806 (range, 200-2400 x 10 9/1) and 781 (range, 140-2530 x 10 9/1), respectively. of the subjects, 87% had serologic evidence (hsv cft) of exposure to hsv, either type 1 or 2. however, in 7% (10/ 126), hsv-2 was isolated from rectal swabs. twenty-eight percent had serologic evidence of previous exposure to a treponemal infection, with reactive tpha in association with either a nonreactive or weakly reactive vdrl. twentytwo percent of the study group (25/113) had anal warts on clinical examination. the prevalence of rectal chlamydial infection was extremely low in this study, with only one case; that patient's rectal biopsy was normal. n. gonorrhoeae was not cultured from any of the rectal swabs. inflammatory changes were noted in the rectal biopsies in 34 patients (24%) and in 17 (12%) were clinically apparent at sigmoidoscopy. these changes were generally mild and consisted of contact bleeding with rectal mucosal congestion; occasionally a follicular appearance or the presence ofmucopurulent exudate was also noted. none of the patients had ulcerated mucosa on macroscopic examination via the sigmoidoscope. almost all patients with proctitis were rated histologic grade ii with chronic inflammatory cells. only one patient had grade iii or acute changes, and the proctitis was not associated with any particular pathogen. potentially pathogenic viruses were carried by 13% (16/ 123): rotavirus in 6, enterovirus in 3 cases, and echovirus type 7, coronavirus, and adenovirus in 2 each. bacterial pathogens were identified in 3%(4/122): yersinia enterocolitica in 3 and shigella flexneri in 1. interestingly, although 34 (26%) of 133 reported occasional and nonspecific gastrointestinal symptoms on direct questioning, there was no association between the detection of any of these organisms and gastrointestinal symptoms or proctitis. in the case of the latter, the only exception was with rectal hsv isolation. factors associated with proctitis. table 1 shows the relationship between sexual practices, hiv antibody, t cell subset measurements, hsv isolation from a rectal swab, tpha, hsv cft, and proctitis. x 2 analysis revealed proctitis to be significantly associated with: hiv antibody (p < .001), cd4+ count <500 x 10 9/1 (p = .01), cd4+:cd8+ ratio < 1.2 (p < .05), rectal hsv isolation (p = .02), and anal warts (p = .02). all the factors significantly associated with proctitis on x 2 analysis were entered into a logistic regression model together with receptive anal intercourse, use of enemas or dildos, hsv cft and tpha results, rectal hsv isolation, and presence of anal warts. the latter were included because of the likelihood of syphilis, anorectal herpes, and trauma from anal intercourse and pleasuring devices playing a contributory role in proctitis. hiv seropositivity (odds ratio [or], 6.5; 95% confidence interval [ci], 2.3-18.6), rectal hsv isolation (or, 5.2; 95% ci, 1.2-23.5), and anal warts (or, 4.9; 95% ci, 1.6-15.1) remained highly significant, with p values of <.001, .01, and .02, respectively. because of collinearity between cd4+ absolute number and hiv seropositivity (r = .68), the significant association between the former and proctitis was lost when hiv antibody result was included in the model. factors associated with hiv seropositivity. the association between sexual practices, t cell subset measurements, proctitis, and hsv cft, tpha, and hiv antibody results was examined using x 2 analysis (table 2) . there was a highly significant association between histologically confirmed proctitis (p < .001) or evidence of past exposure to treponemal infection (p < .01) and less significant associations with past exposure to hsv infection (p < .05) and number of lifetime partners (p < .05). not unexpectedly, there were significant associations between hiv antibody result and cd4+ and cd8+ absolute numbers and cd4+:cd8+ ratio, with p values of <.01, .001, and <.001, respectively. when the variables found significant by x 2 analysis and receptive anal intercourse data were entered into a logistic regression model with hiv antibody result on the basis of their known association with hiv seropositivity, proctitis (or, 8.2; 95% ci, 2.1-31.8), reactive tpha (or, 3.6; 95% ci, 1.4-9.2), and positive hsv cft (or, 5.3; 95%ci, 0.9-31.2) emerged as independently associated with hiv seropositivity, with pvaluesof <.001, .003, and .04, respectively. as described by others, number of lifetime partners (or, 5.5; 95%, ci, 1.8-17.0), cd8+ absolute numbers (or, 3.2; 95% ci, 1.1-9.6), and cd4+:cd8+ ratio (or, 22.1; 95%ci, 6.7-73.7) were also independently associated with hiv seropositivity, with p values of .01, <.01, and <.001 respectively. because of high collinearity between cd4+ absolute number and cd4+:cd8+ ratio (r = .62), the significant association between the former and hiv antibody result was lost when they were both included in the model. both were predictive of hiv seropositivity. we examined first the associations of different variables with proctitis and then the associations of these same variables with the presence ofhi v antibody. as expected, significant associations with hiv seropositivity were found for the number of lifetime sex partners, reactive treponemal serology, and evidence of previous infection with hsv. these results are consistent with those of other investigations [1] [2] [3] 5] . however, the major unexpected finding was the strong association between proctitis diagnosed by histologic criteria and seropositivity for hiv -1. this effect persisted after controlling for a number of potential interactions and confounders such as sexual practices, immunologic status, reactive tpha, positive hsv cft, and rectal hsv isolation. whether recurrent rectal inflammation as described preceded the acquisition of hiv is unclear, since the study design was cross-sectional in nature. conventional explanations for the association between genital ulcer disease and acquisition of hi v usually rely on disruption of the mucosal integrity of the genital (or gastrointestinal) tract [11, 12] . although these explanations appear possible in this group, as conditions associated with a disruption of epithelial integrity (presence of anal warts and rectal hsv) were significantly associated with the detection of histologic features of proctitis, proctitis alone and not these factors was significantly associated with hiv antibody status. in fact, on the basis of clinical and histologic features, there was no mucosal ulceration in any patient in this group. it is possible that not all cases of transmission of hiv are reliant on mucosal ulceration. for example, the presence of inflammatory cells so closely adjacent to infectious virus may be relevant, since it is known that activated lymphocytes are more susceptible to infection [13] , and in recurrent herpes, an inflammatory infiltrate of activated cd4+ lymphocytes and macrophages is found subjacent to the epithelium [14] . the assay we used, which detected antibody to both hsv-l and hsv-2, should in future be replaced by a specific hsv-2 antibody assay, as others have found significant associations between prior hsv-2 infection and acquisition ofhi v [5] . as enteric organisms (other than hsv-2) did not appear to be significantly associated with histologically diagnosed proctitis, other mechanisms contributing to inflammatory changes may need to be investigated. for example, granulomas around foreign body debris were occasionally noted in the histologic sections of biopsies taken from the distal rectum (unpublished data) and may represent localized reactions to oilbased lubricants commonly used by homosexual men during anal intercourse. an alternative explanation for our results could be that the presence of hiv directly or indirectly modified the inflammatory response in the rectal mucosa. we were unable to exclude direct infection of the rectal mucosal cells with hiv; in other studies, this has occasionally been demonstrated in bowel biopsies by molecular techniques [15] . although the gastrointestinal tract appears to be a major target organ late in hiv infection, as indicated by tumors, opportunistic infections, and malabsorption, much remains to be learned about the gastrointestinal mucosal response to hiv. indeed, the mucosal immune response to bowel infections in general has not yet been clarified. in summary, our data suggest a strong association between proctitis diagnosed on histologic criteria and seropositivity for hiv. prospective studies are needed to elucidate this relationship. disseminated intravascular coagulation associated with group a streptococcal infection in pregnancy clinical laboratory and epidemiological investigations ofa streptococcus pyogenes cluster epidemic in a newborn nursery a nursery outbreak of group a streptococcal infection a prolonged nursery epidemic associated with a newly recognized type of group a streptococcus neonatal septicemia caused by group a beta-hemolytic streptococcus a simple sensitive method of analyzing bacterial ribosomal dna polymorphism detection of specific sequences among dna fragments separated by gel electrophoresis fingerprinting" ,6-haemolytic streptococci by their production of and sensitivity to bacteriocin-like inhibitors type 49 streptococcuspyogenes: phage subtypes as epidemiological markers in isolates from skin sepsis and acute glomerulonephritis a serological differentiation of human and other groups of hemolytic streptococci dna fingerprints of streptococcus pyogenes are m type specific ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools a broad-spectrum probe for molecular epidemiology of bacteria: ribosomal rna dna restriction fragment length polymorphism differentiates crossed from independent infections in nosocomial xanthomonas maltophilia bacteremia sexual practices and risk of infection by human immunodeficiency virus. the san francisco men's health study seroepidemiology ofhtlv-ii1 antibody in danish homosexual men: prevalence, transmission, and disease outcome risk factors for seroconversion to human immunodeficiency virus among male homosexuals. results from the multicentre aids cohort study proctitis, proctocolitis, enteritis, and esophagitis in homosexual men the association between genital ulcer disease and acquisition of hi v-i infection in homosexual men proctitis in homosexual men gastrointestinal viral infections in homosexual men who were symptomatic and seropositive for human immunodeficiency virus hiv infection in sexually transmissible disease practice in sydney: the effects of legislation, public education and changing clinical spectrum classification system for human t-iymphotropic virus type iii/lymphadenopathy-associated virus infections revision of the cdc surveillance case definition for acquired immunodeficiency syndrome genital ulcers, other sexually transmitted diseases. and the sexual transmission ofhiy the interaction ofhiy infection and other sexually transmitted diseases and opportunity for intervention cellular tropism of the human retrovirus htly-iii/lay. i. role oft cell activation and expression of the t4 antigen evolution of recurrent herpes simplex lesions. an immunohistologic study human immunodeficiency virus in bowel epithelium from patients with gastrointestinal symptoms we thank geoff holt for assistance in the clinical examinations, coding of information for analysis, and general day-to-day supervision of data collection; w. jones and s. kamath for immunology testing; peter thomson for supervision of the statistical analysis; g. berry for statistical advice on the final manuscript; and, above all, participants in the study for their dedication. key: cord-007295-lq8h1pc6 authors: koudstaal, wouter; koldijk, martin h.; brakenhoff, just p. j.; cornelissen, lisette a. h. m.; weverling, gerrit jan; friesen, robert h. e.; goudsmit, jaap title: preand postexposure use of human monoclonal antibody against h5n1 and h1n1 influenza virus in mice: viable alternative to oseltamivir date: 2009-12-15 journal: j infect dis doi: 10.1086/648378 sha: doc_id: 7295 cord_uid: lq8h1pc6 new strategies to prevent and treat influenza virus infections are urgently needed. a recently discovered class of monoclonal antibodies (mabs) neutralizing an unprecedented spectrum of influenza virus subtypes may have the potential for future use in humans. here, we assess the efficacies of cr6261, which is representative of this novel class of mabs, and oseltamivir in mice. we show that a single injection with 15 mg/kg cr6261 outperforms a 5-day course of treatment with oseltamivir (10 mg/kg/day) with respect to both prophylaxis and treatment of lethal h5n1 and h1n1 infections. these results justify further preclinical evaluation of broadly neutralizing mabs against influenza virus for the prevention and treatment of influenza virus infections that it is unlikely that an effective vaccine will be available in the early stages of a pandemic. for the treatment and/or prophylaxis of influenza virus infections, only 2 classes of drugs are currently available: the adamantanes and the neuraminidase (na) inhibitors. the adamantanes (amantadine and rimantadine) are associated with toxicity and with the rapid emergence of drug-resistant strains [3] . compared with the adamantanes, the 2 licensed na inhibitors-zanamivir (relenza) and oseltamivir (tamiflu)-are associated with little toxicity and are less prone to select for resistant viruses [3] . nevertheless, the emergence of resistance after oseltamivir treatment has been reported ( [4] and references therein). furthermore, oseltamivir-resistant h1n1 viruses are now circulating on all major continents [5] . although at present these viruses are susceptible to zanamivir, the resulting increased use of zanamivir monotherapy may well lead to the development of resistance [6] . consequently, there is an urgent need for the development of new treatments, both prophylactic and therapeutic. monoclonal antibodies (mabs) are attractive biologic drugs given their exquisite specificity and low toxicity. the development of mabs for prophylaxis and treatment of influenza has been inhibited by the lack of candidates with broad neutralizing activity resulting from the virus's tolerance for genetic changes in its immunodominant epitopes. however, a recently discovered class of mabs that are able to neutralize an unprecedented spectrum of influenza virus subtypes by binding to a highly conserved region of the membrane-proximal stem of the viral hemagglutinin holds promise as a future intervention for both seasonal and pandemic influenza [7, 8] . here, we compare the prophylactic and therapeutic efficacies of the mab cr6261, which represents this novel class of anti-influenza virus mabs, with those of the leading antiviral drug, oseltamivir, in mouse models of lethal h5n1 and h1n1 infection. methods. the human mab cr6261 has been described elsewhere [8] . an irrelevant isotype-matched antibody, cr3014 [9] , was used as a control. both antibodies were produced in per.c6 cells (crucell holland). oseltamivir (tamiflu; hoffmann-la roche) was obtained from a local pharmacy. the h5n1 strain a/hongkong/156/97 was originally isolated from a 3-year-old child with respiratory disease [10] . the virus was passaged twice in mdck cells. the stock (8.1 log 10 median tissue culture infective dose [tcid 50 ]/ml) used to infect mice was propagated once in embryonated eggs. the h1n1 strain a/wsn/33 was obtained from the american type culture collection (vr-219). the stock (8.5 log 10 tcid 50 /ml) used to infect mice was propagated once in embryonated eggs. all experiments were approved by the ethical review committee of the central veterinary institute before commencement, in accordance with dutch law. female, 7-week-old, specific pathogen-free balb/c mice (charles river laboratories) were inoculated intranasally with 25 times the median lethal dose of a/hongkong/156/97 (4.5 log 10 tcid 50 ) or a/wsn/33 (6.6 log 10 tcid 50 ), and survival, weight loss, and clinical signs were monitored until 21 days after infection. clinical signs were scored with a scoring system (0, no clinical signs; 1, rough coat; 2, rough coat, less reactive, and passive during handling; 3, rough coat, rolled up, labored breathing, and passive during handling; 4, rough coat, rolled up, labored breathing, and unresponsive). animals with a score of 4 were euthanized. in the prophylactic experiments, 15 mg/kg cr6261 was administered intravenously 1 day before challenge, and 10 mg/kg oseltamivir was administered per os daily for 5 days starting 1 day before challenge. in the therapeutic experiments, cr6261 and oseltamivir were administered as described above on day 4 and for 5 days starting on day 4 after infection, respectively. survival times after viral challenge were analyzed using the log-rank test, and survival proportions were analyzed using the fisher exact test. change in body weight was analyzed using an area under the curve (auc) analysis in which the last ob-served body weight was carried forward if a mouse died or was euthanized during the experiment. the weight per mouse on day 0 was used as baseline, and weight change was determined relative to baseline. the net aucs were compared by 1-way analysis of variance. comparisons to the control group were performed by post-hoc testing with dunnett's adjustment for multiple comparisons. clinical scores were analyzed using the genmod procedure (sas software) to fit a model to repeated measures, with mice as the subject and the data measured on an ordinal scale. statistical analyses were performed using sas (version 9.1; sas institute) and spss (version 15.0; spss) software. the statistical significance level was set at . a p .05 results. figure 1 shows the prophylactic efficacies of the mab cr6261 and oseltamivir against lethal challenge with a/ hongkong/156/97(h5n1) and a/wsn/33(h1n1). all animals that received cr6261 survived challenge with both viruses without showing any signs of disease, except for a decline in body weight of ∼9% during the first 3 days after challenge with a/ wsn/33 ( figure 1e ). in contrast, all control animals rapidly showed signs of disease, lost weight, and either died of the infection or were euthanized within 2 weeks after either challenge. all but one of the oseltamivir-treated mice survived challenge with a/hongkong/156/97 ( figure 1a) . however, in contrast to the cr6261-treated mice, these animals lost weight comparable to the control group until 11 days after infection, when they started to regain weight ( ) ( figure 1b ). fur-p ! .001 thermore, all oseltamivir-treated mice showed signs of illness beginning on day 5 after infection and started to recover on day 15 ( figure 1c) . similarly, treatment with oseltamivir prevented death but not weight loss and signs of disease on challenge with a/wsn/33. mice treated with oseltamivir lost weight similarly to the control group during the first 10 days after challenge, after which they started to regain weight ( figure 1e ), and this loss was greater than that of the group treated with cr6261 ( ). signs of disease were observed in individual p ! .001 animals between days 2 and 19 after challenge, and median clinical scores differed significantly from those of the cr6261treated group between days 2 and 17 ( ) ( figure 1f ). p р .032 the alternating median clinical scores (between 2 and 3) of this group is likely explained by subjectivity in the assessment of clinical scores by different observers. taken together, these data indicate that prophylactic administration of a single intravenous injection with 15 mg/kg cr6261 is more effective against lethal h5n1 and h1n1 challenge than a 5-day course of treatment with oseltamivir at 10 mg/kg/day. figure 2 shows the therapeutic efficacies of mab cr6261 and oseltamivir against lethal challenge with a/hongkong/156/ 97(h5n1) and a/wsn/33(h1n1). treatment with cr6261 four days after challenge with a/hongkong/156/97 did not prevent initial weight loss and signs of disease. however, it did completely prevent mortality, and animals had regained their initial body weight by the end of the experiment (figure 2a and 2b). accordingly, these mice started to recover on day 12, and none showed any signs of disease from day 19 onward. in contrast to the 100% survival in the group treated with cr6261, only 25% of the mice treated with oseltamivir survived infection with a/hongkong/156/97 ( ). these mice lost signif-p p .007 icantly more weight than did those in the group treated with cr6261 ( ), and median clinical scores were signifi-p p .001 cantly higher from day 9 until the end of the study on day 21 ( ). the 2 survivors showed respiratory distress until p р .037 days 19 and 21 and had not regained their initial body weight at the end of the study (ϫ2.4% and ϫ24.9%). although prophylactic administration of cr6261 fully protected mice against lethality from a/wsn/33 challenge, only 3 of 8 animals survived when cr6261 was given 4 days after challenge ( figure 2d) . nevertheless, survival time in this group was significantly longer than that in the group treated with oseltamivir, in which all animals died within 10 days after challenge ( ). however, there was no significant difference p p .031 in relative weight loss between both groups ( ), and p p .592 clinical scores differed significantly only on days 1 and 10 ( and , respectively) ( figure 2e and 2f). p p .021 p p .034 discussion. we have demonstrated that a single intravenous injection with 15 mg/kg cr6261 outperforms a 5-day course of treatment with oseltamivir at 10 mg/kg/day with respect to both prophylaxis against and treatment of lethal h5n1 and h1n1 infections in mice. these encouraging results in these highly stringent models justify further preclinical evaluation of cr6261 as alternative strategy for the control of influenza. the 15 mg/kg dose of cr6261 used in this study was based on previous experiments in mice [8] , but further studies are needed to assess the minimal plasma concentrations of this mab necessary for effective prophylaxis and treatment. a 5-day course of treatment with oseltamivir at 5 mg/kg/day has previously been shown to be protective against lethal challenge of mice with a/wsn/33 [11] , whereas treatments with doses as low as 1 mg/kg/day (and even 0.1 mg/kg/day) have been shown to protect mice against lethal challenge with a/ hongkong/156/97 [12, 13] . in these latter studies, oseltamivir was administered in a twice-a-day regimen, whereas we used a once-a-day regimen. however, when comparing the efficacy of 1 daily administration of 10 mg/kg oseltamivir with that of 2 daily administrations of 5 mg/kg against lethal a/hongkong/ 156/97 infection, we found no difference (data not shown). this result is in line with previously reported data showing no significant difference in the efficacy of oseltamivir at 5 mg/kg/ day against lethal a/wsn/33 infection of mice when given as a single dose or in 2 doses 12 h apart [11] . although therapeutic administration of cr6261 completely prevented death due to a/hongkong/156/97, it only partially prevented mortality due to a/wsn/33. this difference is likely explained by the fact that onset of disease and progression to death were more rapid after challenge with the latter virus. the median clinical scores at the moment of antibody administration (4 days after challenge) were 1 and 3 for the groups of mice challenged with a/hongkong/156 and a/wsn/33, respectively. accordingly, the median survival time in the control group challenged with a/wsn/33 was shorter than that in the control group challenged with a/hongkong/156/97 (7 vs 10 days; ). however, the fact that administration p p .003 of cr6261 to mice that had already lost 24.1% of their initial body weight and that showed severe respiratory distress still partially prevented death demonstrates the fast mode of action. this feature, combined with the relatively long half-life (2-3 weeks) in humans of mabs produced in per.c6 cells [14] , make human mab-based passive immunotherapy a viable option for both treatment and prophylaxis of disease due to influenza virus infection, provided that comparable effi-cacies can be obtained in humans with economically feasible doses of antibody. fact sheet 211: influenza. geneva: world health organization centers for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) medical management of influenza infection neuraminidase inhibitor resistance after oseltamivir treatment of acute influenza a and b in children world health organization. influenza a(h1n1) virus resistance to oseltamivir-2008/2009 influenza season. geneva: world health organization influenza virus resistance to antiviral agents: a plea for rational use antibody recognition of a highly conserved influenza virus epitope heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm+ memory b cells human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness influence of treatment schedule and viral challenge dose on the in vivo influenza virus-inhibitory effects of the orally administered neuraminidase inhibitor gs 4104 comparison of efficacies of rwj-270201, zanamivir, and oseltamivir against h5n1, h9n2, and other avian influenza viruses the neuraminidase inhibitor gs4104 (oseltamivir phosphate) is efficacious against a/hong kong/156/97 (h5n1) and a/hong kong/1074/ 99 (h9n2) influenza viruses first administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity we thank els de boer-luijtze for excellent technical assistance. key: cord-269324-zh1a3gwh authors: mubareka, samira; palese, peter title: human genes and influenza date: 2008-01-01 journal: j infect dis doi: 10.1086/524067 sha: doc_id: 269324 cord_uid: zh1a3gwh nan why some individuals resist infection or recover quickly, whereas others experience severe disease associated with infection, is a fundamental question that medicine has struggled to answer. pathogens and host immune factors have been extensively investigated for many infectious diseases, to address these questions. however, limited information is available concerning the influence of host genetics on the response to viral infections. genetic determinants have the potential to play a role at numerous points during the course of viral infection, including viral attachment and entry, replication, disease progression and development of severity, and, finally, transmission. in this issue of the journal, albright et al. [1] propose that the severity of influenza illness may have a heritable component. to investigate this hypothesis, the authors used as a resource the utah population data base, which contains data from founding families and their descendants, comprised primarily of members of the church of jesus christ of latter-day saints (i.e., mormons), thus representing a relatively ethnically homogeneous population. albright et al. [1] estimated the relative risk (rr) of death for relatives of 4855 individuals (spanning 3 generations) who died of influenza. a substantial proportion of deaths occurred during the 1918 influenza pandemic, when a total of 1937 deaths were documented between 1918 and 1921, and 1293 additional deaths occurred between 1922-1932, followed by a dramatic decrease in the number of deaths occurring annually. the rr of death for first-degree relatives was 1.54 (p ͻ .0001). the rr was higher for spouses (1.98) and for secondand third-degree relatives (1.22 and 1.16, respectively). the timing of the deaths of third-degree relatives suggests that the deaths were not the result of a common exposure. to control for shared environment, the rr of death for spouses' relatives was compared and was found to be lower for first-, second-, and third-degree relatives. excess relatedness among individuals dying of influenza was estimated using the geneological index of familiality, which demonstrated that relatedness among these individuals, including individuals who died during the 1918 pandemic, was greater than expected (p ͻ .001). the analysis was repeated with close relatives excluded (to control for shared environment), and the results were consistent with previous findings. consistent results were not observed when the same analysis was repeated for individuals with diphtheria-associated deaths. for such individuals, excess relatedness was demonstrated; however, when close relationships were excluded, no excess relatedness was detected. specific genes responsible for the host immune response have been invoked as major determinants of the clinical course of hiv-associated disease and hepatitis b and c virus infections [2, 3] . however, there is very little information with respect to genetic determinants as they relate to severe influenza. over the past decade, a greater understanding of the immune evasion strategies of influenza virus has developed. this knowledge can be used to propose several candidate genes that may be responsible for severe illness. clinical and animal studies indicate that cytokine dysregulation is associated with acute respiratory distress syndrome and death among hosts infected with avian influenza virus (h5n1) [4 -6] . toll-like receptors (tlrs), particularly tlr3 (which recognizes double-stranded rna) and tlr7 and tlr8 (which recognize single-stranded rna), are central to antiviral innate immunity [7] . singlenucleotide polymorphisms in tlr genes are not uncommon and vary among populations [8] . tlr4 has been implicated in the innate immune response to respiratory syncytial virus (rsv) infection, and polymorphisms in the tlr4 gene have been associated with severe bronchiolitis in rsv-infected infants, although the significance of the asp299gly polymorphism appears to be a matter of ongoing debate [9 -12] . a missense mutation in the tlr3 gene (f303s) conferring loss of function in in vitro assays has been identified in a japanese child with influenzaassociated encephalopathy [13] . interestingly, better survival rates have been demonstrated for tlr3 knockout mice than for wild-type mice, despite the tlr3 knockout mice having higher lung virus titers after influenza virus infection [14] . tlr genes (in particular, tlr3) would therefore be worthy candidate genes to investigate. in addition, rna helicases, such as retinoic acid inducible gene i (rig-i), recognize double-stranded rna and, thus, contribute to the antiviral state of an infected cell [7] . inhibition of rig-1 by nonstructural protein 1 (ns1) of influenza virus antagonizes interferon (ifn) production and is a means by which the virus evades the host immune system [15] . therefore, the rig-i gene should also be considered to be a candidate for attempts to elucidate aberrations in the immune system that may lead to severe influenza illness. other host gene products involved in ifn production and induction antagonized by the influenza virus ns1 are worthy of consideration [16] . changes in genes involved in other aspects of immunity, such as the complement system, have also been associated with recurrent viral upper respiratory tract infections, and they should not be overlooked [17] . mannose-binding lectins are part of the innate immune system, binding pathogenassociated carbohydrates and activating the complement system through the classical pathway. mutations within the mbl gene have been associated with severe acute respiratory syndrome (sars) [18] . susceptibility to the sars coronavirus has been associated with changes in the 2'5'oligoadenylate synthetase 1 (oas1) and myxovirus resistance 1 (mxa) genes as well [19] . all of these genes would be excellent candidates for an analysis to iden-tify determinants of severity of disease after influenza virus infection. the findings of the study by albright et al. [1] raise many complex questions. the outcome of influenza illness is likely to be dependent on environmental, nutritional, demographic, immunological, and (especially) virologic factors. undoubtedly, host factors, including comorbidities, will also have a bearing on outcome, and the relative contribution of different genetic determinants is likely to vary depending on environmental and virologic contexts. a significant proportion of the data collected in this study emanates from the influenza pandemic of 1918, which occurred in a setting very different from that of interpandemic cases. in addition, the use of death certificates imposes significant limitations. during the 1918 pandemic, anyone dying with respiratory symptoms was likely to have been given a code denoting "pneumonia and influenza," which introduced a potential bias. one could argue that errors related to certification of death would be random rather than systematic and should have minimal bearing on the results. nonetheless, death certificates are notoriously inaccurate, and the absence of microbiological and/or serologic data is a significant limitation of this study. there are also a number of potential confounding factors in this investigation. other heritable factors, such as cardiopulmonary conditions or cystic fibrosis (the most common autosomal recessive lethal disorder in whites), are possible examples. other examples would include common conditions that have a genetic component, such as asthma and coronary artery disease. there is reasonable evidence that the influenza-associated mortality rate is higher for individuals with established coronary artery disease (than for those with other chronic conditions) [20] . coinfections may also confound the data, and the contribution of coinfecting pathogens to mortality cannot be excluded, bearing in mind that genetic susceptibility to such organisms as the pneumococcus has been proposed [21] . environmental and geographic factors, such as socioeconomic variables and environmental and occupational (e.g., mining) exposures, may also cluster in families, and such factors as poverty, malnutrition, and overcrowding may span generations. in addition, access to health care may have been limited for certain families in remote settings, thus contributing to the lethality of influenza virus infection, particularly in individuals at the extremes of age. the implications of this study by albright et al. [1] are that heritable factors for severe influenza illness may well exist and that elucidation of these factors remains a challenge. identification and characterization of genetic determinants of the outcome of infection with influenza virus would lead to further insight into the pathophysiology of the virus and its immune evasion strategies. one would hope that this insight would translate into identification of prognostic factors and improved management of individuals at risk for adverse outcomes after influenza virus infection. identification of genetic determinants of outcome after influenza virus infection would require a substantial number of severe cases of influenza (in which severity of illness is not the result of other factors, such as underlying cardiovascular disease) to gain sufficient power and produce interpretable data. resources stemming from the sequencing of the human genome are now available and render this challenging task more feasible. such approaches as the genomewide and candidate gene study designs are reviewed by burgner et al. elsewhere [22] . the latter approach may be more feasible, given the growing knowledge regarding ifn antagonism and immune evasion by influenza viruses. one must bear in mind that findings may not apply across all host or virus populations. in the event of a pandemic, however, early identification of and intervention for individuals at higher risk for severe illness and death resulting from influenza due to a predisposing genetic determinant would be invaluable. evidence for heritable predisposition to death due to influenza human genes that limit aids a comparative review of hla associations with hepatitis b and c viral infections across global populations acute respiratory distress syndrome induced by avian influenza a (h5n1) virus in mice role of host cytokine responses in the pathogenesis of avian h5n1 influenza viruses in mice fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia toll-like receptors and rna helicases: two parallel ways to trigger antiviral responses genetic polymorphisms of viral infection-associated toll-like receptors in chinese population involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus tlr-4 and cd14 polymorphisms in respiratory syncytial virus associated disease association between common toll-like receptor 4 mutations and severe respiratory syncytial virus disease common human tolllike receptor 4 polymorphisms-role in susceptibility to respiratory syncytial virus infection and functional immunological relevance a missense mutation of the toll-like receptor 3 gene in a patient with influenza-associated encephalopathy detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns1 protein of influenza a virus type 1 interferons and the virus-host relationship: a lesson in détente homozygous deletion of the cyp21a-tnxa-rp2-c4b gene region conferring c4b deficiency associated with recurrent respiratory infections association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection association of sars susceptibility with single nucleic acid polymorphisms of oas1 and mxa genes: a case-control study influenza vaccination as secondary prevention for cardiovascular disease: a science advisory from the american heart association/american college of cardiology mbl genotype and risk of invasive pneumococcal disease: a casecontrol study genetic susceptibility to infectious diseases: big is beautiful, but will bigger be even better? key: cord-007180-pho3miid authors: heine, j.; pohlenz, j. f. l.; moon, h. w.; woode, g. n. title: enteric lesions and diarrhea in gnotobiotic calves monoinfected with cryptosporidium species date: 1984-11-17 journal: j infect dis doi: 10.1093/infdis/150.5.768 sha: doc_id: 7180 cord_uid: pho3miid the pathogenicity of cryptosporidium species was studied by inoculation of two gnotobiotic calves with cryptosporidial oocysts that had been decontaminated by treatment with peracetic acid. two control calves were inoculated with similar material from which the oocysts had been removed by filtration. oocyst-inoculated animals shed cryptosporidium in their feces and developed depression, weakness, anorexia, and diarrhea. at necropsy five days after inoculation, endogeneous stages of cryptosporidium were found in association with epithelial cells throughout the small and large intestines of these animals. the parasites were most numerous in the lower small intestine. atrophic villi, disordered and degenerate villous epithelium, and hyperplastic crypt epithelium were associated with infection in the small intestine. control animals remained normal. extraneous agents were not detected in any of the calves. the results indicate that cryptosporidium can destroy intestinal epithelial cells and cause diarrhea in monoinfected gnotobiotic calves. the pathogenicity of cryptosporidium species was studied by inoculation of two gnotobiotic calves with cryptosporidial oocysts that had been decontaminated by treatment with peracetic acid. two control calves were inoculated with similar material from which the oocysts had been removed by filtration. oocyst-inoculated animals shed cryptosporidium in their feces and developed depression, weakness, anorexia, and diarrhea. at necropsy five days after inoculation, endogeneous stages of cryptosporidium were found in association with epithelial cells throughout the small and large intestines of these animals. the parasites were most numerous in the lower small intestine. atrophic villi, disordered and degenerate villous epithelium, and hyperplastic crypt epithelium were associated with infection in the small intestine. control animals remained normal. extraneous agents were not detected in any of the calves. the results indicate that cryptosporidium can destroy intestinal epithelial cells and cause diarrhea in monoinfected gnotobiotic calves. infections with the coccidian parasite cryptosporidium occur worldwide in the alimentary and respiratory tracts of numerous species of vertebrates. the infection is highly prevalent among cattle; it affects up to 1000;0 of calves during the first four weeks after birth [1] [2] [3] [4] [5] . subclinical infection is common; however, infection is also frequently associated with diarrhea in calves and people [5] [6] [7] [8] [9] [10] . cryptosporidiosis has recently been recognized as a zoonosis [10] [11] [12] . the infection is self-limiting in calves and people with normal immune systems. however, it can become persistent in immunologically compromised individuals, such as patients with the acquired immunodeficiency syndrome [12] [13] [14] . clinically affected calves have atrophy of villi and hyperplasia of crypt epithelium (apparently as a result of the destruction of villous epithelium); those areas of the small intestine that are heavily infected with the parasite become inflamed [7, 9, 10] . some calves also have epithelial damage and inflammation associated with numerous parasites in the large intestine [7] . similar lesions occur in people with cryptosporidiosis [15] [16] [17] . the diarrhea and intestinal lesions associated with cryptosporidiosis in calves and people are presumed to be caused (at least in some cases) by the parasite. however, in calves cryptosporidium frequently coexists with enteropathogenic viruses, bacteria [5] , or other protozoa known to be independently capable of causing these signs and lesions. although the development of signs and lesions during intraspecies and interspecies transmission experiments with cryptosporidium, in the absence of other recognized enteropathogens, suggests that the parasite causes these changes [7, 10] , the possibility of cotransmission of some unrecognized pathogens in such experiments cannot be excluded. moreover, experimental infections in conventional animals have not served to clarify whether or not the intestinal flora is required for colonization and lesion production by the parasite. on the other hand, if confirmed, the reported occurrence of diarrhea and intestinal lesions in gnotobiotic pigs infected with an inoculum treated in a manner that destroys infectious agents other than cryptosporidium [18] provides strong evidence that the parasite can act as a primary enteropathogen in the absence of other enteric flora. the data reported here tend to confirm the report that cryptosporidium can cause diarrhea and intestinal lesions in monoinfected gnotobiotic animals. these studies extend the data to another host species and involve a different isolate of cryptosporidium, a different treatment for the destruction of extraneous agents, a comprehensive search for such agents, and the use of control animals exposed to putative, unrecognized viruses that might have survived the treatment intended to destroy these agents. calves. four gnotobiotic calves were delivered by cesarean section at 270-274 days of gestation and were maintained as described by matthews et al. [19] . the calves were each fed 2 liters of reconstituted condensed milk twice daily. the isolators were kept at 18 c-24 c. inocula. feces from two calves experimentally infected with cryptosporidium of calf origin were suspended in two volumes of 2.5070 potassium dichromate solution. this suspension was passed through a sieve (mesh size, 63 jim) and stored for 10-15 weeks at 4 c. the suspension contained 7.5 x 10 6 cryptosporidial oocysts/ml, as determined by the counting technique reported previously [4] . in preliminary tests cryptosporidial oocysts (in potassium dichromate suspensions of calf feces) that were treated with 3.2% peracetic acid (vol/vol) and held at 22 c for 20 min retained infectivity for mice. oocysts were removed from peracetic acid by centrifugation (500 g for 10 min) and three aseptic washes (by centrifugation) of the sediment in pbs. such peracetic acid-treated oocyst preparations yielded no bacterial or fungal growth when cultured aerobically at 37 c for one week in trypticase soy broth (tsb; bbl microbiology systems, cockeysville, md). in view of these results and the facts that peracetic acid is routinely used as a germicide in gnotobiotic procedures and is effective against a broad spectrum of microbes, this treatment was used for the destruction of putative mi-769 crobes other than cryptosporidium in the inocula given to gnotobiotic calves. each of two calves (no. 1 and no. 2) received the sediment (resuspended in 10 ml of pbs) resulting from peracetic acid treatment of 13.5 ml of potassium dichromate-suspended feces (l08 oocysts per calf). inocula for two control calves (no. 3 and no. 4) were prepared from the same potassium dichromate suspension of feces (27 ml of suspension per calf). the control inocula were passed twice through filters (pore size, 0.8 jim) for the removal of cryptosporidial oocysts before treatment with peracetic acid. the inocula were mixed with the milk fed to the calves at 22 hr of age. aliquots of the oocyst-containing and control inocula were given to baby mice as a test for the presence of infective cryptosporidium and were cultured for aerobic bacteria at 37 c in tsb. none of the inocula produced detectable growth in tsb, and only mice given the oocyst-containing inocula became infected with cryptosporidium. observations. all four calves were observed at 12-hr intervals for five days after inoculation. rectal temperature, appetite, strength, attitude, and character of feces were recorded at each interval. fecal samples were collected at each interval and examined microscopically for oocysts by the carbol fuchsin technique [23] . the percentage of fecal dry weight was determined by drying of each sample to a constant weight at 100 c. in addition, a daily fecal sample was cultured for aerobic bacteria at 37 c for 24 hr in tsb. a fecal sample obtained from each calf on day 5 after inoculation was also cultured anaerobically at 37 c for 48 hr on sheep blood agar and in chopped meat broth. direct smears were made from the final fecal sample obtained from each calf. these smears were stained with gram stain and examined microscopically for bacteria. daily fecal samples were examined for bovine viruses (coronavirus, rotavirus, astrovirus, and breda virus by methods reported previously [20] [21] [22] as well as by direct electron microscopy of negatively stained preparations. no viruses or bacteria were detected in any sample. blood samples were taken from each calf immediately before inoculation and 72, 96, and 120 hr afterwards. the following values were determined by routine procedures: packed cell volume; erythrocyte count; total and differential leukocyte counts; concentrations of hemoglobin, plasma * sections 6 jim thick were prepared 120 hr after calves no. 1 and no. 2 were inoculated with cryptosporidial oocysts. the numbers under the headings "jejunum" and "ileum" represent centimeters distal to the duodenal site (jejunum) or proximal to the ileocecal valve (ileum). key: -== no demonstrable parasites; + == <20 parasites/mm of epithelial surface; + + == 20-80 parasites/mm; and + + + == >80 parasites/mm. protein, albumin, fibrinogen, calcium, inorganic phosphorus, urea nitrogen, na", k+, and ci-; and aspartate aminotransferase level. calves were anesthetized with barbiturate 120 hr after inoculation. two ligated segments of intestine were created at each of the following sites: duodenum (15 em distal to the pylorus), jejunum (100, 320, 450, and 700 em distal to the duodenal site), ileum (50 and 120 em proximal to the ileocecal valve), cecum, apex of spiral colon, and descending colon. the ligated segments were instilled intraluminally with fixative (10070 formalin for one segment and 3% glutaraldehyde for the other), excised surgically, and placed in fixative. calves were killed after these segments were collected. portions of each formalin-fixed segment were embedded in paraffin, sectioned at 6 urn, and stained with hematoxylin and eosin for the preparation of histological sections. portions of formalin-fixed small intestine were also stained by immersion in 2% methylene blue. "whole-mount" preparations of mucosa (one or two villi thick and four to 10 villi long) were dissected from the methylene blue-stained segments with use of a dissecting microscope, a razor blade, and straight pins; the dissection was done underwater by a modification of the procedure of macdonald and ferguson [24] . these preparations were mounted on glass slides in water, with the long axis of villi parallel to the surface of the slides. the lengths of the villi and depths of the crypts in the whole mounts were measured with a dissecting microscope equipped with an ocular micrometer. glutaraldehyde-fixed sections of the small intes-eluded) along segments of epithelium (villous epithelium in the small intestine, crypt and surface epithelium in the large intestine) and for determination of the lengths of the epithelial segments selected. clinical findings. calves no. 1 and 2, which were inoculated with oocysts of cryptosporidium, had a decreased appetite, lost strength, and became depressed beginning 72 and 48 hr after inoculation, respectively. their feces subsequently became watery with clumps of mucus, and its color changed from brown to yellow-beige. fecal dry weights were initially 37% and 18070 for calves no. 1 and 2, respectively; these values fell to 8% at 60 hr and 5070 at 96 hr and remained below 13% in both calves until the experiment was terminated at 120 hr. the body temperature of calf no. 2 increased by 1 c (to 40.4 c) at 72 hr; the body temperatures of calf no. 1 and the two calves given the control inoculum remained at 38.4 c-39.5 c throughout the experiment. no clinical signs developed in control calves. their feces remained formed, semisolid, and brown throughout the experiment. the initial dry weight of their feces was 38%, and this value gradually decreased to levels of 13% and 16%, respectively, by the end of the experiment. neither infected nor control animals underwent marked changes in any of the cellular or chemical hematologic parameters evaluated. parasitological findings. cryptosporidial 00cysts were first detected in the feces of calf no. 1 at 72 hr after inoculation and in the feces of calf no. 2 at 48 hr. both calves continued to shed cryptosporidium in their feces until the experiment was terminated. at necropsy, the parasites were associated with villous epithelium in the small intestine as well as with surface and crypt epithelium in the large intestine. the greatest numbers of parasites per unit (length) of epithelium tended to be in the lower small intestine (table 1) . cryptosporidium was not detected in any fecal sample or histological section from the control calves. pathological findings. no histological or ultrastructural lesions were found in intestinal sections from control calves. in contrast, the villi throughout the jejunum and ileum of infected calves were significantly shorter than those in the jejunum and ileum of control animals (p < .01; figures 1 and 2). crypts from the jejunum and the ileum 120 (i.e., the ileum 120 em proximal to the ileocecal valve) of infected calves were significantly deeper than those from the same sites in controls (p < .01). the epithelium covering the atrophic villi in histological sections from infected calves was basophilic and usually low columnar to cuboidal, with disordered nuclei and an irregular surface (figure 3). in contrast, the crypt epithelium from the small intestine of infected calves remained tall and columnar but contained more mitotic figures than that from controls. the villous epithelium and lamina propria in some sections were infiltrated with a variety of mononuclear and polymorphonuclear inflammatory cells. many villi from the infected calves were fused together. this fusion was apparent either as synechiae formed befigure 5 . transmission electron micrograph of epithelium from an atrophic villus from the ileum of a gnotobiotic calf infected with cryptosporidium (arrows). the epithelial cells are low columnar to cuboidal and have short, sparse microvilli and extensive cytoplasmic vacuolation. portions of two eosinophils can be seen beneath the epithelium; large electron-dense granules are evident at both the left and the right. tween the epithelium of adjacent villi or as villi with an increased width of the lamina propria and with multiple lacteals and central veins. in the large intestine some foci of parasitized epithelium consisted of low columnar to cuboidal cells with nonaligned nuclei and irregular surfaces. the fusion of some atrophic villi in infected calves was confirmed by scanning electron microscopy ( figure 4) . transmission electron microscopy of epithelium from atrophic villi revealed parasites in various endogenous stages (figures 5 and 6). villous absorptive cells frequently had cytoplasmic vacuolation (figures 5 and 6). vacuolation was particularly marked in the ileum (figure 5). occasional epithelial cells had electron-dense cytoplasm and nucleus (figure 6). these "dark" cells were interpreted to be degenerate and shrunken absorptive or goblet cells. absorptive cell microvilli were irregular in length. they were short and sparse on many cells (figure 5); however, those immediately adjacent to cryptosporidium were frequently longer than those further away from the parasite on the same or other absorptive cells ( figure 6 ). occasionally, parasitized epithelial cells were observed, with disrupted apical plasma membranes and cytoplasmic protrusion into the intestinal lumen ( figure 6 ). gnotobiotic calves inoculated with oocysts of cryptosporidium that had been treated with potassium dichromate and peracetic acid became infected with cryptosporidium and developed clinical signs and enteric lesions. in contrast, control gnotobiotic calves remained normal after the inoculation of similar material that had been passed through a filter for the removal of oocysts (but not of any putative viruses). we were unable to cultivate extraneous agents from the inocula or from the feces of any of the calves. we were also unable to demonstrate infectious agents other than cryptosporidium by direct light and electron microscopic examination of feces from the calves or by transmission electron microscopic study of sections of intestine from the animals. these results provide strong evidence that the calves inoculated with oocysts were mono infected with cryptosporidium and that cryptosporidium caused their enteric lesions and diarrhea. the data tend to confirm and extend a previous report that cryp-773 figure 6 . transmission electron micrograph of villous epithelium from the jejunum of a cryptosporidiuminfected gnotobiotic calf. microvilli are irregular in length, and epithelial cell cytoplasm is finely vacuolated. several merozoites (crescentic, with electron-dense granules) have been released into the intestinal lumen at the center of the photograph. the residual body of the schizont and the parasitophorus envelope remain attached to a shrunken, electron-dense epithelial cell. the epithelial cell on the right has a disrupted apical plasma membrane with no visible microvilli, and cryptosporidium (arrow) is attached to a portion of the cell that is protruding into the lumen. tosporidium is pathogenic iii monoinfected gnotobiotic animals [18] . both the enteric lesions (characterized by damage to and loss of epithelial cells, villous atrophy and crypt hyperplasia, and infiltration with a mixture of inflammatory cells) and the signs (diarrhea, depression, and anorexia) were similar to those associated with experimental and naturally acquired cryptosporidial infections in conventional animals [7, 9, 10] and people (10) (11) (12) (13) (14) (15) (16) (17) . in aggregate, the evidence strongly suggests that cryptosporidium causes enteric disease during naturally acquired in-fection. subclinical infections are common. variations in signs and lesions associated with the infection are probably caused in part by variations in the dose (and possibly the virulence) of the cryptosporidium ingested. calves can shed as many as 10 8 cryptosporidial oocysts/ml of feces [4] . we assume that calves sometimes naturally encounter doses of oocysts as large as that used here (l08) because it seems reasonable that calves reared under conditions of poor hygiene could ingest 1 ml of feces. there is also considerble variation in signs and lesions that is dependent on the species, age, and immune status of the host. in our experience, experimentally infected newborn calves usually develop diarrhea [7] , but experimentally infected mice do not [25] . presumably, the amount of mucosal damage is less or the compensatory capacity of the undamaged mucosa is greater in newborn mice than in calves. adult mice are cornparatively resistant to the infection, but immunodeficient (nude) newborn mice developed persistent infection and diarrhea, and some die [25] . calves that recover from experimental cryptosporidial infection are resistant to a second challenge with the organism (author's unpublished data). we suspect that diarrhea occurs in infected animals because extensive damage to epithelial cells and villous atrophy result in malabsorption. the occurrence of hyperplastic crypt epithelium along with damaged villous epithelium and atrophic villi in infected calves represents evidence that the lesions develop as a result of accelerated destruction or loss (rather than decreased production) of epithelial cells. the mechanism(s) by which cryptosporidium destroys or accelerates the loss of epithelial cells is (are) unknown. some evidence suggests that villous atrophy can be induced by cell-mediated immune responses to protozoa and helminths [26, 27] . however, the occurrence of villous atrophy in cryptosporidium-infected nude mice [25] suggests that, although t lymphocytes are required for the development of villous atrophy in some enteric protozoan infections, this is not the case in cryptosporidiosis. perhaps cryptosp oridium damages epithelial cells directly through some toxic, metabolic, or physical effect. cryptosporidial infection in idaho dairy calves kryptosporidioza telat v obdobi mlecne vyzivy die bovine kryptosporidiose. diagnose und therapie der verlauf natiirlicher cryptosporidium-infektionen in vier rinderziichtbetrieben cryptosporidiosis as a probable factor in neonatal diarrhea of calves kryptosporidien-infektionen beim kalb. nachweis, vorkommen und experimentelle ubertragung fecal transmission of calf cryptosporidia between calves and pigs demonstration of cryptosporidia in calf feces: a comparative study bovine cryptosporidiosis: clinical and pathological findings in forty-two scouring neonatal calves cryptosporidiosis in animals and humans cryptosporidiosis in a veterinary student human cryptosporidiosis in immunocompetent and immunodeficient persons cryptosporidiosis: assessment of chemotherapy of males and acquired immune deficiency syndrome (aids) three-step stool examination for cryptosporidiosis in 10 homosexual men with protracted watery diarrhea rubin ceo overwhelming watery diarrhea associated with a cryptosporidium in an immunosuppressed patient acute enterocolitis in a human being infected with the protozoan cryptosporidium intestinal cryptosporidiosis complicated by disseminated cytomegalovirus infection enterocolotis in piglets caused by cryptosporidium sp. purified from calf faeces derivation of gnotobiotic calves by an open cesarian method isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves antigenic relationships among some animal rotaviruses: virus neutralization in vitro and cross-protection in piglets studies with an unclassified virus isolated from diarrheic calves eine einfache nachweismethode fur kryptosporidien im kat hypersensitivity reactions in the small intestine. iii. the effects of allograft rejection and of graft-versus-host disease on epithelial cell kinetics persistent cryptosporidium infection with in congenitally athymic (nude) mice hypersensitivity reactions in the small intestine. i. thymus dependence of experimental 'partial villous atrophy coccidiosis: t-lymphocyte-dependent effects of infection with eimeria nieschulzi in rats key: cord-007264-r1w9a6gc authors: turner, ronald b. title: rhinovirus infection of human embryonic lung fibroblasts induces the production of a chemoattractant for polymorphonuclear leukocytes date: 1988-02-17 journal: j infect dis doi: 10.1093/infdis/157.2.346 sha: doc_id: 7264 cord_uid: r1w9a6gc polymorphonuclear leukocytes (pmnls) appear in the nasal mucosa during rhinovirus colds before the onset of symptoms. this study describes a chemoattractant for pmnls that is elaborated by human embryonic lung fibroblast cells infected with rhinovirus. chemotaxis assays were done in a 48-well microchemotaxis chamber with normal adult pmnls. medium supernatants from rhinovirus-infected cellculture attracted 87 ± 6 (mean ± se) pmnls/10 high-power fields (×450) compared with 38 ± 6 pmnls/10 highpower fields attracted by medium from uninfected cell cultures (p < .0001). elaboration of the chemoattractant was not a result of cell destruction and did not require the presence of infectious virus. this chemoattractant produced by human fibroblast cells may contribute to the influx of pmnls into the nasal mucosa during rhinovirus infection. the pmnls may, in turn, have a role in producing symptoms of the common cold. rhinoviruses are the pathogens most frequently associated with the common cold [1] . the pathogenesis of the symptoms of rhinovirus infection is not known, but it has been suggested that the host response to the virus may cause at least some of the manifestations of infection [2, 3] . recent studies in human volunteers have shown that pmnls appear in the nasal mucosa early in the course of infection, before the appearance of symptoms [4, 5] . the mechanism by which pmnls are attracted to the nasal mucosa is unknown. the production of chemoattractants for pmnls in response to viral infection of cell cultures has previously been reported [6] [7] [8] . herpes simplex virus infection has been shown to be associated with a chemoattractant that is dependent upon the presence of complement [6, 7] . in 1972, ward and co-workers [8] reported that cells infected with mumps virus or newcastle disease virus elaborated a chemotactic factor that was not complement dependent. the purpose of the present study was to determine if, and under what conditions, a chemoattractant for pmnls might be produced in rhinovirus infection. growth ofrhinovirus in cellculture. we used human embryonic lung fibroblast cells (mrc-5; earl-clay laboratories, novato, calif) at passages 23-25 and human foreskin fibroblast cells at passages 6-10. cells were grown in 16 x 125-mm cell culture tubes or in 25-cm 2 cell culture flasks (falcon, oxnard, calif) in eagle's mem (emem) supplemented with 10010 fetal bovine serum, penicillin (100u/ml), and gentamicin (20 j.1g/ml). when the cells were confluent, the growth medium was changed to maintenance medium (emem with 2% fetal bovine serum); cells were used within one week. all sera used in the cellculture media were heat-inactivated to remove complement activity. before cells were inoculated with virus, the maintenance medium was removed, the cell monolayer was washed three times with pbs (ph 7.4), and emem without serum or antibiotics was added to the flasks. the cells were inoculated with rhinovirus type 39 (r39) and incubated at 33 c in a 5% co 2 atmosphere. after 48 h, when cpe was seen in "'80% of the monolayer, the medium was collected from the infected cultures. cell monolayers handled in an identical fashion, except that they were sham inoculated with the diluent for the virus inoculum (serum-free medium) instead of r39, were used as uninfected controls. the media harvested from the infected cells and the uninfected cells were centrifuged at low speed (225 g) in a tj-6 centrifuge (beckman, palo alto, calif) to remove cell debris; they were then assayed for chemoattractant activity. the experiments in hela cells were done by using a rhinovirus-sensitive hela cell line. we used mccoy's medium (whitaker m.a. bioproducts, walkersville, md) supplemented with 10070 and 2% fetal bovine serum as the growth medium and maintenance medium, respectively. titrations of virus. all titrations of virus were done in 96-well microtiter plates (falcon). serial 10fold dilutions of each specimen were made, and 2 x 10 4 hela cells were added to each well. the plates were incubated at 33 c for seven days and then examined for viral cpe. the titers of virus were calculated by the method of reed and muench [9] . chemotaxis assays. pmnls were collected from the peripheral blood of normal young adult volunteers by sedimentation in 2% dextran 70 (mcgaw laboratories, irving, calif) followed by centrifugation with ficoll-paque® (pharmacia, piscataway, nj). the pmnls were then counted and diluted to 2 x 10 6 cells/ml in hepes buffer with i % bovine serum albumin (fraction v powder; sigma, st. louis). chemotaxis assays were done by using a 48-well microchemotaxis chamber (neuroprobe, cabin john, md). each of eight specimens to be assayed for chemoattractant activity was placed into four of the bottom wells of the chamber. cell culture medium that had been incubated with neither cells nor virus was included as a negative control. either 10-8 m n-formyl-tmethionyl-i-leucyl-i-phenylalanine (fmlp; sigma) or 10% zymosan-activated serum prepared as described previously [10] was included as a positive control for each experiment. after placing 30 ul, of the material to be assayed into each of the bottom wells, we covered the wells with a 5-llm filter (millipore, bedford, mass) and placed 50 ul, of the diluted pmnls in the top portion of each well. the chamber was incubated at 37 c for 90 min in a 5% co2 atmosphere, and the filter was fixed in methanol and stained with hematoxylin stain. the cellsthat had migrated through the filter were counted in 10 random fields by using a 5 x 5-mm photographic reticle at a magnification of x 450 (high-power field; hpf). experiments in which the mean cell count in the negative control wells was greater than 15 per 10 hpf or in which the mean counts for the positive control were less than five times those of the negative control were excluded from analysis. approximately 10% of the experiments were excluded by these criteria. time course ofelaboration. the elaboration of the chemoattractant relative to the time of viral infection was determined by infecting mrc-5 cells with r39 at an moi of 0.01 tcid so per cell. experiments 347 were done in 25-cm 2 cell-culture flasks. a 2-ml aliquot of medium supernatant was taken every 24 h and replaced with fresh serum-free emem. one milliliter of each aliquot collected was assayed for chemotactic activity, and the remaining portion was stored at -70 c until the titer of virus was determined. determining the effect ofcelldisruption. medium from uninfected cells, which were disrupted ultrasonically or by multiple freeze-thaw cycles, was assayed to determine if the chemoattractant was a substance preformed within the cell.cell monolayers maintained in serum-free emem were frozen at -70 c and thawed at 37 c three times; the medium was then collected, centrifuged to remove debris, and assayed for chemoattractant activity. sonication experiments weredone by scraping the mrc-5 cell monolayer into serum-free medium and sonicating the specimen with a microtip sonicator (heat systems-ultrasonics,plainview,ny). the specimen was centrifuged to remove debris and assayed for chemoattractant activity. virus-infected cell cultures handled in an identical fashion were assayed to control for destruction of the chemoattractant by freeze-thawing or sonication. associating chemoattractant activity with infectious virus. supernatant from rhinovirus-infected mrc-5 cells was centrifuged at 100 000 g (beckman l5-65 ultracentrifuge, sw65 rotor; beckman) for 24 h. the pellet was resuspended in fresh serum-free medium to the original volume; both the resuspended sediment and the original supernatant were assayed for infectious virus and for chemoattractant activity. attachment of virus. to determine whether viral infection of the cell monolayer was necessary for elaboration of the chemoattractant, we inactivated r39 by exposure to uv light for 30 min. the inactivated virus was then inoculated onto monolayers of mrc-5 cells and incubated at 33 c for 48 h. control monolayers that had been inoculated with infectious virus or with uninfected cell-culture medium were handled identically. after 48 h, medium from the experimental and the control monolayers was assayed for chemotactic activity. chemoattractant activity of other viruses. respiratory syncytial virus (rsv) and coronavirus 229e were inoculated into mrc-5 cell cultures maintained in serum-free medium as described for r39. rsv was also inoculated into hela cell cultures maintained in serum-free medium. after incubation at 33 c for 48 h, media from both infected and control monolayers was assayed for chemoattractant activity. medium from infected cells). treatment by sonication or multiple freeze-thaw cyclesof medium from rhinovirus-infected cells did not alter its chemoattractant effect. after ultracentrifugation of the medium from infected mrc-5 cells, the titer of virus in the supernatant was 3.2 x 10 2 tcidsolml compared with 1.6 x 10 4 tcidso/ml in the sediment. in contrast, the supernatant attracted 102pmnls/lo hpf, compared with 16 pmnls/lo hpf attracted by the sediment. these results suggest that the chemoattractant activity is not directly associated with infectious viral particles. medium collected from mrc-5 cellsthat had been ... comparisons in all other experiments were made by using a two-sided mann-whitney u test. the mean and se were calculated for each of the observations. four measurements were made on each specimen; specimens obtained from different monolayers were considered to be separate experiments. human embryonic lung fibroblast cellsinfected with rhinovirus produced a chemoattractant for pmnls. media from r39-infected and uninfected cells attracted 87 ± 6 (mean ± se) and 38 ± 6 pmnls/ 10 hpf, respectively (p = .0001). the positive controls attracted 122 ± 10 pmnls/lo hpf and the negative controls, 8 ± 1 pmnls/lo hpf. when medium from infected cells was placed above and below the filter to remove the chemotactic gradient, the mean number of cellsmigrating through the filter per 10 hpf was 8 ± 1. incubation of pmnls with the rhinovirus-induced chemoattractant did not affect migration toward wells containing fmlp. chemoattractant activity similar to that induced by r39 was detected when mrc-5 cells were infected with rhinovirus types 2 and 14. medium from cells infected with rhinovirus type 2 attracted 73 ± 15 pmnls/lo hpf, and medium from rhinovirus type 14-infected cells attracted 133 ± 17pmnls/lo hpf. chemoattractant activity was also detected in human foreskin fibroblasts infected with r39 (table 1) . in contrast, the chemotactic activity detected in medium from r39-infected hela cells was not significantly different from the activity in medium from control cells, a result suggesting that elaboration of the chemoattractant is a property of the infected cell. when aliquots of medium were taken from infected cells at 24-h intervals, chemoattractant activity was detectable 24 h after inoculation of virus (figure 1) . the amount of chemoattractant activity increased in each subsequent aliquot collected until the cpe was complete. medium from uninfected cells disrupted by sonication attracted a mean of 20 ± 3 pmnls/lo hpf (p < .0001 compared with medium from infected cells). similarly, medium from uninfected cells disrupted by freezing and thawing attracted a mean of 6 ± 1 pmnls/lo hpf (p < .0001 compared with discussion these experiments indicate that attachment of rhinovirus to human embryonic lung fibroblast cells results in the elaboration of a chemoattractant for human pmnls. this chemoattractant activity is not dependent upon the presence of complement. the absence of chemoattractant activity in medium from disrupted mrc-5 cells and hela cells infected with r39 suggests that the production of this chemoattractant is a specific response of the fibroblast cell to attachment of virus. the production of a chemoattractant for pmnls in response to viral infection has been reported previously. in 1972, ward and co-workers [8] reported that infection of chick embryos with either newcastle disease virus or mumps virus or infection of monkey kidney cells with mumps virus resulted in elaboration of a chemoattractant for pmnls and macrophages. this chemoattractant was produced in the presence of serum and appeared to be dependent upon the cell line infected rather than upon the infecting virus. herpes simplex virus infection of rabbit kidney cells has been reported to produce a chemoattractant for pmnls by two different mechanisms. destruction of the cell monolayer releases a factor that cleavesthe fifth component of complement (c5) and produces c5a, a chemoattractant for pmnls [6] . a chemoattractant is also produced by the interaction of complement with specific antibody to herpes simplex virus [7] .the role of chemotactic factors in the pathogenesis of diseases caused by these viruses is not known. it has been suggested that pmnls may have a role in the pathogenesis of disease caused by viral infection of the respiratory tract. on the basis of studies in a ferret model, sweet et al. [11] have hypothesized that pmnls interact with influenza virus in the upper respiratory tract; the subsequent release of endogenous pyrogen produces the systemic symptoms of these infections. faden et al. [12] have reported thatrsv antibody complexes activate pmnls and have suggested that the products of this activation may be responsible for some of the symptoms of respiratory syncytial virus infection. the pathogenesis of the symptoms produced by rhinovirus infection of the upper respiratory tract is not known. cytopathologic changes in the nasal mucosa appear to be minimal during rhinovirus colds. two different studies of biopsy specimens from human volunteers have found no consistent changes of the nasal epithelium in rhinovirus infection [4, 13] . examination of the cells present in the nasal secretions of infected volunteers revealed that infected ciliated cells are shed from the nasal epithelium during rhinovirus colds [2] ;however,the low number of cells observed is consistent with the absence of findings in the biopsy studies. the absence of histopathologic findings led to the suggestion that the viral infection triggers inflammatory responses that result in the production of symptoms [2, 3] . in 1984, winther et al. [4] reported that the number of pmnls in the nasal mucosa was significantly greater in biopsy specimens taken from volunteers during symptomatic upper-respiratory-tract infections than in specimens taken during convalescence or from asymptomatic control subjects. a study in volunteers with experimental rhinovirus colds re(table 2) . in contrast, medium from hela cellsinfected with rsv attracted 9 pmnls/lo hpf, compared with 7 pmnls/lo hpf attracted by medium from control tubes. these results indicate that infection of fibroblast cells with respiratory viruses other than rhinovirus is also associated with elaboration of a chemoattractant. it remains to be determined whether the chemoattractants produced by infection with these viruses are identical. vealed that the number of pmnls in the nasal mucosa increased significantly following inoculation with virus and that the increase occurred before symptoms were reported [5] . a later study suggests that the concentration of pmnls in nasal secretions also increases during rhinovirus colds (1. o. hendley, personal communication). these data are consistent with the hypothesis that symptoms of the common cold are a result of the host response to the virus and suggest that pmnls may be an important component of this response. the observation that rhinovirus infection of human fibroblast cells results in production of a chemoattractant for pmnls provides a potential explanation for the attraction of pmnls to the nasal mucosa during rhinovirus colds. characterization of this chemoattractant may provide important information about the pathogenesis of viral infection of the respiratory tract. acute respiratory illness in an american community: the tecumseh study shedding of infected ciliated epithelial cells in rhinovirus colds rhinovirus colds: immunology and pathogenesis light and scanning electron microscopy of nasal biopsy material from turner patients with naturally acquired common colds histopathologic examination and enumeration of polymorphonuclear leukocytesin the nasal mucosa during experimental rhinovirus colds inflammation and herpes simplex virus: release of a chemotaxis-generating factor from infected cells inflammation and viral infection: chemotactic activity resulting from the interaction of antiviral antibody and complement with cells infected with herpes simplex virus leukotactic factors elaborated by virus-infected tissues a simple method of estimating fifty per cent endpoints defective monocyte chemotactic responses in diabetes mellitus the local origin of the febrile response induced in ferrets during respiratory infection with a virulent influenza virus activation of oxidative and arachidonic acid metabolism in neutrophils by respiratory syncytial virus antibody complexes: possible role in disease atraumatic nasal biopsy for studies of respiratory virus infection in volunteers key: cord-276005-ifn88mjd authors: da silva filho, luiz vicente ribeiro ferreira; zerbinati, rodrigo melim; tateno, adriana fumie; boas, lucy vilas; de almeida, marina buarque; levi, josé eduardo; drexler, jan felix; drosten, christian; pannuti, cláudio sérgio title: the differential clinical impact of human coronavirus species in children with cystic fibrosis date: 2012-08-01 journal: j infect dis doi: 10.1093/infdis/jis274 sha: doc_id: 276005 cord_uid: ifn88mjd we investigated the clinical impact of human coronaviruses (hcov) oc43, 229e, hku1 and nl63 in pediatric patients with cystic fibrosis (cf) during routine and exacerbation visits. a total of 408 nasopharyngeal aspirate samples were obtained from 103 patients over a 1-year period. samples positive for hcov were submitted for nucleotide sequencing to determine the species. nineteen samples (4.65%) were positive for hcov, of which 8 were positive for nl63, 6 for oc43, 4 for hku1, and 1 for 229e. identification of hcov was not associated with an increased rate of respiratory exacerbations, but nl63-positive patients had higher exacerbation rates than patients who were positive for other hcov species. cystic fibrosis (cf) is an autosomal inherited disease characterized by recurrent and chronic respiratory infections [1] . the role of respiratory viruses in the progression of lung disease in cf is still controversial, although infection with respiratory syncytial virus (rsv) and influenza viruses was shown to carry a significant risk for respiratory exacerbations and hospital admissions [2] . while little is known of the impact of newly described respiratory viruses on lung disease in cf patients, there is evidence that some of these viruses may be more aggressive than others. for example, we recently showed that infection with human rhinovirus c is associated with an increased risk of respiratory exacerbations [3] . human coronaviruses (hcovs) were initially described in the 1960s as the causative agents of upper respiratory tract infections (urtis). although reports of lower respiratory tract infections caused by hcovs were identified thereafter [4] , the coronaviruses were still mainly considered to be the causative agents of urtis [5] until 2002, when a new coronavirus, later named sars coronavirus, was identified during an outbreak of severe acute respiratory syndrome [6] . more recently, other coronaviruses were described, with hcov-nl63 identified in the netherlands in 2004 [7] and hcov-hku1 in hong kong in 2005 [8] . these novel hcov species were hypothesized to cause lower respiratory tract infections at a higher frequency than the previously known hcovs [9] , although recent data suggest that hcov-hku1 does not cause a significantly higher proportion of lrtis than that observed with hcov-oc43 [10] . in this study, we aimed to investigate the clinical impact of all 4 circulating hcovs (oc43, 229e, hku1, and nl63) in pediatric patients with cf who attended an outpatient clinic during routine and exacerbation visits. nasopharyngeal aspirates (npas) or nasal blow (nb) specimens for viral investigation, as well as sputum or oropharyngeal samples for microbiology cultures, were collected during scheduled or unscheduled visits on 408 occasions, with a median ( ± sd) of 4 ± 1.74 visits (range, 1-9 visits) per patient. the local ethics committee approved the study protocol, and all parents/guardians gave informed consent for their children to participate in the study. clinical and functional data were obtained at all visits. exacerbation of respiratory disease was defined as the presence of ≥2 of the following symptoms and signs: fever, increase in the amount of secretion or cough intensity, change in the sputum's aspect, worsening of dyspnea, loss of appetite, a decrease of ≥10% in forced expiratory volume in 1 second (fev1), and weight loss. total nucleic acids were extracted from nasopharyngeal samples with the qiamp viral rna mini kit (qiagen, hamburg, germany), according to the manufacturer's instructions. reverse transcription was performed with a high capacity cdna archive kit (applied biosystems, foster city, ca), using 20 µl of previously extracted rna. identification of respiratory viruses was performed by individual polymerase chain reactions (pcrs) targeting rsv; influenza viruses a and b; human parainfluenza viruses 1, 2, and 3; hcov; human metapneumovirus; adenovirus; human bocavirus; enteroviruses; and the β-actin gene, as previously described [3] . the analytical sensitivity of this assay for hcov detection, determined by quantified plasmid standards, was 100 copies per reaction. all coronavirus-positive samples were confirmed by means of 1-step reverse transcription-heminested pcr, using primers cov2a-f (cttatgggttgggattatcc) and cov2a-r (taataacagacaacgccatcatc) for the first round and the inner primers cov2a-rnest a (ccatcatcactcagaat catca) and cov2a-rnest b (ccatcatcagaaagaatca tca), which generate a 404-base pair amplicon from the rna-dependent rna polymerase (rdrp) gene. nucleotide sequencing was performed using dye terminator chemistry (applied biosystems). nucleic acid alignment was conducted on the basis of the amino acid code by use of the blosum algorithm. phylogenetic classification was performed using the neighbor-joining method, with the percentage distance substitution model and bootstrap values calculated from 1000 replicates with mega 5.0 software (www.megasoftware.net). the nucleotide sequences of the partial rdrp gene of the hcov identified in this study were submitted to genbank (accession numbers: jn251784 -jn251802). the results were expressed as medians or percentages, unless stated otherwise. categorical variables were analyzed by χ 2 or fisher exact tests. the age differences between patients with and patients without hcov or within hcov species were compared by nonparametric kruskal-wallis tests. the proportion of cases with acute respiratory exacerbation among patients infected with different hcov species was compared by χ 2 or fisher exact tests. logistic regression was used to determine whether infection with hcov in general or with one particular hcov species was independently associated with respiratory exacerbation or hospital admission. to account for correlations among samples within the same subject, binomial generalized linear models were used. all analyses were performed using spss for windows (version 18; spss, chicago, il), and the level of statistical significance was set at .05. at least 1 respiratory virus was identified in 203 of 408 samples (49.7%), with rhinovirus being the main identified agent (in 139 samples [34.1%]). hcovs were identified in 19 samples (4.65%) from 17 patients (11 males and 6 females). as shown in figure 1 , 8 of the viruses were nl63, 6 were oc43, 4 were hku1, and 1 was 229e. two patients had 2 separate identifications of hcov in samples collected 2 and 6 months apart, respectively, and different species were identified on each occasion. single hcov infections were found on 13 occasions, while coinfections were observed on 6 occasions, four with the identification of 2 viruses (rhinovirus or enterovirus) and two with 3 viruses identified (rsv + rhinovirus and influenza a virus + enterovirus). overall, 142 episodes (34.8%) of respiratory exacerbations were identified, and hospital admissions occurred in 31 (7.6%) of them. significant associations were found for an increased risk of respiratory exacerbation when rhinovirus c was identified and an increased risk of hospital admission with influenza virus infections (data not shown). no significant difference was found among patients with hcov infection, with exacerbations identified on 6 of 19 occasions (31.5%) and hospital admissions on 2 of 19 occasions (10.5%). the hcov species that were found are displayed in table 1 , as are patient characteristics, including the frequency of symptoms and the clinical status at the time of the visit. the age distribution was similar among patients infected with different hcov species, although a significant difference was found among the ages of patients without viral infections and those infected with hcov or other viruses (table 1) . although no significant differences were seen concerning reported symptoms and clinical findings among patients infected with different hcov species, a significantly greater respiratory exacerbation rate was found among patients infected with hcov-nl63 as compared to those infected with other hcov species (p = .04). in the logistic regression model, however, neither the identification of any hcov nor hcov-nl63 specifically could be demonstrated as being significantly associated with a respiratory exacerbation. this is the first description of the identification of these new hcov species among patients with cf. while no significant clinical impact could be attributed to infection with these species as a whole, the identification of hcov-nl63 was associated with a significantly higher rate of respiratory exacerbation as compared to other hcov species. although hcovs were previously identified as causative agents of lower respiratory tract infections, the medical community commonly knew them as "common cold" viruses. the identification of new species of hcov [7, 8] and the emergence of sars hcov [6] highlighted the potential role of these viruses as causative agents of severe lower respiratory tract infections. following initial descriptions of these new "non-sars" hcov, several other studies describing the prevalence and clinical impact were published, although results were conflicting. while the prevalence values of these new hcov-nl63 and hcov-hku1 oscillate from 0.4% to 9% [5] , reports of severe cases of lower respiratory infections were described in australia for the hcov-nl63 [11] and in germany for the hcov-hku1 [12] . however, most of the studies on the clinical impact of different hcov species were only performed among children who were hospitalized for acute respiratory tract infections, with small sample sizes and short periods of sample collection [10] . more recent studies have included much larger samples. gaunt et al. [9] , who studied 11 600 respiratory samples obtained between 2006 and 2009 in edinburgh, united kingdom, found at least 1 coronavirus in 2.3% of the samples, with marked predominance in winter months. although they found a significant number of coinfections (mainly with rsv), single infections with hcov-oc43, hcov-nl63, and hcov-hku1 were specifically associated with lower respiratory tract disease. they also observed that hcov-229e, which was identified in a smaller proportion of samples, was predominant among immunosuppressed patients. talbot et al. [10] used molecular tools to determine the incidence and clinical features of upper and lower respiratory tract infections that are figure 1 . representative neighbor-joining phylogenetic tree of a partial rna-dependent rna polymerase region of coronavirus (cov), generated with a p-distance model on the basis of a 134-amino acid sequence by use of mega 5.0 software (available at: http://www.megasofware.net). reference cov species data were obtained from the genbank database. viruses identified in this study are indicated in boldface and labeled vfc. bootstrap values >70% in the key branches are depicted. scale bar indicates amino acid substitutions per site. the strain designations, genbank accession numbers, and corresponding hosts are indicated. the respective genera are given on the right. the highly conserved amino acid sequence fragment did not permit differentiation of the 2 nl63 cov clusters. associated with hcov-nl63, hcov-oc43, and hcov-229e during a 20-year period and showed that hcov-oc43 and hcov-nl63 were associated with a significant burden of lower respiratory tract infections in previously healthy outpatient children who manifested with clinical syndromes, such as pneumonia and bronchiolitis, that were similar to those caused by other respiratory viruses [10] . patients with cf represent a specific population that is susceptible to more severe clinical manifestations of respiratory infections overall, because of limitations in mucocilliary transport and anatomic distortions of the bronchial tree that facilitate the retention and infection of respiratory secretions. the impact of respiratory viruses in cf lung disease has been recently reassessed using molecular methods by some researchers, with conflicting results: while olesen et al [13] reported a lack of significant clinical impact of viral infections in lung function or respiratory symptoms, wat et al [2] described an association of viral infections with respiratory exacerbations in cf children, particularly when influenza a virus, influenza b virus, and rhinovirus infections were identified. one possible explanation for the differential clinical impact of respiratory viruses for cf patients is the baseline lung function, and it is possible that patients with worse lung disease would be at more risk to present an exacerbation; we cannot rule out this possibility in the present study, because of the small number of patients infected with hcov. another hypothesis is related to the mechanisms of disease of hcov. both sars coronavirus and hcov-nl63 use the angiotensinconverting enzyme 2 (ace2) as the main pathway for attachment and entry. following infection, a substantial reduction of the ace2 expression in the cell surfaces occurs [14] , but the rate and the intensity of this downregulation seems to be much lower in hcov-nl63 infections as compared to sars coronavirus infections. ace2 downregulation was previously shown to be associated with an increase of the inflammatory process in the respiratory tract and may be one of the determinants of severity in acute respiratory distress syndrome [14] . moreover, genetic polymorphisms associated with an increase of ace expression (which counterbalance ace2 action as a proinflammatory mediator) were shown to be associated with worse lung disease in cf patients [15] . we have previously shown that human rhinovirus c infections were associated with an increased risk of respiratory exacerbations in cf children [3] , and although we could not identify a significant association of hcov infections with respiratory exacerbations in the present analysis, such exacerbations were observed in a significant proportion (62.5%) of hcov-nl63-infected children. it should be mentioned that the mean age of our patients was approximately 9 years, while in the study by talbot et al. [10] , the greatest incidence of lri associated with these viruses occurred between 6 and 23 months of age. therefore, a study of infants with cf could produce very different results. in conclusion, we found all circulating species of hcov in children with cf who were enrolled in this study. symptoms were mainly related to the upper airway, and we did not find an association between hcov infections in general or infections with a given hcov species and an increased risk of respiratory exacerbation or hospital admission, although a significantly higher rate of respiratory exacerbation was observed among hcov-nl63-infected children as compared to children infected by other coronaviruses. cystic fibrosis the role of respiratory viruses in cystic fibrosis rhinovirus c and respiratory exacerbations in children with cystic fibrosis coronavirus infection in acute lower respiratory tract disease of infants human coronavirus nl63: a clinically important virus? identification of a novel coronavirus in patients with severe acute respiratory syndrome identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method the pediatric burden of human coronaviruses evaluated for twenty years new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia two cases of severe obstructive pneumonia associated with an hku1-like coronavirus viral and atypical bacterial infections in the outpatient pediatric cystic fibrosis clinic renin-angiotensin system in human coronavirus pathogenesis end-organ dysfunction in cystic fibrosis: association with angiotensin i converting enzyme and cytokine gene polymorphisms financial support. this work was supported by fundação de amparo à pesquisa do estado de são paulo (grant 05/01625-8).potential conflicts of interest. key: cord-007187-gb1txu1o authors: lindner, juha; zehentmeier, sandra; franssila, rauli; barabas, sascha; schroeder, josef; deml, ludwig; modrow, susanne title: cd4(+) t helper cell responses against human bocavirus viral protein 2 viruslike particles in healthy adults date: 2008-12-01 journal: j infect dis doi: 10.1086/592985 sha: doc_id: 7187 cord_uid: gb1txu1o background. human bocavirus (hbov) was recently described as a new member of the parvoviridae family, and its possible association with respiratory illness in infants has been discussed. to date, hbov genomes have been detected worldwide in respiratory tract samples obtained from children with pulmonary diseases, whereas only limited data on virus-specific immunity are available, mainly because of the lack of recombinant viral antigens. methods. hbov viruslike particles (vlps) were produced in insect cells and characterized by electron microscopy and cesium chloride gradient centrifugation. hbov viral protein 2 (vp2)-specific antibodies and cd4(+) t helper cell responses were analyzed by enzyme-linked immunsorbent assay and enzyme-linked immunospot assay. results. vp2 capsid proteins of hbov were produced in insect cells infected with a recombinant baculovirus, and the formation of icosahedral vlps (diameter, 21–25 nm; sedimentation density, 1.33 g/cm(3)) was demonstrated. a significant increase in secretion of vp2-specific interferon-γ was detected in cultures of peripheral blood mononuclear cells obtained from 69 healthy adults found to be positive for hbov-specific immunoglobulin g antibodies, compared with control stimulations. in parallel, t cell responses against identically expressed parvovirus b19 vp2 vlps were frequently observed in the individuals studied, without there being obvious cross-reactions between hbov and parvovirus b19. conclusions. data suggest the presence of hbov-specific immune responses in adults and strongly support a high prevalence of hbov among humans. human bocavirus (hbov), a newly discovered member of the parvoviridae family, was recently identified in children and infants with infections of the lower respiratory tract [1] . hbov is the second parvovirus currently being discussed as a cause of disease in humans. comprehensive phylogenetic analysis of the hbov genome has revealed a close association between hbov and the canine minute virus and the bovine parvovirus, both of which are members of the bocavirus genus. to date, several hbov isolates with high sequence homology have been described [2] . parvoviruses are small, nonenveloped viruses that are characterized by linear single-stranded dna genomes of 4 -6 kb. common characteristics of parvoviruses are their exceptional stability and the structural simplicity of the virions [3] . the icosahedral virus capsids generally consist of 2 proteins (viral protein 1 [vp1] and viral protein 2 [vp2]) that have identical sequences, except for the aminoterminal domain of vp1 (known as the "vp1 unique region"), which spans 129 and 227 amino acids in hbov and parvovirus b19 (b19v), respectively [4] . b19v vp2 proteins have been shown to possess all the characteristics required for particle formation. viruslike particles (vlps) consisting of vp2 alone or of vp2 and vp1 may be produced in various eukaryotic expression systems (e.g., recombinant baculovirus or yeast) [5, 6] . hbov has been a target of epidemiologic studies since its initial detection. to date, by use of polymerase chain reaction-based techniques, hbov has been detected not only in clinical respiratory tract samples but, also, in fecal excretions from young children in australia, north america, asia, europe, africa, and the middle east, thereby indicating a worldwide distribution of the virus [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . the prevalence of hbov dna in samples obtained from children and infants with symptoms of respiratory illness has been found to be 1.5%-18.3%, and it may vary depending on seasonal fluctuation and the age of the patients studied. because the majority of epidemiologic studies have been performed retrospectively, hbov has not been clearly identified as a sole infectious agent responsible for respiratory diseases. in fact, hbov tends to be associated with a high rate of coinfection with other respiratory viruses (e.g., picornaviruses, adenoviruses, and respiratory syncytial virus) in children. clinical symptoms associated with hbov infections include cough, fever, pneumonia, and bronchitis. to date, only single reported cases of hbov infections in adults have been observed, mainly in immunocompromised individuals [18, 19] . immunologic studies have described the use of recombinant hbov vp1 protein in the detection of virus-specific igg responses in the japanese population [20] . thereby, ubiquitous vp1-specific antibody responses (with a seroprevalence of up to 100%) were detected in adult individuals and in children (age, ͼ6 years), whereas a seronegative status was predominantly noted among young children at 1 year of age. no data are currently available on hbov-specific t cell immunity. numerous studies of b19v, however, have described viral capsid proteins as immunodominant targets of the cellular and humoral immune response [21] [22] [23] [24] , suggesting a possibly similar role for hbov capsid proteins in virus-specific immunity. in the present study, we describe the recombinant expression and characterization of hbov vp2 vlps and their use in the detection of cellular immune responses against hbov in healthy adults. a total of 69 healthy, white individuals (39 men and 30 women; mean age, 39 years [range, 23-73 years]) from the southeastern region of bavaria, germany, were studied to identify hbov-and b19v-specific t cell responses (table 1) and to detect the presence of virusspecific antibodies by means of elisa. for hbov, 100 ng of hbov vp2 vlps were coated on nunc-immuno medisorp plates (nunc) in pbs overnight at 4°c, washed 6 times with washing buffer (pbs containing 0.05% tween 20), and blocked with dilution buffer (pbs containing 2% tween 20 and 3% fcs) for 1 h at 37°c. after incubation with serum samples (1:100 in dilution buffer) was done for 2 h at 37°c, the plates were washed, and an anti-human igg-specific, horseradish peroxidase-coupled secondary antibody (dako deutschland) (1:6000 in dilution buffer) was added for 1 h at 37°c. development was performed using bd opteia substrate (bd biosciences) according to the manufacturer's instructions. b19v-specific igg and igm antibodies were detected in donor serum samples by use of a standardized elisa (biotrin international) and recomblot analysis (mikrogen). a recombinant baculovirus encoding the vp2 gene of the hbov st2 isolate was generated using the bac-to-bac baculovirus expression system (invitrogen). in table 1 . antigen-specific interferon (ifn)-␥ secretion in healthy adults seropositive for human bocavirus (hbov). brief, the vp2 gene was amplified from a pcr 4-topo vector (invitrogen) that included the genome sequence of hbov st2 (genbank accession number nc_007455) (provided by tobias allander, karolinska institute, stockholm, sweden), by use of the primers 5'-gaggagcggccgcatgtctgacactga-cattc-3' and 3'-ccgccctcgagttacaacactttattg-atg-5' with phusion high-fidelity dna polymerase (finnzymes oy). the amplicon was introduced into the pfastbac1 vector (invitrogen) by the xhoi and noti restriction sites. generation of recombinant hbov vp2 bacmid pfastbac/vp2bac and its subsequent transfection into sf9 insect cells were performed according to the manufacturer's instructions. the resulting virus, bacvp2/hbov, was amplified in 3 consecutive passages of infection and subsequently was used for protein production. for the expression of b19v vp2 vlps, the previously established and described recombinant baculovirus bacvp2/b19v was used [25] . production and purification of recombinant hbov vlps. production of both hbov and b19v vlps was achieved in high-5 insect cells. cells were cultured in insect-xpress medium (cambrex bio science walkersville), infected with bacvp2/ hbov or bacvp2/b19v at an moi of 3, and harvested 72 h after infection. recombinant vlps were purified by cesium chloride (cscl) cushion centrifugation. a total of 6 ϫ 10 7 cells were lysed in 5 ml of buffer containing 10 mmol/l tris/hcl (ph 7.4), 10 mmol/l nacl, 15 mmol/l mgcl 2 , and 0.5% triton x-100 with 40 l/ml complete protease inhibitor cocktail (roche applied science) and then were subjected to successive freeze-thaw cycles. the precleared lysates were loaded on double cushions consisting of cscl solutions with densities of 1.52 g/cm 3 and 1.22 g/cm 3 in tris-edta buffer (10 mmol/l tris/hcl [ph 8.7], 1 mmol/l edta, and 0.5% triton x-100). ultracentrifugation was performed at 100,000 g for 4 h at 10°c without brakes. after centrifugation, protein samples were collected in fractions and subjected to sds-page and western blot analysis. endotoxin contaminations of fractions containing antigen were excluded using the qcl-1000 chromogenic lal endpoint assay (lonza), and they were determined to be 1.57 eu/mg and 8.2 eu/mg for hbov and b19v vp2 vlps, respectively. characterized antigens underwent dialysis against pbs and were stored in aliquots at ϫ80°c. freeze-thaw cycles of the purified proteins were avoided. pbmcs were isolated from fresh heparinized blood samples by means of pancoll centrifugation (pan biotech) (at 800 g for 30 min) with the use of leucosep tubes (greiner bio-one). cells were washed twice in pbs and were cultured in rpmi 1640 medium (pan biotech) containing 2 mmol/l l-glutamine, 100 u/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated human ab serum (paa laboratories), in an atmosphere with 5% co 2 at 37°c. a total of 2 ϫ 10 5 pbmcs were seeded in 4 replicate wells on mahan 4550 multiscreen elispot plates (millipore) that had been previously coated with 5 g/ml anti-human interferon (ifn)-␥ monoclonal antibody d1k (mabtech), stimulated with 5 g/ml vp2 vlps of either hbov or b19v, and incubated at 37°c for 60 h. as negative and positive controls, cells were stimulated with 5 g/ml hiv p24-derived murine e10f peptide [26] and 5 ng/ml staphylococcal enterotoxin b (sigma aldrich), respectively. for development, the plates were incubated for 2 h with 1 g/ml biotinylated anti-ifn-␥ 7-b6-1 biotin antibody (mabtech) and for 1 h with streptavidin-alkaline phosphatase (diluted 1:1000 in pbs). staining was performed using the nbt/ bcip stock solution (roche diagnostics) in buffer containing 0.1 mol/l nacl, 0.1 mol/l tris/hcl [ph 9.5], and 0.05 mol/l mgcl 2 . the number of spot-forming cells (sfcs) was determined under magnification, by use of a bioreader 2000 (biosys). t cell depletion assays. depletion of cd4 ϩ or cd8 ϩ t cells from donor pbmc cultures was performed using cd4 and cd8 dynabeads (dynal biotech asa). for control, the purity of depleted cell populations was assessed by staining with the cy-tostat multicolor reagent cd45-fitc/cd4-pe/cd8-ecd/ cd3-pc5 (beckman coulter) and an anti-cd14-pe antibody (bd biosciences) (data not shown). a facs epics xl-mcl flow cytometer (beckman coulter) was used for analysis. all experiments were conducted according to the manufacturer's instructions. statistical data analysis. serologic and cellular responses were statistically evaluated using the mann-whitney u-test for independent samples. seronegative individuals have been described and were found predominantly among infants and young children (those 2 years of age) [20, 27, 28, 29] , serum samples obtained from 10 healthy infants (mean age, 11.8 months [range, 6 -45 months]) without detectable virus-specific antibodies (median od 450 value, 0.056 [range, 0.017-0.104]) were selected and representatively included in the study, to ensure the specificity of the serologic analysis performed. with respect to b19v, 47 of 69 volunteers displayed igg antibodies against the vp1/vp2 proteins, indicating past b19v infection (table 1) . b19v-specific igm antibodies were not detectable in any of the serum samples. because sufficient volumes of blood samples could not be obtained from very young children, only hbov-seropositive adults underwent analysis for hbov-and b19v-specific cellular immune responses by use of the elispot assay. using pbmcs stimulated with either hbov or b19v vp2 vlps (5 g/ml), virus-specific ex vivo ifn-␥ immune responses with median values of 20 and 38 sfcs/2 ϫ 10 5 pbmcs were detected in hbov-and b19vseropositive individuals, respectively (figure 4). the correlation of the number of hbov-specific ifn-␥-secreting cells with anti-hbov igg responses did not reveal any statistical significance (p ϭ .5809). for this analysis, hbov-seropositive individuals were divided in 2 groups: those with od 450 values below the median value and those with od 450 values above the median, respectively. both groups displayed a median of 20 sfcs/ 2 ϫ 10 5 pbmcs. no significant ifn-␥ responses (median response, 3 sfcs/ 2 ϫ 10 5 pbmcs) were measured against the synthetic hiv peptide e10f, which was used as a control for hbov stimulations. control stimulations of b19v-seronegative individuals that involved the use of b19v vp2 vlps resulted only in background levels of ifn-␥ secretion (median response, 6 sfcs/2 ϫ 10 5 pbmcs). . detection of human bocavirus (hbov)-specific igg antibodies in healthy adults. by use of viral protein 2 (vp2) viruslike particles (vlps), all 69 healthy adults who were studied were found to be positive (seropositive) for hbov-specific igg antibodies by elisa. because seronegative individuals have been predominantly described and could only be found among young children and infants, a total of 10 representative (seronegative) children (mean age, 11.8 months) were included in the study to ensure the specificity of the serologic analysis performed. the cutoff value for positive results (dashed line) was defined as an optical density value that was 2.5-fold greater than the median optical density value (horizontal line) noted for infants without hbov-specific igg reactions who were evaluated. to exclude the possibility that cross-reactions between vp2 proteins of hbov and b19v contributed to the positive test results, hbov-specific immune responses were analyzed in association with b19v serologic findings. thereby, no statistically significant correlation (p ϭ .4237) was observed as median values of 17 and 26 sfcs/2 ϫ 10 5 pbmcs were measured in b19vseronegative and -seropositive individuals, respectively, after hbov vp2 vlp stimulation. to associate the hbov vp2-specific cytokine responses with specific cell populations, stimulations of cultures of pbmcs depleted from either cd4 ϩ or cd8 ϩ t cells were performed ( figure 5 ). in cultures of pbmcs obtained from 5 hbov-seropositive individuals who were tested, a substantial reduction in the median number of cells secreting ifn-␥ (from 21 to 2 sfcs/ 2 ϫ 10 5 pbmcs) (p ϭ .0079) was observed after depletion of cd4 ϩ t helper cells, whereas hbov-specific t cell responses remained detectable (median response, 18 sfcs/2 ϫ 10 5 pbmcs) after depletion of cd8 ϩ cytotoxic t lymphocytes (p ϭ .8413). this finding suggests a pivotal role of the cd4 ϩ lymphocyte subset in the cellular immune response against vp2 proteins of hbov. correlation of the hbov vp2-specific immune responses with the age of the study subjects revealed an age-dependent decrease in the median number of cells secreting ifn-␥ (from 35 sfcs/2 ϫ 10 5 pbmcs in young adults [age, 18 -27 years] born between 1980 and 1989 to 17 sfcs/2 ϫ 10 5 pbmcs in elderly adults [age, 68 -77 years] born between 1930 and 1939) ( figure 6 ). in general, when compared with men, women displayed higher or equal numbers of hbov-specific t cells against hbov vp2 vlps, in all age groups. this observation was most prominent among individuals born between 1970 and 1979 (age, 28 -37 years), as was revealed by median responses of 25 sfcs/ 2 ϫ 10 5 pbmcs and 51 sfcs/2 ϫ 10 5 pbmcs for men and women, respectively (p ϭ .0667). hbov vlps consisting of vp2 proteins can be produced using the baculovirus expression system. similar capacities for other parvoviruses, such as b19v, the canine parvovirus, and the porcine parvovirus, have been described elsewhere [5, 30, 31] . by use of electron microscopy and cscl gradient centrifugation, hbov vp2 vlps were shown to possess a diameter comparable to that of native hbov virions [32] and sedimentation densities similar to those of capsids of the canine parvovirus [33] . recombinant vlps are potent reagents for virologic and clinical research and have greatly contributed to the improvement of sensitive diagnostic tests and the development of vaccine against many viruses (e.g., human papillomavirus and hepatitis b virus) [34, 35] . recombinant vp2 particles are known to be crucial in the diagnosis of b19v infections, because virus-specific antibodies lose their affinity against linear antigenic regions in a timedependent manner and are replaced by humoral responses against conformational epitopes [36] . whether similar applications can be established for hbov vp2 vlps needs to be assessed in further studies. using hbov vlps, we observed frequent vp2-specific humoral immune responses in healthy adults, whereas seronegative status was predominantly detected in young children (age, 2 years) [27, 37] . furthermore, this finding is strongly supported by recent epidemiologic data, which describe a seroprevalence of hbov-specific igg antibodies that already reaches up to 100% during early childhood [20] . because of the very young age of the seronegative individuals, it was not possible to obtain blood samples with volumes sufficient for the analysis of hbovspecific t cell immunity. in seropositive adults, frequent cellular immune responses were observed after hbov vp2 vlp stimulation. the median number of cells secreting ifn-␥ against hbov was thereby shown to be slightly lower than the median number of cells secreting ifn-␥ against vp2 particles of b19v, which previously have been shown to serve as targets for ifn-␥-and il-10 -mediated cd4 ϩ t helper cell responses [38, 39] . however, this observation was mainly based on 4 study subjects who exhibited high cytokine responses against the b19v vp2 proteins. these single high responses might be explained by possible recent b19v infections or viral restimulation, because the average seroprevalence of b19v in the german adult population is 77%, and, therefore, acute infections may occur in adults [40] . compared with synthetic peptide controls, slightly elevated ifn-␥ responses against b19v vp2 vlps were observed in seronegative individuals. this finding suggests that minor, unspecific cytokine secretion may be induced in blood cells by means of residual pyrogens (e.g., endotoxin) included in recombinant antigen but not in synthetic peptide preparations [41] . however, hbov vp2-specific t cell responses were not detected in all individuals studied, because, for many donors, only low or undetectable ifn-␥ secretion was observed, suggesting antigen specificity of the detected t cell responses. in the study group, an age-dependent decrease in hbovspecific t cell responses was observed. this might be the result of an age-related decrease in and an impairment of the amount and functions of virus-specific t cell populations, because it has been described for other viral infections (e.g., for populations with adenovirus) [42] . furthermore, it is known that active t cell responses against pathogens undergo confinement to specific memory populations with increasing time after primary infection in the absence of exogenous restimulation events or endogenous reactivations of latent viral infections. because all currently published data on the epide-miologic profile of hbov strongly suggest that primary infection occurs in the very early years of childhood [9 -17, 20] , the time-dependent reduction in the number of virus-specific t cells might be explained by a lack of frequent pathogen contact. this may be considered to provide additional proof of the early time point when hbov infection occurs during childhood. independent of age, female subjects had higher numbers of cells secreting ifn-␥ than did male subjects, especially among younger individuals; this finding possibly indicates more-frequent spontaneous viral restimulation (e.g., by increased contact with children with acute infection), resulting in an overall increase in the hbov-specific cellular immune response. this hypothesis is further supported by the fact that the most prominent difference in sex-specific cellular responses was observed in individuals born between 1970 and 1979, an age group in which women presumably have the most frequent contact with small children. however, because no information on the family status of the study subjects is available, these data will have to be confirmed by additional epidemiologic investigations. cloning of a human parvovirus by molecular screening of respiratory tract samples complete coding sequences and phylogenetic analysis of human bocavirus (hbov). virus stability of minute virus of mice against temperature and sodium hydroxide the vp1 unique region of parvovirus b19 and its constituent phospholipase a2-like activity assembly of empty capsids by using baculovirus recombinants expressing human parvovirus b19 structural proteins parvovirus b19 vp2-proteins produced in saccharomyces cerevisiae: comparison with vp2-particles produced by baculovirus-derived vectors human bocavirus infection human bocavirus infection among children human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus detection of 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cell-mediated in vitro responses of recently and remotely infected subjects to a candidate recombinant vaccine for human parvovirus b19 b cell memory is directed toward conformational epitopes of parvovirus b19 capsid proteins and the unique region of vp1 cd4 ϩ t-cell responses against the vp1-unique region in individuals with recent and persistent parvovirus b19 infection parvovirus b19 does not bind to membrane-associated globoside in vitro influence of polypeptide size and intracellular sorting on the induction of epitope-specific ctl responses by dna vaccines in a mouse model serodiagnosis of human bocavirus infections humoral immune response against human bocavirus vp2 virus-like particles human bocavirus-a novel parvovirus to infect humans identification of domains in canine parvovirus vp2 essential for the assembly of viruslike particles virus-like particle production at low multiplicities of infection with the baculovirus insect cell system electron microscopy observation of human bocavirus (hbov) in nasopharyngeal samples from hbov-infected children canine parvovirus empty capsids produced by expression in a baculovirus vector: use in analysis of viral properties and immunization of dogs efficacy of a quadrivalent prophylactic human papillomavirus (types 6, 11, 16, and 18) l1 virus-like-particle vaccine against high-grade vulval and vaginal lesions: a combined analysis of three randomised clinical trials epitope type-specific igg responses to capsid proteins vp1 and vp2 of human parvovirus b19 clinical and epidemiological aspects of human bocavirus infection t-helper cell-mediated interferon-␥, interleukin-10 and proliferation responses to a candidate recombinant vaccine for human parvovirus b19 t helper cell-mediated interferon-gamma expression after human parvovirus b19 infection: persisting vp2-specific and transient vp1u-specific activity the prevalence of antibody to parvovirus b19 in hemophiliacs and in the general population induction of proliferation and cytokine production in human t lymphocytes by lipopolysaccharide (lps) age-related decrease in adenovirusspecific t cell responses we thank tobias allander and colleagues from the karolinska institute (stockholm, sweden) for the generous gift of the pcr 4-topo vector (invitrogen), including the genome sequence of human bocavirus st2. we also thank klaus hedman and colleagues at the university of helsinki for experimental and theoretical support. the excellent technical assistance of julia mischner and richard klar, as well as the critical proofreading of the manuscript by valeria runza, is appreciated. key: cord-007009-4wbvdg1r authors: takahashi, toru; maeda, ken; suzuki, tadaki; ishido, aki; shigeoka, toru; tominaga, takayuki; kamei, toshiaki; honda, masahiro; ninomiya, daisuke; sakai, takenori; senba, takanori; kaneyuki, shozo; sakaguchi, shota; satoh, akira; hosokawa, takanori; kawabe, yojiro; kurihara, shintaro; izumikawa, koichi; kohno, shigeru; azuma, taichi; suemori, koichiro; yasukawa, masaki; mizutani, tetsuya; omatsu, tsutomu; katayama, yukie; miyahara, masaharu; ijuin, masahito; doi, kazuko; okuda, masaru; umeki, kazunori; saito, tomoya; fukushima, kazuko; nakajima, kensuke; yoshikawa, tomoki; tani, hideki; fukushi, shuetsu; fukuma, aiko; ogata, momoko; shimojima, masayuki; nakajima, noriko; nagata, noriyo; katano, harutaka; fukumoto, hitomi; sato, yuko; hasegawa, hideki; yamagishi, takuya; oishi, kazunori; kurane, ichiro; morikawa, shigeru; saijo, masayuki title: the first identification and retrospective study of severe fever with thrombocytopenia syndrome in japan date: 2014-03-15 journal: j infect dis doi: 10.1093/infdis/jit603 sha: doc_id: 7009 cord_uid: 4wbvdg1r background. severe fever with thrombocytopenia syndrome (sfts) is caused by sfts virus (sftsv), a novel bunyavirus reported to be endemic in central and northeastern china. this article describes the first identified patient with sfts and a retrospective study on sfts in japan. methods. virologic and pathologic examinations were performed on the patient's samples. laboratory diagnosis of sfts was made by isolation/genome amplification and/or the detection of anti-sftsv immunoglobulin g antibody in sera. physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of sfts. results. a female patient who died in 2012 received a diagnosis of sfts. ten additional patients with sfts were then retrospectively identified. all patients were aged ≥50 years and lived in western japan. six cases were fatal. the ratio of males to females was 8:3. sftsv was isolated from 8 patients. phylogenetic analyses indicated that all of the japanese sftsv isolates formed a genotype independent to those from china. most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. conclusions. sfts has been endemic to japan, and sftsv has been circulating naturally within the country. severe fever with thrombocytopenia syndrome (sfts), an infectious disease with a high case-fatality rate, is caused by sfts virus (sftsv), a novel bunyavirus reported to be endemic to central and northeastern parts of china [1, 2] . sftsv, which is classified into the genus phlebovirus and the family bunyaviridae, is suspected to be a tick-borne virus owing to evidence of its presence in 2 species of ticks: haemaphysalis longicornis and rhipicephalus microplus [2, 3] . approximately 2% of h. longicornis organisms collected from sheep, cattle, and dogs in shandong province tested positive for sftsv in virus genome amplification assays [4] . a similar disease, which was named fever, thrombocytopenia and leukopenia syndrome (ftls), was independently reported to be caused by a novel virus, henan fever virus (hnfv) [1] . despite the different names, "sfts" and "ftls" represent the same condition and "sftsv" and "hnfv" represent the same virus. the case-fatality rate of sfts is reported to be approximately 12% [1, 2] . humanto-human transmission of sftsv was reported to occur through close contact with the blood and/or body secretions of infected patients [5] [6] [7] [8] [9] . to our knowledge, there were no published reports of sfts outside of china before we performed the study described here. another tick-borne phlebovirus, the heartland virus, which was detected in missouri, is phylogenetically associated with sftsv, caused severe febrile illness with thrombocytopenia, leukopenia in the total blood cell count, and elevated levels of liver enzymes [10] . we report the first identification of sfts in japan, which was detected in a previously healthy woman aged 50-59 years who died of multiple-organ failure in the autumn of 2012, and findings from a subsequent retrospective study of sfts in japan. culture supernatants were subjected to viral rna extraction using high pure viral nucleic acid kit (roche diagnostics). complementary dna (cdna) was synthesized using super-script iii (invitrogen) with random primers and then randomly amplified using the illustra genomiphi v2 kit (ge healthcare life sciences). a cdna library was prepared using the nextera dna sample prep kit (illumina). a sequencing run for 50 nucleotides was performed with miseq (illumina), using the miseq reagent sequencing kit (illumina). the assembled nucleotide sequences were used to determine homologous sequences by tblast at the national center for biotechnology information web site (available at: http://blast.ncbi.nlm.nih.gov/blast.cgi). the sftsv genome was detected by reverse-transcription polymerase chain reaction (rt-pcr) in total rna extracted from patient sera, using a high pure viral rna kit (roche applied science). reverse transcription was performed with ready-to-go rt-pcr beads (ge healthcare), using random nucleotide hexamers. pcr was performed to amplify sftsv-specific cdna fragments. pcr primer sets were designed to amplify the nucleoprotein (np) region of the sftsv genome (supplementary table 1 ). the pcr conditions were as follows: 1 cycle at 55°c for 30 minutes followed by 95°c for 2 minutes; 45 cycles at 94°c for 30 seconds, 52°c for 30 seconds, and 68°c for 30 seconds; followed by 1 cycle at 68°c for 5 minutes. vero cells were inoculated with rt-pcr-positive patient sera for virus isolation, cultured for 4-7 days, and examined for sftsv antigen detection by indirect immunofluorescence assay (ifa) with a polyclonal antibody raised against sftsv recombinant np (rnp; rabbit anti-sftsv rnp serum), which was produced as follows. sftsv rnp tagged with histidine-tag on the c-terminus was expressed in a baculovirus expression system, as previously described [11, 12] . the np gene of sftsv strain hb29 (genbank accession no. nc_018137) was artificially synthesized. anti-sftsv rnp serum was raised in rabbits by immunization with the purified sftsv rnp, as previously described [13] [14] [15] . the neutralizing antibody to sftsv chinese isolate hb29 strain [2, 16] and japanese isolate yg1 (this study) was detected as reported previously, except for the target virus [15] . with the exception of the antigen preparation, immunoglobulin g (igg) antibody titers to sftsv were determined by indirect ifa, using sftsv hb29-infecting vero cells as previously described [14] . pathologic studies with histopathologic, immunohistochemical, and in situ hybridization at-tailing (ish-at) methods histopathologic studies of formalin-fixed and paraffin-embedded specimens were performed using hematoxylin-eosin stain. immunohistochemical analysis was performed as previously described, with some modifications [17] . the rabbit anti-sftsv rnp serum and the peroxidase-labeled, polymer-conjugated antirabbit immunoglobulin (envision/hrp, dako) were used in the immunohistochemical analysis as the primary and the secondary antibodies, respectively. normal rabbit serum and lymph nodes of necrotizing lymphadenitis without sftsv infection were used as negative controls for the antibody and tissue specimens, respectively. ish-at was used for detection of sftsv genomic rna (negative-strand rna) as reported previously, with the exception of the strand-specific oligonucleotide probes [18] [19] [20] , which were designed for the s and l segments of the sftsv genome (supplementary table 1 ). the sftsv copy number was determined by performing quantitative real-time rt-pcr analysis of rna samples extracted from paraffin-embedded sections as previously described, with some modifications [17] . the amount of human β-actin messenger rna (mrna) in the dnase-treated rna extracted from each section was also determined and used as an internal reference for normalization [21] . the primers and labeled probes are shown (supplementary table 1 ). the culture supernatant from vero cells inoculated with sftsv isolated from the first patient was used for electron microscopic analysis. the samples were fixed with 4% glutaraldehyde. the fixed samples were negatively stained with 2% phosphotungstic acid and then observed using a jem-1400 transmission electron microscope (jeol, tokyo, japan). the first diagnosis of sfts in japan was made public through an announcement from the ministry of health, labor, and welfare of japan on 30 january 2013. physicians were asked to volunteer information if they had treated patients who satisfied the following case definition: (1) fever of >38°c; (2) gastrointestinal tract symptoms, such as nausea, vomiting, abdominal pain, diarrhea, and melena; (3) thrombocytopenia, with <100 × 10 9 platelets/l; (4) leukopenia, with <4 × 10 9 white blood cells/l; (5) elevated levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase; (6) absence of other causes; and (7) death or admission to an intensive care unit because of the severity symptoms. information about patients was collected from their physicians. the retrospective recruitment of patients with suspected sfts was conducted from 30 january to 31 march 2013. written informed consent was obtained from patients or responsible relatives. serum samples, which had been collected from the patients for future analyses to clarify the etiology and had been stored in each hospital by the respective physicians, used for the present study were sent to the department of virology 1 at the national institute of infectious diseases (niid; tokyo, japan) for virologic analyses. clinical and laboratory data of the patients with sfts were also sent to the corresponding author (m. saijo) without information that made it possible to identify individuals. cdnas prepared from the patients' sera samples were used to determine sftsv genome sequences. the terminal sequences of sftsv genomes were determined by rapid amplification of cdna ends (race) at the 3′ and 5′ ends, performed by rt-pcr after 3′ and 5′ linker ligation using a dynaexpress mirna cloning kit, high efficient (biodynamics laboratory, tokyo, japan). purified rna from the culture supernatant of sftsv (yg1)-infected vero cells was subjected to the 3′ linker ligation according to the manufacturer's protocol. next, the 5′ linker was ligated to the 5′-phosphate end of the purified 3′ linker ligated rna, according to the manufacturer's protocol, following rna purification by nucleospin rna clean-up xs (takara bio, shiga, japan). the 3′ and 5′ linker ligated rna was then reverse transcribed according to the manufacturer's protocol by super-script iii (invitrogen, carlsbad, ca), using either the 3′ forward rt primer, which is the antiparallel sequence of the 3′ linker sequence, or the pd(n)6 primer (random hexamer). rt-pcr was performed using q5 hot start high-fidelity dna polymerase (neb, ipswich, ma) and sftsv gene-specific primers (supplementary table 1 ) with the 3′ rt primer or the 5′ primer, which is the antiparallel sequence of the 5′ linker sequence. nucleotide sequences of each full segment of sftsv in patients' sera were aligned using muscle, an en suite program in the molecular evolutionary genetics analysis 5.1 software (mega team, japan). evolutionary distances were estimated using kimura's 2-parameter method, and phylogenetic trees were constructed using the neighbor-joining method. the robustness of the trees was tested using 1000 bootstrap replications. accession numbers of the nucleotide sequences of sftsv l-, m-, and s-segments are described in supplementary table 2 . serum samples were used for virologic analysis after obtaining written informed consent from the patients themselves (for those who survived) or their responsible family members (for those who died). the clinical and laboratory data of the patients with sfts were sent to the corresponding author without personally identifying information. all of the protocols and procedures were approved by the research and ethics committees of the niid. the polyclonal antibody to sftsv rnp was produced by immunizing rabbits with purified sftsv rnp, with approval from the institutional animal care and use committee of the niid (no. 111 124). a previously healthy woman aged 50-59 years who lived in the yamaguchi prefecture of japan was hospitalized with high fever, fatigue, vomiting, and melena in the autumn of 2012. her body temperature was 39.2°c. tick bite wounds were not observed anywhere on her skin. laboratory tests revealed a low platelet count of 89 × 10 9 platelets/l (normal range, 150-250 × 10 9 platelets/l) and a low white blood cell count of 0.4 × 10 9 cells/l (normal range, 4.0-8.0 × 10 9 cells/l). serum levels of alanine aminotransferase, aspartate aminotransferase, and creatine kinase were abnormally high, while the c-reactive protein level was normal. a coagulation study revealed a prolonged activated partial thromboplastin time and a high d-dimer level. the patient's serum ferritin level of >40 000 µg/ l was extremely elevated (normal range, 3-166 µg/l). urinary analysis showed proteinuria and microhematuria. the blood culture was sterile. computed tomography of the chest and abdomen showed right axillary lymphadenopathy and bilateral renal swelling; however, there was no evidence of hepatosplenomegaly. bone marrow aspiration revealed mildly hypocellular marrow with an increase in levels of activated histiocytes and hemophagocytes. the patient's condition deteriorated rapidly on the day following admission with the appearance of macrohematuria and a massive amount of tarry stool. death occurred on the third day of hospitalization. serum was used for virus isolation using vero and felis catus whole fetus (fcwf-4) cells. cytopathic effect appeared in both cells within 5 days. many dna fragments that were homologous to those of sftsv were detected in the culture supernatant of the cells inoculated in next-generation sequencing. dna amplified by conventional rt-pcr using either of 2 primer pairs showed the expected sizes of 458 or 461 bp in agarose-electrophoresis (data not shown). the inoculated vero cells were tested for the presence of sftsv antigen in indirect ifa with rabbit anti-sftsv rnp serum and showed a similar positive reactivity ( figure 1a ). enveloped and spherical virions with approximate diameters of 100 nm were detected by electron microscopy ( figure 1b) . the morphology of the virion is compatible with that of a bunyavirus. the virus isolated from the patient was named sftsv yg1. autopsy findings included right axillary lymphadenopathy (3.5 × 2.0 cm; figure 2a) , bilateral renal swelling, mild retention of pericardial fluid (140 ml), and hepatic steatosis. gastric ulceration was also observed in the pyloric region. severe necrotizing lymphadenitis with massive necrosis, the depletion of small lymphocytes, and severe infiltration of the swollen right axillary and right cervical lymph nodes by histiocytes and immunoblasts were observed ( figure 2b and 2c) . necrosis, which comprised nuclear debris and eosinophilic ghosts but not granulocytes, was distributed throughout the cortical area, the sinuses, and the capsule of the lymph node and had spread to the perinodal adipose tissue. no clusters of epithelioid histiocytes, stellate microabscesses, or granulomas were observed. there were no obvious intranuclear or intracytoplasmic viral inclusions. prominent hemophagocytosis was observed in these lymph nodes, the bone marrow, and the spleen ( figure 2d and 2e). the bone marrow was relatively hypocellular, with no reduction in the number of megakaryocytes. the liver showed mild microvesicular fatty changes in zone 3 and mild inflammation, comprising lymphocytes and macrophages, around the portal tracts. the kidney showed subepithelial hemorrhage within the renal pelvis ( figure 2f ). significant hemophagocytosis were also observed in the mediastinal, hilar, and abdominal lymph nodes with no evidence of necrosis ( figure 3a, 3c, and 3e) . the findings in the remaining visceral organs were unremarkable. positive signals for sftsv np antigen were detected in the cytoplasm of blastic cells and necrotic regions in the cortical area of the right axillary lymph node ( figure 3b ). viral antigen-positive cells were also detected in the right cervical lymph nodes ( figure 3d ) but not in the mediastinal lymph nodes ( figure 3f ) , regardless of the level of immunoblast infiltration and hemophagocytosis ( figure 3e ). relatively few sftsv antigen-positive cells were detected in the bone marrow, adrenal glands, the liver, and the spleen ( figure 3g -j), with no notable cytopathic effects or necrosis. no antigenpositive cells were detected in the heart, lungs, kidneys, gastrointestinal tract, aorta, or iliopsoas muscle. the sftsv genome, sftsv genomic rna (negative-strand rna; figure 4a ), and mrna ( positive-strand rna; figure 4b ) were detected in the blastic cells in the right cervical lymph node by ish-at analysis [18] [19] [20] , while no signals were detected using an irrelevant probe as a negative control ( figure 4c ). no signals showing necrotizing lymphadenitis without sftsv infection were detected in the lymph nodes (negative control; figure 4d ). sftsv rna was present, with high copy numbers, in the right axillary and cervical lymph node sections (supplementary table 3 ). consistent with immunohistochemical analysis results, low copy numbers (100-1000 copies) of sftsv rna were also detected in the bone marrow, the spleen, the liver, and the adrenal glands. a few copies (<100) were detected in other tissue sections that did not contain antigen-positive cells (as assessed by immunohistochemical analysis), suggesting that quantitative real-time rt-pcr detected cell-free circulating sftsv; a high viral load in the serum is characteristic of sfts infection [22] . the number of sftsv rna copies/cell was calculated using the β-actin mrna copy number, estimated at 1500 copies/cell [17] . the sftsv rna copy numbers in the right axillary and cervical lymph node sections were the highest among the tissues tested, while the bone marrow, spleen, and liver showed relatively lower copy numbers per cell (supplementary table 3 ). serum samples collected from 23 patients who were retrospectively suspected of having sfts were sent to the department of virology 1, niid ( figure 5 ). the male-to-female ratio was 18:5. the earliest infection dated back to 2005 ( figure 5b ). serum samples collected from 21 patients (for 2 patients, acute phase serum samples were not available for virologic analysis) were subjected to virus isolation and rt-pcr for amplification of the sftsv genome. serum samples collected from all 23 patients were tested for igg and immunoglobulin m (igm) antibodies to sftsv in the indirect ifa. of the 23 patients, 8 men and 2 women received a diagnosis of sfts: 7 had positive results of virus isolation and genome amplification tests; 1 had positive results of virus genome amplification testing and igm antibody to sftsv but had negative results of virus isolation analysis; and 2 were positive for igg antibody to sftsv. in the present study, the 2 patients who tested positive for sftsv on the basis of detection of igg to sfvsv were regarded as sfts positive because their symptoms were reminiscent of sfts and because reports of asymptomatic cases in china are quite rare [23, 24] . for the purposes of the present study, the first patient was included with the 10 retrospective cases, for a total of 11 diagnosed cases. all of the 11 patients in whom sfts was diagnosed were aged ≥50 years ( figure 5a ) and came from western japan ( figure 5d ). disease onset occurred in all patients between the months of april and december ( figure 5c ). six of the 11 cases were fatal. there was clear evidence of tick bite in 2 cases. the clinical manifestations observed in sfts patients are summarized in table 1 . all patients showed nonspecific febrile symptoms with gastrointestinal tract symptoms in the early phase of the disease. deterioration in consciousness, characterized by dysarthria, disorientation, and alteration in consciousness, was commonly observed. generalized convulsions were seen in the late stages of the disease in 5 of the 6 fatal cases. respiratory symptoms were rarely observed. superficial lymphadenopathy was detected in 5 patients. hemorrhagic symptoms such as petechiae, purpura, melena, bloody vomit, gingival bleeding as a form of discharge, and excessive bleeding at the site of skin biopsy were observed in 9 of the 11 patients. blood urea nitrogen and creatinine levels were elevated in 8 of 11 patients. hematuria and proteinuria were observed in most patients. in all 5 patients for whom bone marrow observation was performed, hemophagocytosis, with or without bone marrow cell dysplasia, was observed. the ferritin level was elevated in the blood of 8 patients, including the 5 in whom bone marrow examination was performed. abnormalities were observed in tests in all of the patients for coagulopathy, prothrombin time, activated partial thromboplastin time, fibrin/fibrinogen degradation products, fibrinogen, and/or d-dimer. the japanese sftsv strains were closely related to the sftsv chinese isolates but formed an independent cluster for each segment ( figure 6) . no geographic or chronological relationship was found between the japanese and chinese strains. a and b) , right cervical (c and d), and the mediastinal (e and f ) lymph nodes. a, c, and e, infiltration by immunoblasts and prominent hemophagocytosis was generally observed in the right axillary, right cervical, mediastinal, hilar, and abdominal lymph nodes; however, necrosis was only observed in the right axillary and cervical lymph nodes. b, d, and f, viral antigen-positive cells were detected in the right axillary and cervical lymph nodes. positive signals for sftsv np neutralizing antibody response to sftsv sera collected from the 5 surviving patients with sfts in the convalescent phase showed neutralizing activities to sftsv japanese isolate yg1 ( figure 1d ). the sera of these 5 patients also showed similar degrees of neutralizing activities to sftsv hb29. the clinical manifestations of japanese sfts were very similar to those of severe cases of chinese sfts [1, 2] , but the casefatality rate (6 of 11 patients [55%]) was apparently higher than that in china, where an average of 12% of cases were fatal. it is worth noting that the characteristics of japanese sfts presented in this study were based on a limited number of patients and a strict case definition. to our knowledge, this may be the first report describing the pathologic findings of a patient with sfts. the local lymph nodes, which were the primary target organ in the first patient, served as the site of virus replication and showed marked pathologic changes, including massive necrosis. no neutrophil infiltration was observed in the patient. the spleen, liver, adrenal glands, and bone marrow contained a few sftsv-infected cells. however, no viral antigens were detected in hepatocytes or in the parenchymal cells of the adrenal glands. viruses belonging to the bunyaviridae family, which includes the rift valley fever virus, hantaan virus, and heartland virus [10] , infect monocytes/macrophages [25, 26] . these cells may control the spread (or containment) of viruses to cells within other organs. the pathologic and virologic observations suggest that sftsv replicated in the blastic cells within the lymph nodes and that replication took place predominantly in the local lymph nodes, not in the major organs. macrophages with phagocytosis of bone marrow cells were observed in all of the patients in whom bone marrow figure 4 . detection of severe fever with thrombocytopenia syndrome virus (sftsv) rna in the right cervical lymph node by the in situ hybridization attailing method. a, sftsv genomic rna was detected in the right cervical lymph node by the in situ hybridization at-tailing method and a sense probe. sftsv genomic rna was detected in the cytoplasm of the blastic cells. b, the in situ hybridization at-tailing method with an anti-sense probe detected a few cells in the right cervical lymph node that were positive for sftsv messenger rna (mrna). sftsv mrna was also detected in the cytoplasm of blastic cells. c, no signals were detected in the right axillary lymph node by the in situ hybridization at-tailing method with an irrelevant probe (negative control). d, a sftsv sense probe detected no signals in lymph node sections showing necrotizing lymphadenitis without sftsv infection. examination was performed. furthermore, pathologic examination of the lymph nodes, bone marrow, and the spleen of the initial patient revealed marked hemophagocytosis, regardless of the presence of sftsv-infected cells ( figure 2d and 2e and figure 3a , 3c, and 3e). ferritin level was extremely elevated in the sera of all patients who were tested. abnormality in coagulopathy-associated indices was also observed in all patients. sftsv infections induced a cytokine storm, the level of which was associated with the severity of sfts [27] . these results indicate that in addition to multiorgan dysfunction, hemophagocytosis and disseminated intravascular coagulation are major factors for poor prognosis. the mode of the natural sftsv lifecycle in japan should be clarified to enable better identification of risk factors for sftsv infection and to address strategies for reducing the risk of infections. further study is necessary to clarify the circulation of sftsv in nature in japan, in terms of tick species, percentages of sftsv positivity for each tick species, and the prevalence of sftsv-positive ticks, to better determine and evaluate the risk factors for sftsv infection in japan. japanese isolates formed a cluster that was independent from chinese isolates (figure 6 ), indicating that sftsv has been circulating in japan naturally for some time. the earliest year for which we have evidence of sfts in patient sera samples was 2005, and to our knowledge, the article by liu et al, which concerns sfts occurrence in china during 2006, reports the oldest cases from china [5] . it is also noteworthy that patients with sfts were reported in south korea in 2013 in conclusion, sftsv is prevalent in japan. japanese sftsv strains have characteristics similar to those of chinese isolates but an independent genotype, which indicates that sftsv has been present in japan for some time. metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (ftls) in henan province, china: discovery of a new bunyavirus fever with thrombocytopenia associated with a novel bunyavirus in china the ecology, genetic diversity, and phylogeny of huaiyangshan virus in china isolation, identification and characterization of sfts bunyavirus from ticks collected on the surface of domestic animals person-to-person transmission of severe fever with thrombocytopenia syndrome virus 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for detection of immunoglobulin g antibodies to crimean-congo hemorrhagic fever virus recombinant nucleocapsid proteinbased igg enzyme-linked immunosorbent assay for the serological diagnosis of sars pathogenesis of emerging severe fever with thrombocytopenia syndrome virus in c57/bl6 mouse model the first autopsy case of pandemic influenza (a/h1n1pdm) virus infection in japan: detection of a high copy number of the virus in type ii alveolar epithelial cells by pathological and virological examination in situ hybridization at-tailing with catalyzed signal amplification for sensitive and specific in situ detection of human immunodeficiency virus-1 mrna in formalin-fixed and paraffin-embedded tissues sars coronavirus-infected cells in lung detected by new in situ hybridization technique application of the hybridization at-tailing method for detection of human immunodeficiency virus rna in cells and simian immunodeficiency virus rna in formalin-fixed and paraffin-embedded tissues vascular endothelial growth factor messenger rna expression level is preserved in liver metastases compared with corresponding primary colorectal cancer detection of a novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome in henan province, china, using real-time reverse transcription pcr severe fever with thrombocytopenia syndrome virus clinical and epidemiological study on severe fever with thrombocytopenia syndrome in yiyuan county, shandong province, china replication of hemorrhagic fever viruses in monocytic cells isolation of haemorrhagic fever with renal syndrome virus from leukocytes of rats and virus replication in cultures of rat and human macrophages host cytokine storm is associated with disease severity of severe fever with thrombocytopenia syndrome acknowledgments. we thank dr de-xin li and dr mi-fang liang, national institute for viral disease control and prevention, chinese center for disease control and prevention, beijing, prc, for providing us with the sftsv hb29 strain; dr roger hewson, virology, pathogenesis, and emerging disease, public health england-microbiology services, porton down, salisbury, united kingdom, for his critical comments; and the municipal officials who supported this work, in the prefectures in which patients with sfts were reported.financial support. this work was supported by the ministry of health, labor and welfare science research (grants in aid h24-shinko-ippan-013, h22-shinko-ippan-006, and h25-shinko-shitei-009).potential conflicts of interest. all authors: no reported conflicts. key: cord-007277-86lynlxn authors: kenneth, mcintosh title: coronaviruses in the limelight date: 2005-02-15 journal: j infect dis doi: 10.1086/428510 sha: doc_id: 7277 cord_uid: 86lynlxn nan for ∼35 years after their first description by tyrrell and byneo in 1965 [1] , the field of human coronaviruses (hcovs) was pretty dull. there were classic early descriptions of their respiratory pathogenicity in volunteer studies [2, 3] , and there were seroepidemiologic studies of the 2 most easily studied strains, hcov-229e and hcov-oc43 [4] [5] [6] . efforts to implicate hcovs in diseases of the gastrointestinal tract were largely unsuccessful, with the possible exception of a postulated role in necrotizing enterocolitis of newborns [7] . during this time, the fields of animal covs and of the molecular biology of covs were, in contrast, buzzing. covs were discovered in large numbers and were implicated in a rich variety of animal diseases in multiple species. diseases as widely varying as progressive peritonitis, nephritis, acute and chronic hepatitis, and subacute encephalitis were described, along with the more traditional respiratory and gastrointestinal syndromes, and pathogenesis was explained through broad mixtures of viral cytopathogenicity, immunologic damage, and genetic susceptibilities. the cov genome proved to be the largest of all of the rna viruses and to have a unique strategy of replication, with transcription and protein production occurring through a nested set of mrna molecules [8] . then, in 2003, the appearance of severe acute respiratory syndrome (sars) suddenly brought the field of hcovs back into the limelight. it seemed clear that this disease, unique in its clinical spectrum, resulted from the movement of an animal cov across species lines, and it seemed possible that the virus spread in the human population through a process of adaptation by deletion and mutation [9, 10] . the rapid recognition of the etiology of sars depended heavily on genomic sequence data assembled from the study of multiple animal covs, allowing the sars agent to be quickly identified and classified and leading to the development of detection methods that would guide the containment of the epidemic. it is in this context that the article and brief report by esper et al. that appear in this issue of the journal of infectious diseases should be read [11, 12] . in the first of these papers, esper et al. use the accumulated knowledge of the coronaviral genomic sequence to search for new hcovs in children with respiratory disease [11] . the authors' discovery of a previously undescribed hcov was accomplished through the design of a polymerase chain reaction assay that was based on the common region of the polymerase gene. this method was logical, intelligent, and highly original-and a new vi-rus, designated "new haven coronavirus" (hcov-nh), did appear. in fact, esper et al.'s finding was not surprising. the reason for this is not that a very similar hcov was being described by 2 independent groups of virologists in the netherlands [13, 14] at the same time (that virus was not known when esper et al. started their work), but rather that, in some of the earliest work on covs during the 1960s, viruses were reported that were then forgotten-viruses that came from adults with respiratory illness, that grew only in human embryonic tracheal organ culture, that caused illness in volunteers, and that were not, or were only distantly, antigenically related to the 2 hcov species that were subsequently the best studied, hcov-229e and hcov-oc43. one of these forgotten viruses, b814, was the first hcov to be described [1] . the others-hcov-oc16, hcov-oc37, and hcov-oc48-were 3 of the 6 strains recovered from organ culture in my laboratory [15] . all 4 of these strains produced colds in volunteers [2, 3] , but none grew in tissue culture, and none could be adapted to grow in animal models. thus, the subsequent neglect of these potentially important viruses stemmed from the fact that, essentially, no methods were available to study them at the time. esper et al.'s findings on the clinical impact of hcov-nh infection, although limited, are consistent both with those from europe on the novel hcov reported in the netherlands and with the available information on hcov-229e and hcov-oc43 infection in children hospitalized with acute respiratory disease [5, 13, 14] . however, the details from esper et al.'s study-the seasonal distribution, the percentage of positive samples, the associated respiratory syndromes, and the numbers of infected children at various ages, for example-were heavily influenced by both the particular population that was investigated and the clinical setting, so it is essentially impossible to draw conclusions on the epidemiology, pathogenicity, and relative importance of hcov-nh in relation to other respiratory viruses. on the other hand, it seems to be likely that this is not the last chapter in the story of respiratory hcovs and that additional studies will clarify the picture. moreover, it seems quite possible that other strains will be found, by use of similar methods. in contrast, the findings reported in esper et al.'s brief report were, to this reader, quite surprising [12] . esper et al. have shown a temporal association with quite strong statistical significance between infection with hcov-nh and kawasaki disease, establishing this association by means of a case-control study in hospitalized children. until there are corroborating studies, it would seem wise to retain a healthy level of skepticism with regard to the significance of this association. nevertheless, the findings are extremely interesting and set off in me a series of thoughts as to what might be going on. first, the skepticism. the linking of an agent to kawasaki disease follows a long trail of previously failed or still-struggling attempts to identify the etiologic agent of this important syndrome, ranging from an unidentified retrovirus [16] , to parvovirus b19 [17] , to epstein-barr virus [18] , to chlamydia pneumoniae [19] , and to toxinproducing staphylococcus aureus or streptococcus pyogenes [20] , with a scattering of others along the way. although the association shown by esper et al. was statistically compelling, previous initial find-ings were equally so (the first descriptions of both parvovirus b19 and the toxin-producing bacteria included similarly significant associations) and have been difficult to confirm. there are, however, some tantalizing facts about both covs and kawasaki disease that might allow for cautious optimism with regard to esper et al.'s reported association. first, there was early epidemiologic evidence [21] , subsequently confirmed [22] , that a respiratory syndrome preceded the onset of kawasaki disease. (incidentally, the interval between the onset of the respiratory syndrome and the onset of kawasaki disease appeared to be ∼2 weeks [21] , which seems a long time for the shedding of a respiratory hcov, although pertinent data on infants are lacking [23] [24] [25] .) second, kawasaki disease is frequently seasonal, with peaks during the winter and spring; its seasonality is roughly similar to that of infection with respiratory hcovs [5, 6] . third, and more recently, there has been evidence from molecular immunopathologic studies by rowley et al. indicating that, during kawasaki disease, some external agent triggers a powerful iga response in the respiratory tract as well as in other organs (including medium and large muscular arteries), suggesting that the target of the extensive immunologic reaction during kawasaki disease is a specific microbe (rather than an nonspecific stimulus, such as a superantigen) and that this microbe enters the body through the respiratory tract [26] [27] [28] . if this is, in fact, the case, then a respiratory hcov might be the inciting agent. fourth, the sars story reminds us of what veterinarian virologists have known for many years: that covs, with their huge genome, are capable of enormously varied pathogenicity, causing diseases that affect multiple organs through a variety of pathogenetic mechanisms. also, sars-cov crossed species lines and was genetically quite distant from the 3 known cov groups, whereas hcov-nh (along with its companion novel virus reported in the netherlands) appears to be a member of the group 1 covs and has other features that make it appear to be closer to hcov-229e and hcov-oc43 in its pathogenicity [13] . if hcov-nh is, in fact, the agent responsible for kawasaki disease and is acting alone, then we have to postulate that it has acquired a pathogenicity that is quite different from that of its close relatives and of other respiratory viruses. clearly, a lot more work needs to be done. because esper et al.'s study of kawasaki disease was epidemiologic, confirmation in broader epidemiologic terms (other places, other times, other detection methods, other populations) is required, as is nonepidemiologic confirmation through the demonstration of an immunologic response to hcov-nh and of its presence in biopsy specimens. if the association is confirmed, then the pathophysiologic mechanism will need to be further worked out. kawasaki disease has a complex pathogenesis and has been the subject of much study by microbiologists, immunologists, rheumatologists, cardiologists, and molecular biologists [29] . much is known about many of the mechanisms of kawasaki disease, and in some way these must be linked to the presumed microbial etiology. a broad question would be: is the pathogenesis of kawasaki disease the product of hcov-nh infection by itself? there are several animal cov diseases that are models of complex pathophysiologic mechanisms-the multiple sclerosis-like disease of mice caused by certain neurogenic strains of mouse hepatitis virus [30] and the complex, immunologically mediated, progressive feline peritonitis caused by the cov of that name [31] , for example. in these diseases, genetics, the immune system, and the complex cov genome all interact. alternatively, might there be another microbial pathogen involved in the pathogenesis of kawasaki disease, such that it is a 2-hit disease requiring both a virus and a toxin? we will watch this field with great interest. cultivation of a novel type of common-cold virus in organ cultures effects of a "new" human respiratory virus in volunteers coronative antibody titres in sera of healthy adults and experimentally infected volunteers virologic studies of acute respiratory disease in young adults. v. coronavirus 229e infections during six years of surveillance seroepidemiologic studies of coronavirus infection in adults and children the tecumseh study of respiratory illness. vi. frequency of and relationship between 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epstein-barr virus infection associated with kawasaki disease-like coronary artery aneurysms demonstration of chlamydia pneumoniae in cardiovascular tissues from children with kawasaki disease toxic shock syndrome toxin-secreting staphylococcus aureus in kawasaki syndrome kawasaki syndrome: description of two outbreaks in the united states investigation of kawasaki syndrome risk factors in colorado effect of specific humoral immunity and some non-specific factors on resistance of volunteers to respiratory coronavirus infection detection of human coronavirus 229e in nasal washings using rna:rna hybridisation the time course of the immune response to experimental coronavirus infection of man iga plasma cell infiltration of proximal respiratory tract, pancreas, kidney, and coronary artery in acute kawasaki disease oligoclonal iga response in the vascular wall in acute kawasaki disease detection of antigen in bronchial epithelium and macrophages in acute kawasaki disease by use of synthetic antibody kawasaki syndrome cutting edge: cd8 t cell-mediated demyelination is ifn-gamma dependent in mice infected with a neurotropic coronavirus natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease key: cord-290277-ndfoppoq authors: bahl, prateek; doolan, con; de silva, charitha; chughtai, abrar ahmad; bourouiba, lydia; macintyre, c raina title: airborne or droplet precautions for health workers treating covid-19? date: 2020-04-16 journal: j infect dis doi: 10.1093/infdis/jiaa189 sha: doc_id: 290277 cord_uid: ndfoppoq cases of covid-19 have been reported in over 200 countries. thousands of health workers have been infected and outbreaks have occurred in hospitals, aged care facilities and prisons. world health organization (who) has issued guidelines for contact and droplet precautions for healthcare workers (hcws) caring for suspected covid-19 patients, whilst the us centre for disease control (cdc) has recommended airborne precautions. the 1 – 2 m (≈3 – 6 ft) rule of spatial separation is central to droplet precautions and assumes large droplets do not travel further than 2 m (≈6 ft). we aimed to review the evidence for horizontal distance travelled by droplets and the guidelines issued by the world health organization (who), us center for diseases control (cdc) and european centre for disease prevention and control (ecdc) on respiratory protection for covid-19. we found that the evidence base for current guidelines is sparse, and the available data do not support the 1 – 2 m (≈3 – 6 ft) rule of spatial separation. of ten studies on horizontal droplet distance, eight showed droplets travel more than 2 m (≈6 ft), in some cases more than 8 meters (≈26 ft). several studies of sars-cov-2 support aerosol transmission and one study documented virus at a distance of 4 meters (≈13 ft) from the patient. moreover, evidence suggests infections cannot neatly be separated into the dichotomy of droplet versus airborne transmission routes. available studies also show that sars-cov-2 can be detected in the air, 3 hours after aeroslisation. the weight of combined evidence supports airborne precautions for the occupational health and safety of health workers treating patients with covid-19. m a n u s c r i p t coronaviruses are respiratory pathogens, and the sars-cov-2 has been identified in both upper and lower respiratory tract samples from patients [3] . fever, dry cough, malaise, lethargy, shortness of breath, myalgia are the commonest symptoms [2] . less common symptoms are headache, productive cough and diarrhoea. mild cases may present with a common cold like syndrome, whilst severe cases may develop severe acute respiratory distress syndrome and pneumonia. according to the who 21% of cases in china have a severe illness [2] . early estimates of the reproduction number, r0, give values around 2.2 with a mean incubation period of 5.2 days [4] , and a range up to 24 days. a review found the average r0 value for covid-19 to be up to 3.28 and median value to be around 2.79 [5] . a more recent study estimated the maximum-likelihood (ml) value of r0 to be 2.28 for the diamond princess cruise ship [6] . all these estimates are similar to r0 estimates for sars [7] . in the past epidemics of sars and mers coronavirus, health care workers (hcws) have paid a heavy toll. during sars, hcws comprised 21% of all cases and in some countries, such as hong kong, singapore and canada, more than half the cases were hcws, with deaths reported among them [8] . hcw deaths have already been reported with a c c e p t e d m a n u s c r i p t 5 the who has issued guidelines for protection of hcws which recommend contact and droplet precautions for hcws caring for suspected covid-19 patients [9]. specifically, a medical mask is recommended for routine care, while a respirator (airborne precautions) is recommended if hcws are conducting an aerosol-generating procedure such as endotracheal intubation, bronchoscopy or airway suctioning, along with droplet precautions [9] . droplet precautions includes the recommendation to maintain spatial separation of 1 m (≈3 ft) with an infected patient, in the belief that large droplets can only spread horizontally to a maximum of 1 m (≈3 ft) [10] . the initial guidelines released by us centers for disease control recommended a more precautionary approach, which includes the use of a mask by the patient (source control [11] ), and airborne precautions for hcws [12] . we aimed to review the evidence supporting the rule of 1 m (≈3 ft) spatial separation for droplet precautions in the context of guidelines issued by the world health organization (who), us center for diseases control (cdc) and european centre for disease prevention and control (ecdc) for hcws on respiratory protection for covid-19. a systematic review was conducted for evidence of horizontal distance travelled by respiratory droplets, using the prisma criteria [13] we found 393 papers in the initial search. after reviewing the titles and abstracts 28 papers were selected for full text review. finally, 10 papers were included in the review (figure 1 ). eight of the ten studies discussed a horizontal trajectory greater than 2 m (≈6 ft) for a range of droplet sizes of less than 60 µm [14] [15] [16] [17] [18] [19] [20] [21] . seven out of ten studies are based on modelling and a c c e p t e d m a n u s c r i p t 7 among them the extent of horizontal spread of droplets vary between 2 -8 m (≈6 -26 ft) [14] [15] [16] [17] [18] [19] [20] , highlighting differing findings between them, which can be partially attributed to the methodologies employed. specifically, four of these studies rely on computational fluid dynamics (cfd) approaches that do not account accurately for the multiphase particle-flow interaction physics [14, 15, 18, 20] and three of them model cough as a turbulent jet (continuous ejection with conservation of momentum flux) instead of a turbulent puff (short sudden ejection with conservation of momentum) [15, 18, 20] . the fourth study used lagrangian modelling for the droplet dispersion and it was acknowledged that this approach assigns a larger momentum to air hence, making it difficult to translate the results into relevant settings for hospital infection control [14] . two studies used analogous water tank experiments to validate the mathematical modelling developed and reported distances up to 1.4 m (≈4.5 ft) and 2.5 m (≈8.2 ft) [17, 22] . one of these two studies modelled coughs as turbulent jets (continuous emission) [22] despite contrary evidence showing that the physics of violent exhalations is captured by puffs, sudden high momentum emission of moist and hot air [17] . five studies performed experiments on human subjects [14, 17, 19, 21, 23] , four of them generated undisturbed/natural sneezes and coughs, without injestion of fluid or powders by the human subjects [17, 19, 21, 23] . out of five, two studies used the human subject measurements to develop and validate the mathematical modelling of the droplet dispersal and showed the importance of the exhaled gas cloud of hot and moist air in trapping and extending the range of all droplets [17, 19] . one involved injection of powder in the mouth of the human subject potentially shifting the natural droplet sizes ejected [14] . the other two used still photographs [23] and particle counters [21] and the distance reported among these two vary from 1 -3 m (≈3 -10 ft). table 1 the interim guidelines for covid-19 appear to assume only droplet and contact spread and the general risk limit defined for healthcare workers is 1 m (≈3 ft) from the patient [10, 31] . the transmission of covid-19 is not well characterised, but is likely to be similar to sars, which was spread by contact, droplet and airborne routes [32] . given the presence of sars-cov-2 viral loads in both the lower and upper respiratory tract [3] , as well as persistence of the virus in the air 3 hours after aeroslisation [33] , airborne transmission is possible. a recent study showed that seasonal coronaviruses were more commonly emitted in aerosols than in droplets, a c c e p t e d m a n u s c r i p t 9 even through normal tidal breathing [34] . it is timely to review the evidence informing the 1 -2 m (≈3 -6 ft) rule of infection control, which drives guidelines for droplet precautions. most studies of horizontal transmission of droplets show distances of greater than 2 m (≈6 ft). the maximum distance recorded in the few available studies is 8 m (≈26 feet) [19, 35] . we note, although the studies employed very different methodologies and should be interpreted cautiously, they still confirm that the spatial separation limit of 1 m (≈3 ft) prescribed for droplet precautions, and associated recommendations for staff at ports of entry [10] , are not based on current scientific evidence. the horizontal distance of droplet spread depends on various factors such as viscoelasticity of the expiration fluid, type of ventilation, velocity of expiration, rate of evaporation and the dynamics of turbulent cloud generated during exhalations, sneezing, or coughing [15, [17] [18] [19] . the 1 -2 m (≈3 -6 ft) limit is based on very limited epidemiologic and simulated studies of some selected infections [36] . some studies cite jennison (1942) [23] as the evidence in support of the 1 -2 m (≈3 -6 ft) risk limit. this study used high speed exposure to capture still photographs of the atomising secretions generated by human sneezing, coughing and talking, imaged very close to the mouth. it was concluded that the distance to which the majority of droplets were expelled is 2 -3 ft (≈1 m) but, no details were provided about how they reached this conclusion. the study acknowledges that the motion picture film used for the experiments was not sensitive enough to capture all the droplets. the lighting technique used inherently selects for the largest sizes of droplets and fluid ligaments, not capturing the rest of the emissions and gas cloud carrying them. the author used still photographs, in which many droplets move out of focus and become unrecordable very quickly, especially using photographic technology from the 1940s. more recent studies have shown the extent of droplet spread to be greater than 2 m (≈6 ft) [16] [17] [18] [19] [20] [21] 35] , and that infection risk exists well beyond the recommended range of spatial separation. a c c e p t e d m a n u s c r i p t 10 further, there is no agreement on the definition of "droplet" route of transmission. there is some agreement that particles with diameters less than 5 µm are airborne particles but, there is significant variation in the literature when it comes to the classification of the lower size limit of droplets. wells (1934) [37] considered 100 µm as the cut-off limit for the droplet route. but, later studies considered a cutoff particle diameter of more than 10 µm to more than 100 µm [14, 15, 20] . the world health organization (who) employs a cut off limit of 5 µm to differentiate between aerosols (≤5 µm) and droplet (>5 µm) [38] transmission routes. however, even particles with a diameter of more than 10 µm can remain airborne long enough to not fall under the framework of classification of "droplet" route [39] . in addition, the size of a droplet is dynamic and changes within seconds during the transit from the respiratory tract to the environment due to evaporation [39] . a large droplet expelled during coughing or sneezing can become an airborne particle in less than a second [39] and that timescale changes depending on the cloud dynamics of exhalation [17, 19] . hence, it is not possible to characterize droplet and airborne spread as separate, mutually exclusive modes of transmission and further studies of the risks accounting for combined ambient conditions and patient exhaled cloud are needed. indeed, another important consideration is the effect of temperature, relative humidity, ventilation etc. on the extent of droplet spread which has been examined by only a few studies. to summarise, they have shown that relative humidity plays an important role in the evaporation of the droplets and the distance a droplet can travel. they report that as the relative humidity increases the extent of droplet spread decreases [18, 20] , yet the horizontal range of the cloud propelling the drops was found to increase with increase in relative humidity, due to the role of buoyancy of the exhaled cloud [17] . for droplets less than 20 µm in diameter, local airflow field due to body heat is an important factor in determining the extent of spread since it can lift the droplets upwards into the breathing zone [40] . studies have also shown that depending on the flow direction and airflow pattern, increasing ventilation rate can effectively a c c e p t e d m a n u s c r i p t 11 reduce the risk of long range airborne transmission [41] . most patients spend the majority of time in normal breathing and can saturate the room air with airborne particles expelled during breathing. moreover, despite negative pressure isolation conditions, airflow due to door motion can cause breakdown in isolation conditions and as a result pathogen can escape the room and there is probability of infection spread outside the room [42] . in general recent studies show distances reached by potentially pathogen-laden droplets of a continuum of sizes to be far greater than 2 m (≈6 ft) [16] [17] [18] [19] [20] , therefore the probability of infection well beyond the defined risk limit can be significant. for example, sars was classified as predominantly transmitted through contact and droplet modes, but, aerosolised transmission well beyond 2 m (≈6 ft) was reported in the amoy gardens outbreak [32] . the ability of countries to respond effectively depends on the safety and confidence of the health workforce, especially in low income countries with low ratios of hcws per head of population and protective measures are crucial to ensure a functional health workforce. we have previously shown that masks do not have clinical efficacy against respiratory infections [43, 44] , and that intermittent use of respirators (which depends on hcws to assess their own risk and use the device when they judge they are at risk) is as equally ineffective as mask use [44] . a recent trial confirmed there is no difference between targeted respirator use and surgical mask use, but did not have a control arm and so may have shown equal efficacy or inefficacy [45] . proven efficacy of a respirator is seen when the device is worn continually during the shift [43] . the sars-cov-2 has been found in both upper and lower respiratory tract specimens, often early in the upper and later in the lower respiratory tract [3] , which means it can potentially be dispersed in fine, airborne particles. influenza studies show that in a busy emergency department or hospital ward, airborne particles with viable virus can persist for hours in the air [46] . a study of sars-cov-2 in a hospital in wuhan found virus at least 4 m (≈13 ft) within a hospital ward, and virus was identified in air samples and on multiple air outlet a c c e p t e d m a n u s c r i p t 12 vents [47] . other studies have also found sars-cov-2 on air vents in a patient room [48] . another study found virus in air samples three hours after aersolisation [33] . we have also shown that airborne precautions are more efficacious in protecting hcws even against infections assumed to be spread by the droplet route [49] . this further supports the conclusion that infections cannot be neatly separated into droplet versus airborne transmission routes, and that it is likely both airborne and large droplets, carried by the respiratory cloud, are emitted close to the patient and further away. in light of the lack of definitive transmission data for sars-cov-2 , as well as persistence of the virus in the air 3 hours after aeroslisation [33] , the precautionary principle in the initial cdc guidance was justified. this includes use of a mask by the patient, for which the limited evidence is supportive [11] . guidelines should be precautionary in ensuring protection of the occupational health and safety of health workers treating covid-19 [50] . although the majority of the studies reviewed point towards horizontal spread of more than 2 m (≈6 ft), these results cannot be translated directly to hospital settings, as the studies used varying range of assumptions. the recent data on sars-cov-2 in a hospital ward shows a distance travelled by the virus of at least 4 m (≈13 ft), double the assumed safe distance [47] . this review reveals the limited scientific data to inform spatial separation guidelines, and a growing body of evidence that droplet precautions are not appropriate for sars-cov-2. hence, future works on carefully documenting and studying the mechanisms shaping transmission distances are warranted, particularly with experiments over a large number of subjects and a variety of conditions, to update current spatial separation guidelines and the current paradigm of droplet and airborne respiratory transmission routes. m a n u s c r i p t 21 novel coronavirus (2019-ncov) situation reports virological assessment of hospitalized patients with covid-2019 early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the reproductive number of covid-19 is higher compared to sars coronavirus estimation of the reproductive number of novel coronavirus (covid-19) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis transmission dynamics and control of severe acute respiratory syndrome infection prevention and control during health care when novel coronavirus (ncov) infection is suspected world health organization. management of ill travellers at points of entryinternational airports, seaports and ground crossings -in the context of covid-19 outbreak: interim guidance world health organization cluster randomised controlled trial to examine medical mask use as source control for people with respiratory illness interim healthcare infection prevention and control recommendations for patients under investigation for 2019 novel coronavirus preferred reporting items for systematic reviews and meta-analyses: the prisma statement study on transport characteristics of saliva droplets calm indoor environment how far droplets can move in indoor environments -revisiting the wells evaporation-falling curve theoretical analysis of the motion and evaporation of exhaled respiratory droplets of mixed composition violent expiratory events: on coughing and sneezing enhanced spread of expiratory droplets by turbulence in a cough jet evaporation and dispersion of respiratory droplets from coughing. indoor air quantity, size distribution, and characteristics of coughgenerated aerosol produced by patients with an upper respiratory tract infection human cough as a two-stage jet and its role in particle transport atomizing of mouth and nose secretions into the air as revealed by high-speed photography. aerobiology. 17th ed. american assn. for the advancement of science interim domestic guidance on the use of respirators to prevent transmission of sars infection prevention and control during health care for probable or confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection: interim guidance: updated interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) rapid risk assessment: severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) cdc updates guidance on ppe for health care personnel; covid-19 declared a pandemic interim infection prevention and control recommendations for patients with suspected or confirmed coronavirus disease 2019 (covid-19) in healthcare settings infection prevention and control and preparedness for covid-19 in healthcare settings -second update evidence of airborne transmission of the severe acute respiratory syndrome virus aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 respiratory virus shedding in exhaled breath and efficacy of face masks turbulent gas clouds and respiratory pathogen emissions guideline for isolation precautions: preventing transmission of infectious agents in health care settings on air-borne infection. ii. droplets and droplet nuclei world health organization. infection prevention and control of epidemic-and pandemic-prone acute respiratory diseases in health care aerosol technology: properties, behavior, and measurement of airborne particles thermal effect of human body on cough droplets evaporation and dispersion in an enclosed space ventilation control for airborne transmission of human exhaled bioaerosols in buildings door-opening motion can potentially lead to a transient breakdown in negative-pressure isolation conditions: the importance of vorticity and buoyancy airflows a cluster randomized clinical trial comparing fit-tested and non-fit-tested n95 respirators to medical masks to prevent respiratory virus infection in health care workers a randomized clinical trial of three options for n95 respirators and medical masks in health workers n95 respirators vs medical masks for preventing influenza among health care personnel measurement of airborne influenza virus in a hospital emergency department aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient the efficacy of medical masks and respirators against respiratory infection in healthcare workers. influenza other respi viruses uncertainty, risk analysis and change for ebola personal protective equipment guidelines m a n u s c r i p t 23 wei and li key: cord-288821-nalulzfo authors: bastien, nathalie; anderson, kelly; hart, laura; caeseele, paul van; brandt, ken; milley, doug; hatchette, todd; weiss, elise c.; li, yan title: human coronavirus nl63 infection in canada date: 2005-02-15 journal: j infect dis doi: 10.1086/426869 sha: doc_id: 288821 cord_uid: nalulzfo the isolation of human coronavirus nl63 (hcov-nl63) in the netherlands raised questions about its contribution to respiratory illness. in this study, a total of 525 respiratory specimens, collected in canada primarily during the winter months of 2001–2002, were tested for hcov-nl63; 19 tested positive for hcov-nl63, demonstrating virus activity during january–march 2002. patients with hcov-nl63 were 1 month-100 years old (median age, 37 years). the main clinical presentations were fever (15/19), sore throat (5/19), and cough (9/19), and 4 patients were hospitalized. these results provide evidence for the worldwide distribution of hcov-nl63. hcov-oc43, are responsible for up to 30% of the commoncold syndrome [3] . however, several studies have found that these viruses are associated with more-severe respiratory infections [4] [5] [6] [7] . a novel coronavirus, hcov-sars, has been associated with severe atypical pneumonia and caused 774 deaths worldwide during november 2002-july 2003 [8] . coronaviruses are divided into 3 serotypes, on the basis of both their host range and their genome sequence. group 1 and 2 viruses are mammalian coronaviruses and include hcov-229 and hcov-oc43, respectively, whereas group 3 consists of avian coronaviruses. hcov-sars does not closely resemble viruses in any of the 3 known groups of coronaviruses. analyses of the complete genome sequence of hcov-nl63 have revealed that the virus is a new group 1 coronavirus that is closely related to hcov-229 [2] . the relative importance of hcov-nl63 in viral respiratory-tract illnesses is still not known. in the present study, we retrospectively looked for the presence of hcov-nl63 in canadian patients suffering from acute respiratory-tract infection (ari) during the winter months of 2001-2002, to assess the impact that hcov-nl63 infections have on ari and to describe the presenting signs and symptoms of the ari caused by this virus. materials and methods. primers used for amplification and sequencing were based on the published hcov-nl63 genome sequence [2] . two nested sets of primers, described by van der hoek et al. [2] , were used in this study. the primer set based on the 1b gene-primers repsz-1f-(15973) (5 -gtgatgcata-tgctaatttg-3), repsz-3r-(16210) (5 -ctcttgcaggtat-aatccta-3 ), repsz-2f-(16012) (5 -ttggtaaacaaaagat-aact-3 ), and repsz-4r-(16181) (5 -tcaatgctataaacag-tcat-3 )-were used for diagnosis; the primer set based on the 1a gene-primers ss5852-5pf-(5777) (5 -cttttgataacggt-cactatg-3 ), p4g1m-5-3pr-(6616) (5 -ctcattacataaaa-catcaaacgg-3 ), p3e2-5pf-(5788) (5 -ggtcactatgtag-tttatgatg-3 ), and ss6375-3pr-(6313) (5 -ggtcactatgt-agtttatgatg-3 )-were used for confirmatory purposes and sequence analysis. the primers used for amplification of the rnase p housekeeping gene were based on the published sequences (genbank accession number nm_006413) 5 -agatt-tggacctgcgagcg-3 (forward primer) and 5 -gagcgg-ctgtctccacaagt-3 (reverse primer). viral rna was extracted from 100 ml of either original samples or tissue-culture fluid, by use of the rneasy mini kit (qiagen). viral rna was amplified by use of a 1-step reverse transcription-polymerase chain reaction (rt-pcr) kit (qiagen), according to the manufacturer's recommendations. in brief, 5 ml of rna was added to the rt-pcr mixture, which the dna sequences were assembled and analyzed with the seqman, editseq, and megalign programs in the lasergene suite (dnastar). phylogenetic trees were generated by the neighbor-joining method using the mega program [9] . to monitor the efficiency of nucleic-acid extraction, each sample was also tested for the presence of the rnase p housekeeping gene by use of a 1-step rt-pcr kit under the following conditions: 30 min at 50њc, for reverse transcription; 15 min at 95њc, for the activation of the hotstart dna polymerase; 50 cycles of 15 s at 94њc, 30 s at 50њc, and 30 s at 72њc; and an extension for 7 min at 72њc. of the 525 specimens tested, all were positive for the rnase p gene. the hcov-nl63 sequences described in the present study have been deposited in genbank, under accession numbers ay675541-ay675553. results. the national microbiology laboratory received a total of 525 specimens collected from patients with ari, from provincial public health laboratories in manitoba (377 specimens), saskatchewan (104 specimens), and nova scotia (44 specimens). these representative specimens were selected to be negative for (1) swabs, 3 from throat washes, and 22 other, miscellaneous specimens from the respiratory system. of the 525 specimens tested, 19 (3.6%) were positive, by rt-pcr, for hcov-nl63, and they were from all 3 provinces: 13 (68%) from manitoba, 4 (21%) from saskatchewan, and 2 (11%) from nova scotia. hcov-nl63 activity was found during january (4 specimens [21%]), february (2 specimens [11%]), and march (13 specimens [68%]) 2002 and subsided during late spring; in contrast, no specimens positive for hcov-nl63 were retrieved for the same period during 2001. the sex distribution of these 19 specimens was 68% (13) male and 32% (6) female (table 1) . patients with hcov-nl63 were 1 month-100 years old (median age, 37 years). the proportion of positive specimens was significantly higher in the 0-5-year-old age group than in the 15-year-old age group (table 1) (8/110 vs. 11/402; ). p p .05 seven patients from the 150-year-old age group were involved in an outbreak of ari in a personal-care home in manitoba (table 1) . hcov-nl63 was found in 7 of the 8 patients from this outbreak whose samples were analyzed in this study. (table 1) . other observed symptoms included bronchiolitis (2 patients), rhinitis (1 patient), and lymphadenitis (1 patient), and 2 patients died-1 because of unrelated causes (this patient, a male infant, was smothered) (table 1). of the 4 patients (21%) with hcov-nl63 who were hospitalized, 3 (75%) were in the 0-5-year-old age group, and 1 was in the 150-year-old age group (table 1) . for 13 of the 19 specimens positive for hcov-nl63, nucleotide sequences were determined for nucleotides 5856-6280 of the 1a gene. comparison of these sequences with those published for dutch isolates of hcov-nl63 showed that the 1a genes were relatively well conserved, with 98.1%-100% nucleicacid identity between specimens. the phylogenetic tree of the hcov-nl63 isolates showed the existence of 2 major groups or clusters that contained both canadian and dutch strains of hcov-nl63 (figure 1). similar canadian hcov-nl63 strains were isolated from adults and children in all 3 provinces and during different outbreaks. strains isolated from the outbreak of ari in a personal-care home in manitoba were grouped together into cluster 2. discussion. although, in a study like this one, the inclusion of a control group of healthy individuals is necessary to clearly demonstrate a causal relationship, the detection of hcov-nl63 in respiratory-tract specimens from patients suffering from ari of unknown causes strongly suggests that it is associated with respiratory illness. this finding supports the association, found by van der hoek et al. [2] , of hcov-nl63 with ari. it also demonstrates for the first time that hcov-nl63 was present in several canadian provinces during the 2002 winter season, suggesting that hcov-nl63 may be circulating worldwide. we detected the presence of hcov-nl63 in 19 (3.6%) of the 525 analyzed specimens that were negative for (1) influenza viruses a and b; (2) piv 1, 2, and 3; (3) adenovirus; (4) rsv; and (5) hmpv; and these results provide further evidence of the contribution of hcov-nl63 to ari-and of the significant burden that it therefore may present to health-care systems. for the time period that was analyzed in the present study, peak activity of hcov-nl63 was found to occur during january-march 2002. although sampling occurred only during the winter season (october-april), our results are consistent with van der hoek et al.'s [2] finding that hcov-nl63 appears to be transmitted predominantly during the winter season. other hcovs, as well as influenza and rsv, which are involved in a substantial number of hospitalizations for ari, were also shown to be present in patients with ari during that time of the year [7] . the clinical symptoms associated with hcov-nl63 infection are also comparable to those observed to be associated with influenza, rsv, and other hcovs, making it impossible, at least on the basis of seasonality and clinical manifestations, to differentiate between these viral infections. previous estimates of the contributions of rsv and other etiological agents to ari based on these parameters may have been biased because the involvement of other new respiratory viruses such as hmpv and hcov-nl63 were overlooked [10] . thus, the systematic detection of hcov-nl63 in respiratory specimens may improve the understanding of the etiology of ari; however, the possibility of dual infection cannot be excluded, because the present study utilized samples from patients with ari who were tested-and found to be negative-for only (1) influenza viruses a and b; (2) piv 1, 2, and 3; (3) adenovirus; (4) rsv; and (5) hmpv. nor can it be ruled out that hcov-nl63 can exist asymptomatically in an individual, because samples from healthy individuals were not included. in addition, the present study provides evidence that hcov-nl63 may have been associated with an outbreak of ari in a personal-care home. all hospitalizations occurred in the 0-5-year-old and 150-year-old age groups, which suggests that, like the other hcovs and rsv, hcov-nl63 may cause more-severe ari in frail patients, such as infants and the elderly, than in other patients [4, 6, [11] [12] [13] . phylogenetic analysis based on the 1a gene confirms previously published results and shows the presence of viruses with different molecular markers [2] . the increased number of isolates detected provides further evidence of genetic diversity and the presence of 2 genetic clusters. close clustering of hcov-nl63 isolates recovered from different canadian provinces and from the netherlands suggests that the evolutionary pattern of hcov-nl63 does not presently correlate with geographic location. in summary, our data suggest that hcov-nl63 may play a significant role in ari, especially in young children and the elderly. more-comprehensive studies, which would include data on prevalence, risk factors, and use of health services, are necessary to determine both the importance of hcov-nl63 in ari and its impact on the health-care system. editorial board of respiratory disease in canada. respiratory disease in canada identification of a new human coronavirus fields virology spectrum of clinical illness in hospitalized patients with "common cold" virus infections rhinovirus and coronavirus infection-associated hospitalizations among older adults impact of respiratory virus infections on persons with chronic underlying conditions an outbreak of coronavirus oc43 respiratory infection in normandy, france world health organization, department of communicable disease surveillance and response. summary of probable sars cases with onset of illness from 1 mega2: molecular evolutionary genetics analysis software contribution of rsv to bronchiolitis and pneumonia-associated hospitalizations in english children respiratory syncytial virus coronavirus infection in acute lower respiratory tract disease of infants coronavirus 229e-related pneumonia in immunocompromised patients key: cord-001764-njzyu4mv authors: hofmann-winkler, heike; gnirß, kerstin; wrensch, florian; pöhlmann, stefan title: comparative analysis of host cell entry of ebola virus from sierra leone, 2014, and zaire, 1976 date: 2015-10-01 journal: j infect dis doi: 10.1093/infdis/jiv101 sha: doc_id: 1764 cord_uid: njzyu4mv the ongoing ebola virus (ebov) disease (evd) epidemic in western africa is the largest evd outbreak recorded to date and requires the rapid development and deployment of antiviral measures. the viral glycoprotein (gp) facilitates host cell entry and, jointly with cellular interaction partners, constitutes a potential target for antiviral intervention. however, it is unknown whether the gps of the currently and previously circulating ebovs use the same mechanisms for cellular entry and are thus susceptible to inhibition by the same antivirals and cellular defenses. here, we show that the gps of the ebovs circulating in 1976 and 2014 transduce the same spectrum of target cells, use the same cellular factors for host cell entry, and are comparably susceptible to blockade by antiviral interferon-induced transmembrane proteins and neutralizing antibody kz52. thus, the viruses responsible for the ongoing evd epidemic should be fully susceptible to established antiviral strategies targeting gp and cellular entry factors. ebolaviruses, from the family filoviridae, cause a severe and frequently fatal disease in humans [1] . the first documented outbreak of ebola virus (ebov, formerly zaire ebolavirus) disease (evd) occurred in zaire and was associated with a case-fatality rate of 88% [2] . subsequently, 22 evd outbreaks were recorded in sub-saharan africa [2] . these outbreaks generally occurred in relatively remote areas, were contained by quarantine measures, and caused a maximum of 425 cases per outbreak (during the 2000-2001 evd outbreak in uganda). the ongoing evd epidemic, which originated in guinea [3] , differs markedly from previous outbreaks: it affects western africa and is associated with a higher death toll than all previously recorded outbreaks jointly [4] . the massive scale of the epidemic is likely due to the delayed public health response. however, the currently circulating viruses harbor >30 unique amino acid substitutions [5] , and it is currently unclear whether these mutations allow for more-efficient spread in and between humans, compared with viruses responsible for past outbreaks. the ebov glycoprotein (gp) is inserted into the viral envelope and facilitates viral entry into target cells [6, 7] . moreover, gp is the sole target for the neutralizing antibody response. gp is synthesized in the constitutive secretory pathway of infected cells and consists of a surface unit, gp1, and a transmembrane unit, gp2 [6, 7] . the gp1 subunit contains an n-terminal receptor binding domain (rbd) [8, 9] , which binds to host cell receptors, and a c-terminal mucin-like domain (mld), while the gp2 subunit contains functional elements required for fusion of the viral envelope with a host cell membrane. cellular entry of ebovs commences with uptake of virions into host cell endosomes. this process can be promoted by recognition of phosphatidylserine in the viral envelope by tim proteins [10] and, via bridging molecules, tam receptor tyrosine kinases (tyro3, axl, and mer) [11] on the host cell surface or by binding of gp to host cell lectins [12, 13] . within the endosome, cysteine proteases remove the mld [14] , which allows the subsequent interaction of gp with the cholesterol transporter npc-1 [15, 16] , and drug-induced accumulation of endosomal cholesterol blocks infectious entry [17, 18] . finally, an incompletely characterized stimulus triggers the membrane fusion reaction [19] , which allows the release of the ribonucleoprotein complex into the host cell cytoplasm. all viral and cellular factors involved in the entry of ebovs into host cells are potential targets for antiviral intervention. however, the gp of the ebov, which is currently circulating in west africa harbors 8 unique substitutions in gp [5] , and it is unknown whether these changes alter the entry cascade and/or render the virus resistant to inhibitors or neutralizing antibodies. the present study addressed this question by comparing host cell entry mediated by gps of the ebovs circulating in 1976 and 2014. the adherent human and simian cell lines cells were cultivated in dulbecco's modified eagle's medium (paa laboratories) and the suspension cells in roswell park memorial institute 1640 medium (paa laboratories), supplemented with 10% fetal bovine serum (biochrom) and antibiotics. the following cell lines were used: 293t (human embryonic kidney), huh7 (human hepatoma), u373 (human glioblastoma), hos (human osteosarcoma), hela (human cervix carcinoma), rpe (human retinal pigment epithelial), ea-hy (human endothelial hybrid), raji (human b cell), jurkat (human t cell), cemx174 (human t-cell/b-cell hybrid), smagi (rhesus macaque mamma carcinoma), llc-mk2 (rhesus macaque kidney), vero (african green monkey kidney), and cos (african green monkey kidney). differentiation of human thp-1 monocytes into thp-1 macrophages was achieved by incubation with phorbol-12-myristate-13-acetate (pma; 10 ng/ml) for 48 hours. bat cell lines were propagated as described elsewhere [20] . all cells were grown in a humidified atmosphere at 37°c and 5% co 2 . the nucleotide sequence encoding the gp of the currently circulating ebov (makona variant; genbank accession number km233105; ebola virus/h.sapiens-wt/sle/2014/makona-g3838) was synthesized (geneart, life technologies) and inserted into pcdna3.1zeo via hindiii and xbai sites. expression plasmids for the gps of ebov strain mayinga from 1976 (ebov-gp 1976), lassa fever virus, influenza a virus (strain a/wsn/33), and vesicular stomatitis virus (vsv), as well as 3-component human immunodeficiency virus type 1 (hiv-1)and mlv-based vector systems for analysis of gp-driven transduction have been described elsewhere [18, 18, [21] [22] [23] . plasmids encoding the lectins dc-sign, dc-signr, lsectin, asgpr-1, clec5a, and folate receptor α were also described previously [21, [24] [25] [26] . monoclonal antibodies kz52 and 3b11 directed against ebov-gp [27, 28] were kindly provided by prof s. becker, marburg. monoclonal antibody 183-h12-5c recognizing hiv-1 gag was obtained from the national institutes of health (nih) aids reagent program. the generation of retroviral vectors pseudotyped with heterologous viral gps ( pseudotypes) was carried out as described before [18] . in brief, 293t cells underwent calcium phosphate transfection with an expression plasmid encoding the gp of choice in combination with plasmids encoding hiv-1 gag-pol and plasmid pcswf-luc [29] or plasmids encoding mlv gagpol and packaging plasmid mlv-luc [23] . the culture supernatants were harvested 48 hours after transfection, passed through filters ( pore size, 0.45 µm), aliquoted, and stored at −80°c. for production of rhabdoviral particles, a protocol described elsewhere [18] was followed. vlps were produced by expression of hiv-1 gag in combination with the gp of interest in 293t cells, followed by centrifugation of the vlp-containing supernatant through a 20% sucrose cushion [30] . the presence of gag and gp in vlp preparations was analyzed by western blot, using antibodies against hiv-p24 (nih) and monoclonal antibody 3b11 directed against ebov-gp [31] . for all transduction experiments, target cells were seeded in 96-well plates at a density of 3 × 10 4 cells/well. cells were then incubated for 8 hours with 50 µl of medium containing pseudoparticles bearing the gp of interest. transduction efficiency was determined by quantification of luciferase activities in cell lysates at 30 hours (rhabdoviral pseudotypes) or 72 hours after transduction (retroviral pseudotypes), using commercially available kits (promega, pjk). in some experiments, target cells were transduced to express interferon-induced transmembrane (ifitm) proteins or were transfected to express cellular lectins, as described previously [18] , before transduction with gp-bearing pseudotypes. alternatively, expression of ebov entry factors was inhibited by small interfering rnas (sirnas). for this, sirna knock down in target cells was performed 24 hours prior to transduction by transfection of 5 pmol of sirna (all santa cruz), using lipofectamine 2000 (life technologies). to determine whether cationic amphiphiles (u18666a, merck; clomiphene or terconazole, sigma-aldrich) or protease inhibitors (catl inhibitor iii, merck; ca074me, calbiochem; ca074; sigma-aldrich; aebsf, roth) influence transduction efficiency, the inhibitors were diluted in appropriate solvent as recommended by the manufacturer. target cells were preincubated with inhibitor for 60 minutes at 37°c before addition of pseudotypes, and the culture medium was replaced by fresh culture medium without inhibitor after 8 hours. to assess virion stability, pseudotypes bearing ebov-gp or the gp of vsv (vsv-g) were normalized for comparable infectivity. subsequently, the pseudotypes were incubated at defined temperatures for increasing periods, frozen at −80°c at a given time point, and used for transduction of 293t target cells. seventy-two hours after transduction, transduction efficiency was quantified by a luciferase assay. pseudotypes carrying the gps of the respective ebov isolate or vsv-g as a control were normalized for comparable infectivity and incubated with monoclonal antibody kz52 at indicated dilutions for 1 hour at 37°c. thereafter, the antibody/pseudotype mixtures were added to 293t cells, the cells were incubated for 72 hours, and luciferase activities in cell lysates were determined. ebolaviruses infect a broad spectrum of cell types in cell culture [32] , but macrophages and dendritic cells constitute early and sustained targets in the infected host [33] . in contrast, lymphocytes are refractory to infection, both in vitro and in vivo [32, 33] . to analyze whether previously and currently circulating ebovs show differences in host cell tropism and entry, we comparatively analyzed pseudotypes bearing the gps of ebov, strain mayinga, 1976 (ebov-gp 1976) and ebov variant makona (ebov-gp 2014) [34] , which differ in 1 amino acid residue in the rbd and 16 amino acid residues in the mld (supplementary figure 1) . pseudotypes bearing vsv-g served as positive control, while pseudotypes bearing no gp were used as negative control. both ebov gps were efficiently expressed in transfected cells and incorporated into retroviral particles ( supplementary figure 2) , and both mediated entry into the human cell lines 293t, huh7, u373, rpe, hos, and ea-hy with comparable efficiencies ( figure 1a) . similarly, transduction of macrophages obtained upon pma treatment of thp-1 cells was comparable ( figure 1a ). in contrast, both gps were unable to facilitate transduction of a human b-cell line (raji), a human t-cell line (jurkat and a human t/b hybrid cell line (cemx174; figure 1b ). ebov is highly pathogenic for nonhuman primates (nhps), which are used as animal models for evd in humans [35] . therefore, we also analyzed whether ebov-gp 1976 and ebov-gp 2014 transduce nhp-derived cell lines. transduction of the llc-mk2 and smagi cell lines was comparable to transduction of the vero cell line ( figure 1c ). in contrast, ebov-gp 1976 was more efficient than ebov-gp 2014 at transducing cos7 cells. finally, the ability of ebov-gp 1976 and ebov-gp 2014 to transduce bat cell lines (derived from rousettus aegyptiacus and hypsignathus monstrosus) was examined, since h. monstrosus, epomops franqueti, and myonycteris torquata can serve as natural reservoir for ebovs [36] . however, no appreciable differences were observed ( figure 1d ). in sum, these results suggest that ebov variants circulating in 1976 and 2014 exhibit a comparable cell tropism. lectins, tim-1, and tam kinase axl can augment entry of ebovs into certain target cells [6] . in contrast, npc-1 is universally required for entry (dahlmann et al, submitted) . we addressed whether ebov-gp 1976 and ebov-gp 2014 show differential dependence on these factors for host cell entry. the lectins dc-sign, dc-signr, asgpr1, and lsectin are known to augment gp-driven entry [12, 13, 37] and promoted transduction driven by ebov-gp 1974 and ebov-gp 2014 to similar extents while expression of clec5a, which binds dengue virus [38] , had no effect (figure 2a) . moreover, both ebov-gp 1976 and ebov-gp 2014 used tim-1 and axl for entry into certain target cells ( figure 2b ), while expression of npc-1 was universally required for entry ( figure 2b and 2c) . finally, entry driven by both gps was comparably inhibited by u18666a, which induces an npc-1 knockout phenotype in cells [39] , and related compounds ( figure 2c ), in the absence of unwanted cytotoxic effects (not shown). thus, our studies revealed no obvious difference in entry factor use by ebov-gp 1976 and ebov-gp 2014. the enzymatic activity of the ph-dependent endosomal/lysosomal cysteine proteases cathepsin b (catb) and cathepsin l (catl) is required for cellular entry of pseudotypes bearing ebov-gp 1976 and authentic ebov 1976 [14] . we used a panel of inhibitors previously employed to study ebov-gp protease use [40] , to determine whether ebov-gp 1976 and ebov-gp 2014 exhibit differences in their protease requirements. entry driven by both proteins was comparably inhibited by the cysteine protease inhibitors ca074me and ca074, which efficiently inhibit catb but have little or no effect on catl and cats activity [40] . in contrast, the serine protease inhibitor aebsf had no effect on gp-driven entry, and none of the compounds tested modulated entry driven by vsv-g ( figure 3a ), as expected [40] . these observations indicate that ebov-gp 1976 and ebov-gp 2014 both depend on catb activity for efficient transduction of 293t cells. however, ebov-gp 2014 was less susceptible than ebov-gp 1976 to blockade by a third inhibitor, catl ( figure 3b ), which inhibits catb and catl activity to similar extents [40] , suggesting subtle differences in the protease dependence of these gps. hiv-derived particles rapidly lose infectivity when stored at room temperature [41] , and this loss might be due to inactivation of the viral envelope protein. therefore, we asked whether retroviral particles bearing ebov-gp 1976 and ebov-gp 2014 exhibit different stabilities. incubation of viral particles bearing vsv-g, ebov-gp 1976, and ebov-gp 2014 for up to 8 hours at 4°c, room temperature, and 37°c modestly and comparably reduced particle infectivity, and this loss occurred slightly more rapidly at room temperature and 37°c as compared to 4°c (figure 4) . a more profound loss was observed upon incubation at 42°c, but again no differences were observed between ebov-gp 1976 and ebov-gp 2014 (figure 4) , suggesting that the stability of these gps is comparable. comparably inhibited by ifitm proteins and neutralizing antibody kz52 ifitm proteins 1, 2, and 3 inhibit cellular entry of ebov [42] and several other viral pathogens [42, 43] and might reduce ebov amplification in the infected host, raising the question of whether viruses circulating in 2014 are less susceptible to ifitm protein inhibition than those responsible for the 1976 outbreak in zaire. engineered expression of ifitm proteins in 293t cells had no appreciable impact on transduction mediated by lassa fever virus gpc but reduced influenza virus hadriven transduction, with inhibition by ifitm protein 3 being most efficient ( figure 5 ), in keeping with published data [42] . transduction by the 2 ebov-gps tested was also inhibited by ifitm protein expression, and no appreciable differences in inhibition efficiency were observed ( figure 5) . thus, the virus host cell entry of ebovs can be inhibited by gp-specific antibodies, and some neutralizing antibodies were shown to exert antiviral activity in the host. we assessed inhibition of ebov-gp 1976-driven and ebov-gp 2014-driven entry by antibody kz52 (obtained from an evd survivor), which targets gp2 [44] and displays antiviral activity in guinea pigs but not monkeys [27] . entry driven by both gps was comparably inhibited by preincubation of particles with kz52, while this antibody had no effect on entry driven by vsv-g (figure 6) , indicating that the epitope recognized by kz52 is conserved between ebov 1976 and ebov 2014. the ongoing evd epidemic in western africa is of unprecedented proportions and calls for the large-scale assessment of existing options for antiviral intervention and the development of new antiviral strategies. however, amino acid substitutions unique to the gp of the currently circulating ebov [5] might render the viruses nonsusceptible to antivirals and cellular defenses targeting gp or cellular factors promoting gp-driven entry. the present study indicates that this scenario does not apply: no major differences in host cell entry driven by ebov-gp 1976 and ebov-gp 2014 were observed, and both gps were comparably susceptible to blockade by inhibitors and antiviral host cell proteins. ebov-gp 2014 contains a unique amino acid substitution in the rbd [5, 8] , which may alter the requirement for host cell receptors and could modulate viral cell tropism. our results suggest that this is not the case: entry mediated by ebov-gp 1976 and ebov-gp 2014 was dependent on expression of the cholesterol transporter npc-1 and was inhibited by compounds that induce accumulation of endosomal cholesterol. moreover, expression of endogenous tim-1 and axl, which can augment gp-driven entry by binding directly (tim-1) or via a bridging molecule (axl) to ptdser in the viral envelope [45, 46] , promoted entry driven by both ebov-gp 1976 and ebov-gp 2014. the mld of gp is heavily modified with olinked glycans and is required for binding to certain host cell lectins [47] . the observation that the mld of ebov-gp 2014 contains 16 amino acid substitutions relative to the mld of ebov-gp 1976 suggested that engagement of cellular lectins might differ between these gps. however, again no appreciable differences were observed. in keeping with the comparable entry factor use, both gps facilitated entry into an identical spectrum of cell lines derived from bats, nhps, and humans, including thp-1 cell-derived macrophages. the only consistent difference observed was a reduction of ebov-gp 2014-mediated transduction of cos7 cells, relative to that mediated by ebov-gp 1976. this effect might point toward subtle differences in entry factor use, which may not be detectable in the assay system used in the present study. the removal of the mld by endosomal cysteine proteases, catb and l, is required for subsequent binding of gp to npc-1 [14, 19] . several studies indicate that vectors bearing ebov-gp, as well as authentic ebov, depend on the activity of catb/l for infectious entry into cell lines [14, 48, 49] , although catb/l dependence seems to vary between ebolavirus species [48, 50] , and the activity of these particular proteases is dispensable for ebov spread in a murine model [48] . the present study suggests that catb/l dependence extends to viruses currently circulating in west africa. however, reduced susceptibility of ebov-gp 2014 to the inhibitor catl, relative to that of ebov-gp 1976, points toward minor differences in the protease requirements of the respective gps. the gps of ebovs are cleaved by proprotein convertases in the golgi apparatus of infected cells, and evidence for cleavage of ebov-gp 2014 was obtained (data not shown). the cleavage products, the surface unit gp1 and the transmembrane unit gp2, remain covalently associated via a disulphide bond. one could speculate that the stability of this association might be different between ebov-gp 1976 and ebov-gp 2014, which could translate into different stability of retroviral particles bearing these gps. however, a comparable time-and temperaturedependent loss of infectivity of ebov-gp 1976-bearing and ebov-gp 2014-bearing particles was observed, arguing against substantial differences in gp stability, although it cannot be disregarded that gp stability might differ in the context of retroviral and filoviral particles. interferon-induced antiviral host factors and neutralizing antibodies can contribute to viral control in the infected host. the present study suggests that ebov variants from 1976 and 2014 are susceptible to both defense mechanisms. expression of ifitm proteins comparably inhibited host cell entry driven by ebov-gp 1976 and ebov-gp 2014, potentially by increasing endosomal cholesterol levels [17, 18] . moreover, entry driven by both gps was blocked by neutralizing antibody kz52, which was obtained from a human evd survivor and targets gp2 [44] . collectively, these results and the observations discussed above suggest that previously and presently circulating ebov variants might use similar mechanisms for host cell entry, infect a similar spectrum of target cells, and might be comparably susceptible to inhibition by innate and adaptive immune responses. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. ebola haemorrhagic fever world health organization (who) emergence of zaire ebola virus disease in guinea ebola virus disease in west africathe first 9 months of the epidemic and forward projections genomic surveillance elucidates ebola virus origin and transmission during the 2014 outbreak host cell factors in filovirus entry: novel players, new insights filovirus entry: a novelty in the viral fusion world conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor comprehensive analysis of ebola virus gp1 in viral entry t-cell immunoglobulin and mucin domain 1 (tim-1) is a receptor for zaire ebolavirus and lake victoria marburgvirus tyro3 family-mediated cell entry of ebola and marburg viruses c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells endosomal proteolysis of the ebola virus glycoprotein is necessary for infection ebola virus entry requires the cholesterol transporter niemann-pick c1 small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterolindependent mechanisms ebola virus entry requires the host-programmed recognition of an intracellular receptor comparative analysis of ebola virus glycoprotein interactions with human and bat cells dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus proteolytic activation of the 1918 influenza virus hemagglutinin infectious hepatitis c virus pseudoparticles containing functional e1-e2 envelope protein complexes lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection structural flexibility of the macrophage dengue virus receptor clec5a: implications for ligand binding and signaling preand postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody complex of a protective antibody with its ebola virus gp peptide epitope: unusual features of a v lambda x light chain the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies the ebola virus glycoprotein and hiv-1 vpu employ different strategies to counteract the antiviral factor tetherin the signal peptide of the ebolavirus glycoprotein influences interaction with the cellular lectins dc-sign and dc-signr characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection nomenclature-and databasecompatible names for the two ebola virus variants that emerged in guinea and the democratic republic of the congo in 2014 animal models for ebola and marburg virus infections fruit bats as reservoirs of ebola virus differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr clec5a is critical for dengue-virusinduced lethal disease cholesterol synthesis inhibitor u18666a and the role of sterol metabolism and trafficking in numerous pathophysiological processes cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss2 expression survival of hiv-1 activity after disinfection, temperature and ph changes, or drying distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus structure of the ebola virus glycoprotein bound to an antibody from a human survivor tim-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine the role of phosphatidylserine receptors in enveloped virus infection human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry cathepsin b & l are not required for ebola virus replication role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences acknowledgment. we thank dr bruce chesebro (aids reagent program, division of aids, national institute of allergy and infectious diseases, national institutes of health) for the kind gift of hiv-1 p24 hybridoma (183-h12-5c).financial support. this work was supported by the german research foundation (grant po 716/8-1 to s. p.), the german ministry for research and education (subproject within ebocon; to s. p.), the leibniz graduate school for emerging infectious diseases (to s. p.), and the leibniz foundation.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-002926-7ereip3x authors: yoon, sun-woo; wong, sook-san; zhu, huachen; chen, rirong; li, long; zhang, yu; guan, yi; webby, richard j title: dysregulated t-helper type 1 (th1):th2 cytokine profile and poor immune response in pregnant ferrets infected with 2009 pandemic influenza a(h1n1) virus date: 2018-02-01 journal: j infect dis doi: 10.1093/infdis/jix328 sha: doc_id: 2926 cord_uid: 7ereip3x pregnancy has been associated with severe influenza, an association highlighted during the 2009 pandemic of influenza a(h1n1) virus (a[h1n1]pdm09) infection. to assess the underlying mechanism, we infected pregnant and non-pregnant ferrets with a(h1n1) pdm09 virus. a(h1n1)pdm09-infected pregnant ferrets also had higher levels of inflammatory cytokines in their pulmonary tracts. systemically, total cd8(+) t cell counts and a(h1n1)pdm09-specific b-cell responses in blood were significantly lower in pregnant ferrets. this model predicts that the poorer outcome for pregnant women during the a(h1n1)pdm09 pandemic was due to an elevated level of viral replication and to a cytokine imbalance that led to a less effective immune response. pregnancy has been associated with severe influenza, an association highlighted during the 2009 pandemic of influenza a(h1n1) virus (a[h1n1]pdm09) infection. to assess the underlying mechanism, we infected pregnant and non-pregnant ferrets with a(h1n1) pdm09 virus. a(h1n1)pdm09-infected pregnant ferrets also had higher levels of inflammatory cytokines in their pulmonary tracts. systemically, total cd8 + t cell counts and a(h1n1)pdm09-specific b-cell responses in blood were significantly lower in pregnant ferrets. this model predicts that the poorer outcome for pregnant women during the a(h1n1)pdm09 pandemic was due to an elevated level of viral replication and to a cytokine imbalance that led to a less effective immune response. keywords. pregnancy; pandemic influenza a virus; immune response. pregnant women are at risk of increased mortality and morbidity due to influenza virus infection [1] . during the 2009 pandemic of influenza a(h1n1) virus (a[h1n1]pdm09) infection, pregnancy was associated with an increased risk of hospitalization [1] , disease susceptibility [2] , and influenza-related death in the united states. in particular, women in their third trimester were especially vulnerable. several studies have shown that pregnant women also had increased mortality and hospitalization rates during previous influenza pandemics: the [3] . in these cases, higher risk was not restricted to infection during the third trimester, and virus exposure during early gestation was also associated with adverse outcome for the fetus [2, 4] . pregnancy is considered to be a state of immune suppression, with cytokine profiles biased toward a t-helper type 2 (th2)-type response [5] . peripheral blood mononuclear cells (pbmcs) isolated from pregnant women responded less robustly to innate immune stimulation than did those isolated from nonpregnant women. although pregnant women are known to be a high-risk group for influenza virus infections, little is known about the underlying mechanisms. while some studies have assessed the impact of pregnancy during influenza virus infection in mice, studies in more-relevant animal models are warranted. we therefore developed a pregnant ferret model and analyzed the pathologic and immunologic changes following infection with a(h1n1)pdm09. all experiments were approved by the shantou university medical college (reference number: sumc2013-130) in compliance with their policies for animal ethics and welfare and use of animals in research, the guidelines of the world health organization, and the international council for laboratory animal science. eighteen-to 20-month-old ferrets (mustela putorius furo) were obtained from wuxi sangosho co. ltd. (jiangsu province, china). in this study, we used 10 pregnant ferrets (6 infected with a[h1n1]pdm09 and 4 uninfected) and 8 nonpregnant ferrets (4 infected with a[h1n1]pdm09 and 4 uninfected). all ferrets were serologically negative for a(h1n1) pdm09 and seasonal influenza a(h3n2) virus as determined by hemagglutination inhibition assays. on day 14 after mating, ferrets were infected intranasally with 500 μl of 10 5 50% tissue culture infective doses (tcid 50 )/ml of the a(h1n1)pdm09 virus a/california/07/09 (ca07), each diluted in phosphate-buffered saline (ph 7.4), or received 500 μl of phosphate-buffered saline alone, after sedation with isoflurane. all inoculated animals were monitored daily. for viral titration, nasal washes from each ferret were collected on days 1, 3, 5, 7, 9, 11, and 13 after inoculation, and organs were collected on 5 days after inoculation (2 or 3 ferrets per group). all virus titers were expressed as tcid 50 in madin-darby canine kidney cells. for histopathologic analysis, tissue specimens were processed for hematoxylin and eosin staining and stained with a mouse anti-nucleoprotein (np) monoclonal antibody for analysis of pbmc responses, cells were collected on 5 days after inoculation and isolated by density centrifugation by using histopaque-1077 medium (sigma, ca) according to the manufacturer's instructions. for quantification of peripheral cd8 + cells, pbmcs were stained with allophycocyanin-labeled cd8 antibody (ebioscience, ca) and data acquired on a bd facsaria ii (bd biosciences, nj). all samples were analyzed by flowjo software (tree star, or). to measure the influenza virus-specific b-cell response, 96-well multiscreen ha plates (millipore, ma) were coated with 1 µg of recombinant hemagglutinin (ha) from a(h1n1)pdm09 (sino biological, china), and cells were plated and incubated at 37°c for 5 hours. after binding, cells were stained with alkaline phosphatase-conjugated goat anti-ferret immunoglobulin g antibody (southern biotechnology, al) and incubated overnight at 4°c. spots were visualized by colorimetric analysis, using the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (bcip/nbt, kpl, md), and positive spots were counted by using a ks enzyme-linked immunospot (elispot) system (carl zeiss). cytokine and chemokine expression levels were measured using quantitative real-time polymerase chain reaction analysis (roche, ca). to determine whether pregnancy in ferrets results in enhanced influenza severity, as seen in humans, we examined the virologic and pathologic changes following ca07 infection. owing to the significant weight gain in pregnant ferrets, weight loss, which is typically used as a clinical marker of disease in ferrets, was not a feasible measure for this study. upon infection, we observed that there were no significant differences in the temperature changes between pregnant and nonpregnant groups (p = .302; data not shown). although our study was not designed to specifically measure this outcome, we noticed differences in the number of newborns between uninfected and infected pregnant ferrets by the end of the gestational period. two newborns were produced by the noninfected pregnant ferrets, but none were produced by the infected pregnant ferrets. to determine the pattern of virus shedding in pregnant and nonpregnant animals, we collected nasal wash samples every other day. viruses were detected in the nasal wash specimens from all inoculated ferrets, with significantly higher titers in pregnant ferrets (p < .0001) that were maintained for longer periods ( figure 1a ). by day 9, all animals fully recovered, and virus was cleared in all infected ferrets. to evaluate the histopathologic changes upon ca07 infection, tissue samples were collected 5 days after inoculation from pregnant and nonpregnant ferrets. viruses were detected in turbinate, tracheal, and lung tissues from all animals, with significantly higher titers (p < .001) seen in the pregnant group (mean titers ranged from 2.5 to 4.1 log 10 tcid 50 /ml; figure 1b ). viral np staining confirmed virus replication in lungs (figure 1dc and 1 dd) and tracheas (figure 1dg and 1 dh) of inoculated ferrets. no evidence of viral infection was observed in nonpulmonary tissues ( figure 1b) , including the transplacental tissues, suggesting a general lack of systemic spread of viruses in ferrets and no evidence of intrauterine infection. pathologically, a difference in lung damage was observed between pregnant and nonpregnant groups. this was characterized by more-pronounced alveolar damage, widespread edema in the lungs (figure 1cc and 1 cd), and severe degeneration of epithelial cell walls in trachea (figure 1cg and 1 ch) of the pregnant animals. the numbers of viral np-positive cells were also higher in pulmonary tissues of pregnant ferrets (figure 1dd and 1 dh) than in those of nonpregnant ferrets (figure 1dc and 1 dg) . no evidence of np positivity or tissue damage was observed in the control mock-infected ferrets (figure 1ca, 1cb, 1ce , and 1cf and figure 1da, 1db, 1de, and 1df) . overall, these results showed that during pregnancy, both virus replication and subsequent tissue damage are elevated following influenza virus infection during pregnancy. influenza virus infection in pregnant women is associated with increased levels of respiratory tract inflammatory cytokines and chemokines [5] . to determine whether a similar phenotype is present in pregnant ferrets, we measured the expression levels of inflammatory cytokines and chemokines by quantitative real-time polymerase chain reaction in the trachea (representative of the upper respiratory tract), lungs (representative of the lower respiratory tract), and bronchoalveolar lavage to evaluate responses in resident or infiltrating immune cells lining the airways [6] of pregnant and nonpregnant animals following infection. in general, although not exclusively, when differences in the levels of cytokines and chemokines were noted, levels were higher in pregnant animals, consistent with observations in humans. in lung tissues, levels of interferon α (ifn-α) and interleukin 4 (il-4) were significantly higher in pregnant animals; levels of interleukin 10 (il-10) and interleukin 12p40 (il-12p40) were higher in nonpregnant animals. in tracheal tissues, messenger rna (mrna) levels of tumor necrosis factor α, ifn-γ, and interleukin 6 (il-6) were higher in pregnant animals. in bronchoalveolar lavage, pregnant animals had higher mrna levels for ifn-α, ifn-γ, il-6, and il-12p40. in pbmcs, nonpregnant animals had higher levels of il-4 and il-12p40 (figure 2a) . in pregnant mice, influenza virus infection has been associated with alterations in the immune response [7, 8] . correspondingly, we assessed the numbers of cd8 + t lymphocytes and influenza virus-specific b cells in total pbmcs 5 days after inoculation in our infected ferrets. in a(h1n1)pdm09-infected pregnant ferrets, the percentage of cd8 + t lymphocytes among pbmcs was significantly lower than in nonpregnant ferrets (p < .001; figure 2b ). elispot measurements of a(h1n1) pdm09-specific b cells also showed reduced numbers in the pregnant ferrets (p < .05; figure 2c ). together, these results suggest that both innate and adaptive immune responses are impaired in pregnant ferrets during influenza virus infection. although pregnancy is associated with increased susceptibility to pathogens owing to immunologic suppression, it is not entirely clear how this manifests as increased severity after influenza virus infection. while informative studies have been conducted in mice, the nature of influenza virus-mediated disease in this host is distinctly different than what is seen in humans; we thus turned to the ferret model. the pregnant ferret model has been used since the 1970s to study the teratogenic potential of influenza viruses [9] [10] [11] . these studies showed that fetal infection and pathology were only possible through virus inoculation directly into the amniotic space or maternal bloodstream, leading the authors to suggest that the teratogenic risk to the fetus due to natural influenza virus infection was low. however, these studies did not focus on the virologic or immunologic outcome of infection in the pregnant ferret itself. thus, our study aimed to address this knowledge gap. in our study, we infected the ferrets on day 14 after mating, which, in the ferret's gestational cycle, loosely corresponds to early stages of human pregnancy. the cytokine profile in the infected ferrets was consistent with that observed in infected humans, notably the elevation levels of ifn-α, il-6, and ifn-γ [12] . this suggests that the immune response in infected ferrets to some extent mimics the response in humans [13] . when we compared the cytokine mrna levels between pregnant and nonpregnant ferrets, however, there were striking differences in responses of several key cytokines in the trachea, lung, and bronchoalveolar lavage. stronger proinflammatory responses were observed in the trachea (ie, upper respiratory tract) of pregnant ferrets, which also had extended and higher levels of virus shedding as compared to nonpregnant ferrets. interestingly, levels of il-6 and tnf-α in the upper respiratory tract of influenza virus-infected humans were correlated with virus shedding [12] . in contrast, mrna levels of the antiinflammatory cytokine il-10 were increased in the lungs of nonpregnant ferrets. this suggests that nonpregnant ferrets may have more capacity to regulate excessive inflammatory responses and tissue repair in the lungs, compared with pregnant ferrets, as was previously shown in the murine model [8] . however, as was shown in same study, cytokine levels may change over time, and because of limitations of the ferret model, our results may only represent a snapshot of these changes. severe cases of influenza in pregnant women were also associated with decreased levels of immunoglobulin in sera [14] and dysregulated t lymphocytes [15] . consistent with these observations, we found that the numbers of a(h1n1)pdm09-specific b cells and cd8 + t cells in peripheral blood in pregnant ferrets were lower than those in nonpregnant ferrets. pbmcs from pregnant ferrets produced less il-4 and il-12p40 than those from nonpregnant ferrets after a(h1n1)pdm09 infection, which could account for the decreased number of influenza virus-specific b cells and cd8 + t cells. unlike the murine model, one major limitation of our ferret model was the lack of reagents for detailed immunologic analyses. for example, the cytokine responses could only be profiled at the mrna level and not at the protein level. at the time of the experiments, no suitable assay for the assessment of ferret-specific cd8 + t-cell function and specificity was available. for the same reason, we were also unable to assess the cellular innate responses or the cd4 + t-cell responses. these are also critical immune regulators that can modulate disease severity during influenza virus infection. our findings here will have to be interpreted with these caveats in mind. in our combined virologic and immunologic analyses, we showed that a(h1n1)pdm09 infection during pregnancy induces more-severe disease as a consequence of elevated viral loads and innate responses, combined with a diminished adaptive response. these factors likely contributed to the increased morbidity and mortality rates observed during the 2009 influenza pandemic in pregnant women. novel influenza a(h1n1) pregnancy working group. h1n1 2009 influenza virus infection during pregnancy in the usa influenza and congenital anomalies: a systematic review and meta-analysis california pandemic (h1n1) working group. severe 2009 h1n1 influenza in pregnant and postpartum women in california influenza in pregnancy: the case for prevention the th1:th2 dichotomy of pregnancy and preterm labour bronchoalveolar lavage constituents in healthy individuals, idiopathic pulmonary fibrosis, and selected comparison groups. the bal cooperative group steering committee wild type and mutant 2009 pandemic influenza a (h1n1) viruses cause more severe disease and higher mortality in pregnant balb/c mice fatal outcome of pandemic h1n1 2009 influenza virus infection is associated with immunopathology and impaired lung repair, not enhanced viral burden, in pregnant mice the effects of maternal influenzal viraemia in late gestation on the conceptus of the pregnant ferret association of foetal wastage with influenza infection during ferret pregnancy the pregnant ferret as a model for studying the congenital effects of influenza virus infection in utero: infection of foetal tissues in organ culture and in vivo local and systemic cytokine responses during experimental human influenza a virus infection. relation to symptom formation and host defense local innate immune responses and influenza virus transmission and virulence in ferrets association between severe pandemic 2009 influenza a (h1n1) virus infection and immunoglobulin g(2) subclass deficiency regulatory t cells mediate maternal tolerance to the fetus key: cord-289255-qwzg7prx authors: seligman, stephen j. title: evidence for quasi species in severe acute respiratory syndrome-associated coronavirus deletion mutants date: 2007-02-15 journal: j infect dis doi: 10.1086/510917 sha: doc_id: 289255 cord_uid: qwzg7prx nan using specimens obtained during the 2003 epidemic in hong kong, tang et al. have reported data on a 386-nt deletion in severe acute respiratory syndrome-associated coronavirus (sars-cov) [1] . because 1 patient had both the l386del variant and the wild-type variant in the same specimen, they raised the possibility that sars-cov exists as a quasi species, at least in some patients. previous authors studying single-nucleotide variants from beijing-area isolates in 2003 [2] and from the singapore 2004 outbreak [3] have also found multiple viral sequences in the same sample that they attributed to quasi species. although these 3 studies clearly establish the presence of a diversity of sars-cov genomes in individual patients, the issue of whether sars-cov quasi species exists remains open, particularly with respect to the 386-nt deletion. the concept of quasi species was described by eigen et al. to indicate a "complex, self-perpetuating population of diverse related entities that act as a whole" [4, p. 42; for review, see 5]. the earliest experimental verification of the theory was obtained with the bacteriophage qb, in which the wild type existed only as the weighted average of different sequences [6] . eigen has acknowledged, however, that in instances in which the characteristics of a heterogeneous population had not been established, it might be more appropriate to call the population a "swarm" rather than a quasi species [7] . this distinction could be particularly pertinent to the large deletion described by tan et al., because the resulting swarm lacks a prom-inent mechanism for the maintenance of quasi species, the ability to back-mutate point mutations. the finding of the l386del variant along with the intact genome has at least 3 possible implications. the first is the issue of whether the gene products of the deleted segment are either virulence factors or essential for viral replication. the 386-nt deletion disrupts open reading frame (orf) 9 and eliminates orf 10 and 11. the functions of the products of these 3 orfs are currently unknown. however, they could be essential and involved with virulence, the functionality being supplied by other genomes in the swarm. the second is the possibility that the deletion variants produce truncated protein that acts as a decoy for the immune system [8] . last, although the deletions are usually much larger, the deletion mutant could be an early stage in the development of defective interfering particles. because of safety considerations, additional experiments involving growth and separation of viruses with the different genomes may not be advisable. however, if sufficient patient material is available, multiple clones could be obtained from polymerase chain reaction products and the percentage of genomes with the deletion determined for various stages of both infection in the individual patient and the course of the epidemic. the finding of patients with severe disease who did not have full-length variants would support the idea that the deletion mutants were both fully infectious and virulent. on the other hand, as is consistent with the data in figure 2 of the tang et al. article [1] , the finding of progressive increase in the percentage of genomes with the de-letion mutants would support the hypothesis of defective interfering particles. the large 386-nt deletion in sars-associated coronavirus: evidence for quasispecies? sars-associated coronavirus quasispecies in individual patients sars transmission pattern in singapore reassessed by viral sequence variation analysis viral quasispecies what is a quasispecies? nucleotide sequence heterogeneity of an rna phage population on the nature of virus quasispecies long-term transmission of defective rna viruses in humans and aedes mosquitoes department of microbiology and immunology, new york medical college, valhalla key: cord-007234-hcpa8ej5 authors: renwick, neil; schweiger, brunhilde; kapoor, vishal; liu, zhiqiang; villari, joseph; bullmann, reinhard; miething, robert; briese, thomas; lipkin, w. ian title: a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany date: 2007-12-15 journal: j infect dis doi: 10.1086/524312 sha: doc_id: 7234 cord_uid: hcpa8ej5 acute respiratory infection is a significant cause of morbidity and mortality in children worldwide. accurate identification of causative agents is critical to case management and to prioritization in vaccine development. sensitive multiplex diagnostics provide us with an opportunity to investigate the relative contributions of individual agents andmayalso facilitate the discovery of new pathogens. recently, application of masstag polymerase chain reaction (pcr) to undiagnosed infuenza-like illness in new york state led to the discovery of a novel rhinovirus genotype. here we report the investigation, by masstag pcr, of pediatric respiratory-tract infections in germany, studying 97 cases for which no pathogen was identified through routine laboratory evaluation. respiratory viruses were identified in 49 cases (51%); of the 55 identified viruses, 41 (75%) were rhinoviruses. the novel genotype represented 73% of rhinoviruses and 55% of all identified viruses. infections with the novel genotype were associated with upper-respiratory-tract symptoms but, more frequently, with bronchitis, bronchiolitis, and pneumonia. human rhinoviruses (hrvs) are the most frequent cause of acute respiratory illness worldwide. although hrvs are most commonly associated with mild upperrespiratory-tract disease [1] [2] [3] , infection of lower airways does occur [4 -7] . lower-respiratory-tract infections (lrtis) related to hrv are increasingly being reported in infants, elderly persons, and immunocompromised patients [8] . hrvs are also implicated in exacerbations of asthma [9, 10] , chronic bronchitis [11] , and acute bronchiolitis [12] . taxonomically, hrvs are currently grouped into 2 species, human rhinovirus a (hrv-a) and human rhi-novirus b (hrv-b), in the genus rhinovirus of the family picornaviridae ( [13, 14] ). these nonenveloped, positive-sense, single-stranded rna viruses have been classified serologically [15, 16] and on the basis of their antiviral susceptibility profiles [17, 18] , their nucleotidesequence relatedness [19, 20] , and their use of receptors (intercellular adhesion molecule 1, low-density lipoprotein receptor, and decay-accelerating factor) [21] [22] [23] . phylogenetic analyses of the vp4/vp2 and vp1 coding regions have indicated the presence of 76 serotypes in genetic group a and 25 serotypes genetic group b [18 -20, 24 ]. an agent is commonly not implicated in up to 50% of cases of severe respiratory disease, despite the application of polymerase-chain-reaction (pcr) assays as well as classical diagnostic methods, including antigen tests, serology, and culture methods. broad-range molecular assay systems, such as multiplex pcr (hexaplex [25] , genescan [26] , and masstag [27] ), or microarrays (vi-rochip [28] and panmicrobial greenechips [29] ) may therefore allow us to gain new insights into epidemiology and clinical associations [30, 31] . with respect to hrv, recent studies employing sensitive pcr systems for these difficult-to-isolate organisms have shown an increased detection rate, compared with culture methods [1, [32] [33] [34] [35] . applying a multiplex masstag pcr platform, we recently detected numerous agents of respiratory illness in samples that had been submitted for laboratory diagnosis but that had tested negative during routine diagnostic assessment [30] . hrvs were identified at high frequency in this set of samples. detailed genetic analysis indicated that a large fraction of these viruses represent a previously uncharacterized genotype of rhinovirus, one that diverges from either hrv-a or hrv-b. in an attempt to gather additional information on the potential pathogenicity, as well as temporal and geographic distribution, of rhinoviruses, including the recently identified genotype, we evaluated specimens collected, during the 2003-2006 seasons in bad kreuznach, germany, from children hospitalized because of severe lrti. nasopharyngeal aspirates were obtained from children admitted, because of acute respiratory-tract infection, to the kreuznacher diakonie hospital (bad kreuznach, germany) during the interval of 2003-2006. individuals ranged in age from 2 weeks to 5 years (mean age, 5 months; median age, 10 months); 46% were male, 54% female. specimens collected at the time of admission were forwarded undiluted to the robert koch institute (berlin, germany) for laboratory evaluation. rna extraction was performed by use of qiaamp viral rna kits (qiagen). the 97 samples for which no pathogen had been diagnosed after assessment by realtime reverse-transcription (rt)-pcr assay for influenza virus [36] and respiratory syncytial virus infection were stored at assay procedures. the 97 rna samples representing cases of undiagnosed respiratory diseases were employed as a template for cdna synthesis by use of superscript ii kits with random hexamer priming (invitrogen), and they were analyzed by masstag pcr by using a viral primer panel [27] that targeted influenza virus a and b (fluav and flubv, respectively), respiratory syncytial virus a and b (rsv-a and rsv-b, respectively), human parainfluenza virus 1, 2, 3, and 4 (hpiv-1, hpiv-2, hpiv-3, and hpiv-4, respectively), human coronavirus 229e and oc-43 (hcov-229e and hcov-oc43, respectively), human metapneumovirus, entero-and rhinoviruses, and adenoviruses. the fidelity of the masstag pcr signal was verified by reamplification of products and by sequence analysis for all positive specimens. in instances in which masstag pcr indicated the presence of a picornavirus, the vp4/vp2 region was amplified [37] . amplification products were purified from agarose gels (qia gel extraction kit; qiagen), and nucleotidesequencing reactions were performed on both strands by use of the abi prism big dye cycle sequencing kits and the abi prism genetic analyzer systems (applied biosytems). identical results were obtained with duplicate aliquots processed at the new york and berlin laboratories. sequence analyses, alignments, and phylogenetic reconstructions were performed by use of programs from the wisconsin gcg package (accelrys) and by mega 3.1 software [38] . nucleic-acid sequences generated during this work are available at genbank, under the accession numbers eu081778 -eu081816. we used masstag pcr to investigate 97 nasopharyngeal aspirates from children hospitalized because of acute respiratory illness for which no pathogen was identified through routine laboratory testing. masstag pcr identified at least 1 candidate respiratory-viral pathogen in 49 specimens. although there was variability across the 3 seasons clinical associations. hrvs were the viruses most frequently detected in our set of samples, representing 75% (41/55) of the identified viruses; coinfection with another virus was observed in only 12% (5/41) of these cases (table 2). the frequency of fever or cough in infections with hrv (82%) was comparable to that in infections with the other viruses (89%); the frequency of rhinitis or pharyngitis with hrv (79%) was comparable to that with other viruses; and the frequency of lrti symptoms (bronchitis, bronchiolitis, and pneumonia) with hrv (71%) was comparable to that with the other viruses (67%). whereas pneumonia was more common in infections with hrv a/b (56%) than in infections with hrv x (36%), the frequency of bronchiolitis with hrv a/b (11%) was comparable to that with hrv x (12%), and the frequency of bronchitis with hrv a/b (67%) was comparable to that with hrv x (60%). lrti was recorded in 72% of hrv x infections; however, some cases were related to milder disease (table 2) . molecular epidemiology of identified picornaviruses. masstag pcr targets conserved sequences in the 5'untranslated region of entero-and rhinoviruses; thus, to facilitate phylogenetic analysis of hev and hrv, we amplified and sequenced the vp4/vp2 gene region. however, when we used the basic local alignment search tool for analysis at the nucleotide level, we did not find, for 30 of the vp4/vp2 sequences, a significant match with hrv-a, hrv-b, or hev sequences; analysis at the amino-acid level revealed homology to enteroand rhinoviral sequences, with a sequence identity of 60%-65%. high similarity at both the nucleotide-and amino-acid levels was evident when sequences were aligned with an unclassified genetic clade of picornaviruses recently identified in new york state [30] . however, detailed phylogenetic analysis indicated significant sequence diversity among the 30 viruses ( in this study of samples collected, during a 3-year interval, from hospitalized children with severe undiagnosed respiratory infection, masstag pcr allowed us to detect viral pathogens in 49 (51%) of 97 cases. the pathogens most commonly identified were hrvs. these findings are consistent with other studies, which have indicated that rhinoviruses or picornaviruses ac-count for 20%-80% of acute respiratory infections [1, 32, 33, 39 -41]-exceeding, in some instances, even the frequency of rsv infection in pediatric-patient populations [34, [41] [42] [43] . the presence of hrv is not sufficient to prove causation. asymptomatic hrv infection has been described; however, the extent to which infection without disease represents carriage, incubation, or convalescence is unknown [35, 39, 40, 42, 44] . although we did not have samples to test for the presence of hrv in the lower respiratory tract, the high frequency at which hrv was identified as being the sole virus detected suggests a correlation between the agent and the observed lrti symptoms. support for the plausibility of hrv being pathogenic in lrti comes from the facts that (1) in situ hybridization has demonstrated that hrvs exist in lower airways and (2) hrvs have been shown to trigger inflammatory processes in the infected cells and tissues [5] [6] [7] [45] [46] [47] [48] . among the hrvs identified in the present study were representatives of the novel genetic clade recently discovered in new york state [30] ; indeed, these viruses comprised the majority of hrvs detected. hrv-a and hrv-b have been implicated in common colds as well as in severe lrti. in our patients, viruses of the novel genetic clade were also associated with a wide range of diseases, ranging from rhinitis to bronchitis to severe pneumonia, necessitating supplemental oxygen in ϳ50% of cases. a seasonal pattern of hrv infections has been described [2, 3, 43] ; however, data regarding (43) pathogens detected (no.) hpiv-2 (1) rsv-b (1) hpiv-1 (3) hadv (1) hpiv-3 (1) hpiv-2 (1) hev/hrv (12) hpiv-4 (1) hmpv (1) hcov-oc43 (1) hev/hrv (15) hadv (2) hev/hrv (15) specific identification (no. either serotype-or genotype-specific patterns of seasonality or disease symptoms are limited [49 -51] . a temporal trend of sequence diversity or of correlation between genotype (within the novel hrv clade) and clinical diagnosis was not apparent in our data (figure 1). no detailed information is available yet concerning the history of the novel hrv clade; nonetheless, the sequence diversity observed within it (figure 1) is not consistent with a re-cent introduction. this clade may account, in part, for earlier reports of nontypeable rhinoviruses [41, 44] . indeed, its discovery may reflect the implementation of new technologies rather than novelty of the agent itself. we anticipate that future work will define more than 1 serogroup. our findings reinforce other groups' recent work indicating the significance of hrvs in pediatric lrti. the presence of novel hrvs in 2 disparate geographic locations, in association with serious respiratory disease in children as well as in adults, mandates further work in epidemiology and pathogenesis. frequency and natural history of rhinovirus infections in adults during autumn viruses and bacteria in the etiology of the common cold the seasonality of rhinovirus infections and its implications for clinical recognition characterization and classification of echo 28-rhinovirus-coryzavirus agents detection of rhinovirus rna in lower airway cells during experimentally induced infection rhinoviruses infect the lower airways quantitative and qualitative analysis of rhinovirus infection in bronchial tissues rhinovirus and the lower respiratory tract interleukin-10 gene expression in acute virus-induced asthma rhinovirus viremia in children with respiratory infections viral infections of human association of rhinovirus infection with increased disease severity in acute bronchiolitis international committee on taxonomy of viruses. universal database of the international committee on taxonomy of viruses virus taxonomy: eighth report of the international committee on taxonomy of viruses antigenic groupings of 90 rhinovirus serotypes a collaborative report: rhinoviruses-extension of the numbering system from 89 to 100 two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity alignment of capsid protein vp1 sequences of all human rhinovirus prototype strains: conserved motifs and functional domains molecular relationships between 21 human rhinovirus serotypes genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70 many rhinovirus serotypes share the same cellular receptor the major and minor group receptor families contain all but one human rhinovirus serotype human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features vp1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (hexaplex) genescan reverse transcription-pcr assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness diagnostic system for rapid and sensitive differential detection of pathogens microarray-based detection and genotyping of viral pathogens panmicrobial oligonucleotide array for diagnosis of infectious diseases masstag polymerase-chainreaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influeza-like illness in new york state during microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr molecular diagnosis of human rhinovirus infections: comparison with virus isolation detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season application of a fluorogenic pcr assay for typing and subtyping of influenza viruses in respiratory samples simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms novel rhinovirus genotype in lrti • jid respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care: ige and eosinophil analyses rhinovirus-associated hospitalizations in young children persistence of rhinovirus and enterovirus rna after acute respiratory illness in children lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects low grade rhinovirus infection induces a prolonged release of il-8 in pulmonary epithelium rhinovirus replication causes rantes production in primary bronchial epithelial cells rhinovirus infection increases 5-lipoxygenase and cyclooxygenase-2 in bronchial biopsy specimens from nonatopic subjects rhinovirus infections in an industrial population. v. change in distribution of serotypes the seattle virus watch. v. epidemiologic observations of rhinovirus infections, 1965-1969, in families with young children rhinoviruses in britain 1963-1973 we thank ingrid zadow, bettina bauer, manuela friedrich, marlies hartwig, and sven tietze for their excellent technical assistance. key: cord-273356-1ius4ksa authors: sauceda, john a; neilands, torsten b; lightfoot, marguerita; saberi, parya title: findings from a probability-based survey of united states households about prevention measures based on race, ethnicity, and age in response to severe acute respiratory syndrome coronavirus 2 date: 2020-08-29 journal: j infect dis doi: 10.1093/infdis/jiaa554 sha: doc_id: 273356 cord_uid: 1ius4ksa we investigated individual behaviors taken by white, african american, and latino united states (us) households in response to severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and likelihood of using digital tools for symptom surveillance/reporting. we analyzed cross-sectional week 1 data (april 2020) of the coronavirus disease 2019 (covid-19) impact survey in a large, nationally representative sample of us adults. in general, all groups engaged in the same prevention behaviors, but whites reported being more likely to use digital tools to report/act on symptoms and seek testing, compared with african americans and latinos. individual behaviors may not explain covid-19 case disparities, and digital tools for tracking should focus on uptake among race/ethnic minorities. there have been 21.7 million reported cases of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection and >776 000 deaths due to coronavirus disease 2019 (covid-19) worldwide through 17 august 2020. more than one-fourth of cases are in the united states (us), with african americans and latinos being disproportionately impacted in case counts and death rates [1] [2] [3] [4] [5] . prevention control messages and efforts, such as sheltering in place and quarantining, may not have been as successful among african americans and latinos for numerous reasons, such as needing to work outside of the home, living in large households in close quarters, and including the effects of structural racism (ie, access to health insurance and care, limited health literacy) [6, 7] . little is known about individual prevention measures that were taken in response to covid-19 or how people may engage with surveillance/reporting strategies as we enter phase 2 of the pandemic. we analyzed data collected from 20 to 26 april 2020 in the covid-19 impact survey [8] . data are from a probabilitybased panel representative of us households. we tested for differences between non-latino white, african american, and latino respondents on prevention control measures, likelihood of using surveillance/reporting strategies, and household size. the national opinion research center (norc) at the university of chicago (www.norc.org) collects weekly responses to health, economic security, and social dynamics questions. all survey data are available at www.covid-impact.org [8] . norc's probability-based panel is designed to be representative of the us household population. adults aged ≥18 years representing 50 states and the district of columbia were randomly sampled from the amerispeak panel. surveys were conducted online or by telephone with a norc interviewer. the sample weights in the dataset were calculated with information from demographic weighting variables acquired from the 2020 current population survey reflecting the us population [9] . this manuscript meets criteria to be self-exempt from the university of california, san francisco institutional review board. participants responded yes (1) or no (0) to 19 measures taken in response to sars-cov-2 (eg, worn a facemask, kept distance of 6 feet). respondents rated their likelihood of "installing an [mobile] app that (1) asks you questions about your own symptoms and provides recommendations about covid-19" and (2) "tracks your location and sends push notifications if you might have been exposed to covid-19, " and (3) "using a website to log your symptoms and location and get recommendations about covid-19. " respondents also rated their likelihood of "testing for covid-19 infection using a q-tip to swab your cheek or nose" and "testing you for immunity or resistance to covid-19 by drawing a small amount of blood. " ordinal response options were as follows: 1 (not likely at all), 2 (not too likely), 3 (moderately likely), 4 (very likely), and 5 (extremely likely). the norc field report contains complete details on the statistical weights, design effect, and margin of sampling of error [10] . the statistical weights we used were computed by norc and included as an additional variable in the dataset. the weights are the inverse of probability of selection from the sampling frame used to sample housing units for amerispeak. both analyses we ran were conducted with mplus statistical software and used the sampling weights. the first analysis was a multivariable logistic regression model with all 19 prevention control measures regressed on a dummy-coded race/ethnicity variable: non-latino whites, african americans, and latinos, with age (18-29, 30-44, 45-59, and ≥60 years) and sex (female vs male) as covariates. the second analysis was a multivariable ordinal logistic regression model where the app-and internetbased and testing ordinal responses were regressed on race/ethnicity, with age and sex as covariates. ordinal regression results are interpreted as odds of responding to each outcome level (5 = extremely likely) vs all lower options (1 = not likely at all through 4 = very likely). the sample included 1395 white, 265 african american, and 369 latino respondents. table 1 shows the full sample characteristics and descriptive statistics of key variables. there were no differences in the reporting of 19 prevention control measures between white and african american respondents ( table 2 ). latinos were less likely than whites to report keeping physical distance with people outside of their household (odds ratio [or], 0.49 [95% confidence interval {ci}, .28-.86]; p < .04) ( table 2) . females were consistently engaging in more prevention control measures than males (table 2) , whereas age was not associated with any measure. relative to whites, african americans and latinos reported a lower likelihood of installing an app that asks about symptoms (or, 0.52 [95% ci, .38-.70] and 0.53 [95% ci, .40-.70], respectively; p < .001 for both). relative to 18-to 29-year-old respondents, those ≥60 years of age reported a lower likelihood of installing an app that asks about symptoms (or, 0.66 [95% ci, .49-.91]; p < .04). relative to whites, latinos reported a lower likelihood of installing an app that tracks location and sends push notifications if users were possibly exposed to sars-cov-2 (or, 0.71 [95% ci, .55-.92]; p < .03). no differences emerged between african americans and whites. african americans and latinos reported a lower likelihood of using a website to log symptoms and get recommendations for covid-19, relative to whites (or, 0.47 [95% ci, .33-.65] and 0.54 [95% ci, .40-.71], respectively; p < .001 for both). relative to 18-to abbreviations: ci, confidence interval; or, odds ratio. a age was not associated with any coronavirus disease 2019 prevention control measure and thus is not shown here. in a probability-based household survey, non-latino white, african american, and latino respondents engaged in nearly identical patterns and frequencies of individual prevention measures taken in response to sars-cov-2. only 1 difference emerged between latinos and whites, which was keeping social distance. thus, individual behavior may not be driving in the spread of sars-cov-2 in racial and ethnic minority communities. these data show that there is a consistent difference in behaviors by sex but not by age. male respondents' odds for engaging prevention control measures were consistently lower compared with female respondents, which may partially explain higher covid-19 cases in males vs females [11] . there was a general low endorsement of technology-based reporting strategies, but consistent differences between groups. first, latinos and african americans reported being less likely to use these strategies compared to whites. for sars-cov-2 testing, latinos reported being less likely to test compared with whites. these differences may be in part due to immigration status and wanting to avoid involvement in the health system, medical racism, and unwillingness to contribute to an effort with indirect benefit (ie, helping public health monitoring). furthermore, younger respondents reported a higher likelihood of using technology-based strategies and testing compared with older respondents. there are several implications from these findings. first, between march and may 2020, unemployment climbed from 6.7% to 16.8% among african americans and from 6.0% to 17.6% among latinos [12] . this could have led to greater crowding within homes if person were unable to work, but also does not exclude the possibility of working informally, all of which could have increased exposure to sars-cov-2. and as evidenced by an analysis of the medical expenditures panel survey, african americans and latinos were more likely than whites to be "essential workers" (ie, food sector) or to work in health settings [13] , further adding to the risk of exposure. in our data, while latinos reported greater likelihood for not keeping social distance, relative to whites (the only difference between the 2 groups), it is likely that they did not have the opportunity to keep social distance (eg, dimensions of work environment), rather than a notion that they disregarded this prevention control measure. while characteristics such as age and comorbidities (eg, diabetes) were identified as predispositions to severe covid-19 complications and death [6] , the magnitude of disparities cannot be explained by individual behavior, as we have shown, or individual risk factors noted as predispositions [13] . it is likely that other social and structural drivers of health disparities, such as racism, may better explain the disproportionate impact of covid-19. second, technology-based strategies will be less effective if they do not account for medical mistrust, lack of familiarity with technology, and privacy concerns [14] . digital tracking of a pandemic affecting racial and ethnic minorities will likely miss these key populations unless critical acceptability and outreach are established. last, if exposed and/ or diagnosed, quarantining may be difficult for individuals in households with people who cannot work from home, that are multigenerational, or that experience overcrowding [15] . in our sample, 32% of latinos, compared to 13.3% of whites, lived with 5 or more people. thus, density is a considerable risk factor for sars-cov-2 exposure to address among populations who live in crowded settings. study limitations include the survey questions not being previously validated and not applicable to all persons (eg, people who are unable to work from home could not opt out of the question whether they did or not). between 20 and 26 april 2020, there were differences between states and cities in sars-cov-2 mandates; therefore, some variations in prevention control measures may have depended on region. despite these limitations, we believe that documenting individual behaviors is critical to plan for strategies that effectively mitigate current and future problems. we also believe that our findings show that disparities in covid-19 may not be explained by individual behavior. as new surveillance tools and testing strategies become available, we must ensure equal acceptability and access among all racial/ethnic groups. our data can help in planning for dealing with the current pandemic, future outbreaks of sars-cov-2, and potential future pandemics. covid-19 and african americans failing another national stress test on health disparities variation in covid-19 hospitalizations and deaths across new york city boroughs the disproportionate burden of covid-19 for immigrants in the bronx hospitalization rates and characteristics of patients hospitalized with laboratoryconfirmed coronavirus disease 2019-covid-net, 14 states pérez-stable e. covid-19 and racial/ethnic disparities structural racism and health inequities in the usa: evidence and interventions current population survey data tables. 2019. www.census.gov/programs-surveys/cps. html. accessed 15 does gender influence clinical expression and disease outcomes in covid-19? a systematic review and metaanalysis the employment situation covid-19 and race/ethnic disparities in health risk, employment, and household composition the digital divide in health-related technology use: the significance of race/ethnicity the number of people in the average u.s. household is going up for the first time in over 160 years we thank the national opinion research center (norc) at the university of chicago. the data analyzed are publically available and free to use through the norc at the university of chicago.potential conflicts of interest. all authors: no reported conflicts of interest.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-007188-tcq8lnwg authors: cunningham, anthony l.; grohman, gerhard s.; harkness, john; law, carmela; marriott, deborah; tindall, brett; cooper, david a. title: gastrointestinal viral infections in homosexual men who were symptomatic and seropositive for human immunodeficiency virus date: 1988-08-17 journal: j infect dis doi: 10.1093/infdis/158.2.386 sha: doc_id: 7188 cord_uid: tcq8lnwg gastrointestinal viruses, predominantly rotaviruses and adenoviruses, were detected by enzyme-linked immunosorbent assay, electron microscopy, or cell culture in >50% of two groups of homosexual men with symptomatic human immunodeficiency virus (hiv) infection, who did (54%) or did not (50%) have diarrhea. lower detection rates were observed in hiv-seronegative (15%) and asymptomatic hiv-seropositive (16%) men. in the patients with diarrhea, 95% of the isolates of virus were found in the most immuno suppressed patients, those patients with aids-related complex or opportunistic infections associated with aids. high excretion rates of these viruses are probably associated with both anal-oral transmission and immunosuppression. these viruses apparently cause acute episodes or relapses of diarrhea in some patients but may be co-pathogens or noncontributory to chronic diarrhea in others. or parasitic etiology. chronic diarrhea in the absence of cryptosporidium or other pathogens has been described as a common presenting feature of aids in africa [2] . hiv has been shown to infect colonic cell lines and has been suggested as the main cause of most idiopathic cases [3] . viruses may be significant gastrointestinal pathogens in other groups of patients with severe t lymphocyte deficiencies, such as bone marrow transplant recipients, although this group has the additional problem of graft-versus-host disease and chemoradiation damage that may involve the gut. yolken et al. [4] and we (0. m., j. h., g. s. g., k. atkinson, and j. c. biggs, unpublished observations) have observed that adenoviruses, coxsackieviruses, rotaviruses, and clostridium difficile toxin are detectable in the feces of marrow transplant recipients and hematology patients receiving chemotherapy. in yolken's study, patients infected with these organisms had significantly increased rates of diarrhea and abdominal cramps. the infections with rotaviruses and adenoviruses occurred at the same time that these viruses were prevalent in the normal pediatric population. the presence of these pathogens was associated with a marked increase in mortality [4] . adenoviruses have also been associated with diarrhea or colitis in patients with aids [5] . we have recently detected sporadic, as well as epi-demic, cases of norwalk-like viruses in adults [6] . immunosuppressed patients (including the marrow transplant recipients) have not been previously examined for these pathogens because of a lack of available antibody specific for norwalk virus. we report here the detection of viruses from the stools of a large proportion of patients with symptomatic hiv infection (aids, arc, and pol) and acute or chronic diarrhea when no other microbial pathogen could be identified. hiv-seronegative and -seropositive homosexual men presenting for hiv antibody screening provided control groups to examine the effects of transmission via anal-oral contact and infection with hiv, with or without significant depression of concentration of circulating cd4 lymphocytes. we studied stool samples from two groups of patients for the presence of viral and other microbial pathogens. group 1. all patients with symptomatic hiv infection (aids, arc, or pol) who presented with acute or chronic diarrhea to the st. vincent's hospital aids service between january 1986and january 1987were entered in the study. diarrhea was defined as more than three fluid stools (i.e., assuming the shape of the container) per day and as chronic if it persisted for more than three weeks. exacerbations of chronic diarrhea were defined as a transient marked increase in stool frequency and volume. fecal specimens were collected early in the course of acute diarrhea, during exacerbations of chronic diarrhea, or on several occasions during persistent chronic diarrhea. the specimens were examined (microbiology laboratory, st. vincent's hospital) by microscopy and culture, using standard methods, for the full range of potentially pathogenic bacteria and parasites occurring in these patients (including salmonella spp., shigella spp., campylobacter jejuni, yersiniaenterocolitica, aeromonas hydrophila, plesiomonas shigelloides, vibrio parahaemolyticus, atypical mycobacteria, cryptosporidium spp., isosopora belli, giardia lamblia, amoebae, strongyloides stercoralis, and c. difficile; toxigenic and enteropathogenic escherichia coli were excluded). c. difficile toxin was detected by a standard cytotoxic assay. group 2 (controls). homosexual men presenting to a sexually transmitted diseases (stds) center 387 for routine examinations for stds and for hiv antibody screening during the same period provided a comparison group. they were all examined by one of us (c. l.). on examination, 7010 of these men had acute diarrhea, and 18% had proctitis. stool specimens were obtained on three separate days over a one-to two-week period from all men in the group. all three specimens were screened for protozoa and helminths at the school of public health and tropical medicine (sydney university). two specimens were cultured for the same range of bacterial pathogens, and the first specimen was examined for viruses. all specimens for virological examination were transferred immediately to the virology department (institute of clinical pathology and medical research, westmead hospital), where they were examined for viruses. fecal specimens were processed for viral diagnosis as follows: a 10% solution was prepared in pbs (ph 7.2), held overnight at 4 c, and clarified by low-speedcentrifugation. aliquots of supernatant were inoculated into cell cultures (primary monkey kidney, hep-2, and human diploid fibroblasts) and also tested for rotavirus antigen by elisa (enzygnost'"; calbiochem-behring, sydney). the elisa-positive samples were later examined by electron microscopy (em) and tested in a blocking elisa (kallestad, shaska, minn) for rotavirus by using goat antibody to rotavirus (kallestad). the remainder of the supernatant was treated with arklone (1 ,1,2-trichlorotrifluoroethane; sigma, st. louis), held for i h at 4 c, and then ultracentrifuged. the pellet was resuspended, negatively stained with phosphotungstic acid, and examined by em. to detect norwalk virus by immune em, we incubated the resuspended pellet, before ultracentrifugation, with antiserum specific for norwalk virus. in the group of 137 homosexual men presenting to the std clinic for hiv antibody screening, the proportions of hi v-seronegative (12of 82) and asymptomatic seropositive men (seven of 43) with virus in their stools was not significantly different (15% vs. lamblia) did not significantly alter these proportions (12.5% vs. 14.3%; 'y} = 0.08). in the hivseropositive group, rotaviruses and adenoviruses were the predominant viruses detected (table 1) , and none of these viruses were associated with acute diarrhea. in the hlv-seronegative group, one of three rotaviruses identified and one of five enteroviruses was associated with acute diarrhea. in the group of 68 hospital patients with diarrhea, 107 stool specimens were examined for viral and other pathogens. twenty-eight patients had one episode of acute diarrhea, six had acute exacerbations of chronic diarrhea, and 34 had persistent chronic diarrhea. as shown in table 2, pathogens were isolated from 75% (51 of 68) of the patients, most commonly from those with arc (cdc class iva) and aids-associated opportunistic infections (aids-oi; t two patients progressed from arc to aids-oi and are included in both classes. (table 3) . almost all of these isolates were obtained from patients with arc or aids-o!. fifty percent of the adenoviruses detected were noncultivable and probably pathogenic. isolation of these strains in graham 293 cells, and subsequent typing, is in progress. none of the cultivable adenoviruses were type 35 [7] . in the hospital group (patients with diarrhea), 11 of the 37 patients with gastrointestinal viruses isolated had dual infections (five with adenovirus and five with rotavirus), whereas these viruses were found much less commonly in the randomly screened group (one of 25; ' 1. 2 = 4.79, p < .05). no seasonal clustering of rotavirus or adenovirus was observed. fifteen of the 26 rotavirus and 8 of the 18 adenovirus (3 noncultivable) isolates were associated with acute diarrheal episodes or exacerbations of chronic diarrhea. the remaining isolates of virus weredetected in patients with persistent chronic diarrhea, often in association with likely primary pathogens such as cryptosporidium or mycobacterium avium-mycobacterium intracellulare (table 2 ). in seven patients from whom three or more serial stool specimens were examined, rotaviruses or adenoviruses were found in two consecutive specimens in three patients. rotavirus was detected in specimens that were obtained 14and 33 d apart, and adenovirus was detected in specimens obtained nine days apart. all the rotavirus-positive stool specimens reported in this study were initially detected by elisa (enzygnost) and later examined by em. the elisa and em results correlated well, with all but three elisapositive stool specimens being confirmed by em. one specimen was em-positive for rotavirus but elisa negative. twenty-threeof the elisa-positive (7) note. for an explanation of cdc classes see table 2. * data are reported as the mean ± sd cd4 + cell concentration/mm" (normal concentration~400/mmj); n = no. of patients. stool specimens were available for retesting in a blocking elisa assay for rotavirus (kallestad). all were confirmed positive. no reoviruses were isolated in cell culture. other common enteric viruses were, however, isolated, as shown in tables i and 3. as reported above, the prevalence of viruses in stools showed a marked increase from asymptomatic to symptomatic hiv-infected patients, and 950/0 of the diarrheal patients with virus in their stools were found in the two most immunosuppressed classes (aids-oi and arc). the mean cd4 + lymphocyte concentrations for patients who did or did not have virus in their stools were not significantly different in any of the groups of patients (table 4). as expected there was a significant difference between the mean cd4 cell concentrations of asymptomatic and symptomatic hiv-seropositive men (t = 2.66; dj = 53, p < .02). persistent, profuse, watery diarrhea; malabsorption; or colitis are the most dramatic gastrointestinal manifestations of aids. most studies of gastrointestinal pathogens in patients with aids have focused on the microbial causes of these syndromes, including cryptosporidium spp., i. belli, m. avium-m. intraeellu/are, and cmv. in addition there are the usual pathogens that also cause diarrhea in hiv-seronegative homosexual men: campy/obaeter, salmonella, shigella, yersinia, giardia, and e. histo/ytiea. the common occurrence of acute diarrhea or exacerbations of chronic diarrhea in symptomatic hiv-seropositive patients and the possible association of this diarrhea with gastrointestinal viral pathogens other than cmv has often been overlooked in the literature, although a recent association between colitis and adenovirus has been reported [5] . in this study we showed that patients with aids or arc may present with acute diarrhea or exacerbations of chronic diarrhea and that in patients with symptomatic hiv infection and diarrhea, >50% excreted gastrointestinal viruses. the majority of these were rotaviruses and adenoviruses. these high detection rates for rotavirus and adenovirus in patients with arc or aids-oi are similar to those observed in marrow transplant recipients who also have a t cell immunodeficiency and often have gastrointestinal mucosal damage from graft-versus-host disease [4] . prolonged diarrhea and fecal excretion of rotavirus for more than six weeks have also been observed in children with t cell immunodeficiency. comparisons of stool isolation rates in hiv-seronegative homosexual men and asymptomatic or symptomatic hiv-seropositive men strongly suggested there were two factors responsible for the high detection rate: (1) life style factors involving transmission by direct or indirect anal-oral contact, a possibility supported by the lack of seasonal variation, and (2) immunosuppression. the latter factor was further supported by the predominance of gastrointestinal viruses (particularly adenoviruses and rotaviruses) in patients with arc or aids-o!. the demonstration of a high detection rate of gastrointestinal viruses (especially rotavirus) in patients with symptomatic hiv infection but no diarrhea and the transient association of rotaviruses and cultivable adenoviruses with other more-certain microbial pathogens in patients with aids-oi or arc and diarrhea suggest that these viruses may often be "passengers." however, the presence of rotaviruses and the usually pathogenic, noncultivable adenoviruses as the only significant microbes in the stools of patients with acute diarrhea (a finding that could not be readily attributed to other causes such as drug toxicity) strongly suggests a pathogenic role. this observation needs to be confirmed in future studies by testing serial stool samples during the course of acute episodes of diarrhea and by excluding other pathogens, including hiv itself, by using intestinal biopsies. the serial studies should also assist in determining the role of dual viral infection (especiallywith rotaviruses and adenoviruses) in the etiology of diarrhea in these patients. malabsorption and mucosal abnormalities of the small intestine in the acquired immunodeficiency syndrome slim disease: a new disease in uganda and its association with htlv-iii infection productive persistent infection of human colorectal cell lines with human immunodeficiency virus infectious gastroenteritis in bonemarrow-transplant recipients cytomegalovirus" colitis: can it be caused by adenovirus [abstract no. th8. 3j? in: program and abstracts of the iii international conferece on aids viral diarrhoea in children in australia molecular epidemiology of adenovirus type 35 infections in immunocompromised hosts chronic rotavirus infection in immunodeficiency intestinal infections in patients with the acquired immunodeficiency syndrome (aids) note added in proof in a recent study of 20 homosexual men with aids and diarrhea and 10 homosexual men with aids but without diarrhea, smith et at. [9] were unable to detect rotavirus antigen in any patients' stools by using elisa (abbott laboratories, north chicago, ill).in contrast, in the year after our study ended (1987), rotavirus continued to be the predominant virus detected in the stools of homosexual men with aids and diarrhea in sydney (21 [18%] of 116 patients). such disparate results suggest marked geographic variations in gastrointestinal viral infections in these patients. key: cord-001401-f29y8vh5 authors: nelson, martha i.; njouom, richard; viboud, cecile; niang, mbayame n. d.; kadjo, hervé; ampofo, william; adebayo, adedeji; tarnagda, zekiba; miller, mark a.; holmes, edward c.; diop, ousmane m. title: multiyear persistence of 2 pandemic a/h1n1 influenza virus lineages in west africa date: 2014-07-01 journal: j infect dis doi: 10.1093/infdis/jiu047 sha: doc_id: 1401 cord_uid: f29y8vh5 our understanding of the global ecology of influenza viruses is impeded by historically low levels of viral surveillance in africa. increased genetic sequencing of african a/h1n1 pandemic influenza viruses during 2009–2013 revealed multiyear persistence of 2 viral lineages within west africa, raising questions about the roles of reduced air traffic and the asynchrony of seasonal influenza epidemics among west african countries in the evolution of independent lineages. the potential for novel influenza virus lineages to evolve within africa warrants intensified influenza surveillance in africa and other understudied areas. our understanding of the global ecology of influenza viruses is impeded by historically low levels of viral surveillance in africa. increased genetic sequencing of african a/h1n1 pandemic influenza viruses during 2009-2013 revealed multiyear persistence of 2 viral lineages within west africa, raising questions about the roles of reduced air traffic and the asynchrony of seasonal influenza epidemics among west african countries in the evolution of independent lineages. the potential for novel influenza virus lineages to evolve within africa warrants intensified influenza surveillance in africa and other understudied areas. keywords. human influenza a virus; pandemic; phylogenetic analysis; africa. despite strong seasonal bottlenecks, influenza a viruses (iavs) persist in humans through continual global migration, which repeatedly reseeds viral diversity on local scales [1, 2] . it has been proposed that southeast asian countries are the most important global sources of antigenically novel iavs, supplying europe, north america, africa, latin america, and oceania with novel variants on a continual basis [3] . subsequent studies added complexity to this model, including roles for north america and other regions in the genesis of novel diversity [4] that are more consistent with a shifting meta-population model [5] . however, the lack of viral sequence data from a number of global regions, including latin america, south asia, and africa, remains a major barrier to understanding the complex global ecology and evolution of iavs. the emergence of novel pandemic a/h1n1 ( ph1n1) viruses in early 2009 resulted in a global expansion in iav sequencing. currently, >8000 full-length sequences of the main antigenic protein, the hemagglutinin (ha), are available through the global initiative on sharing all influenza data (gisaid; www.gisaid.org). notably, influenza surveillance increased in a number of african countries during 2009-2013 in response to the pandemic and as part of a larger trend of increased recognition of the seasonal influenza virus burden in africa (see reviews [6, 7] ). to elucidate the evolution of influenza viruses in africa, we conducted a large-scale phylogenetic analysis of global ph1n1 influenza virus diversity during 2009-2013, including 299 ph1n1 ha sequences collected in 18 african countries. our analysis identified 2 well-supported clades of ph1n1 viruses that each persisted for >1.5 years in west africa, highlighting the need to further understand the ecology and evolution of iavs in this understudied and relatively geographically isolated region. table 1 ; genbank accession numbers kj026367-kj026452). because cameroon shares a long border with nigeria and no other central african countries had viral sequence data available that met the criteria for this study, for simplicity cameroon was considered to be within west africa in this analysis. the entire data set of 8712 ph1n1 ha (h1) sequences was aligned using muscle alignment software (version 3.3.81) [8] , with manual correction. a maximum likelihood (ml) tree of these data was inferred by using raxml software (version 7.2.6) [9] and a general time-reversible (gtr) model of nucleotide substitution with a gamma-distributed (γ) rate variation among sites (data available on request). given the extremely large size of the data set, 100 bootstrap replicates were generated using raxml software, with the same gtr + γ model of nucleotide substitution, to assess the robustness of individual nodes. the resulting phylogeny was visualized and midpoint rooted using figtree software (version 1.4.0; available at: http://tree. bio.ed.ac.uk/software/figtree/), and amino acid substitutions were identified using a customized r script. similarly, an ml phylogeny also was inferred for 7644 full-length (1407 nt) ph1n1 na (n1) sequences that were obtained and analyzed using identical methods (supplementary figure 1) . for the purposes of visualization ( figure 1 ), an ml tree also was inferred from a subset of the ha sequence data (1643 ha sequences) that included all 299 african viruses, 9 viruses identified within west african clades i and ii (described below), and 300 ha sequences randomly sampled from each year during 2009-2013 (all sequences from 2013 were used because <300 sequences were available). the full tree used for figure 1 , including tip labels, is available in supplementary figure 2 . the majority of ph1n1 influenza viruses collected in africa during 2009-2013 (256 of 299; 86%) are phylogenetically interspersed with viruses collected from other continents, as expected given the ability of influenza viruses to rapidly disseminate between continents. however, 43 african ph1n1 viruses (14%) are positioned within 2 phylogenetically distinct clades that comprised viruses collected predominantly in west africa (labeled as west african clades i and ii in figure 1 ). both west african clades i and ii are defined by high bootstrap values (≥80%) and separated by long branch lengths on the ha tree, indicating that the lineages have evolved independently. a monophyletic cluster of 21 kenyan viruses also was identified that contains viruses collected from march 2010 (a/nairobi/ 20/2010) through november 2011 (a/kenya/190/2011), indicative of 20 months of persistence ( figure 1 ). however, this cluster was not supported by high bootstrap values and therefore was not considered in our analysis. both west african clades i and ii are relatively small-15 and 37 isolates, respectively. however, the small size of these clades is more likely to be explained by the sparse sampling of influenza viruses in west africa than by their low prevalence (table 1) . west african clade i comprises14 isolates collected from 3 west african countries (côte d'ivoire, ghana, and senegal) figure 3) . otherwise, the s145t and r276k substitutions were observed globally at extremely low levels, at <1% among the other 8790 pandemic h1 sequences. all 14 west african clade i isolates also clustered together on the na global phylogeny, and 11 of 14 isolates in this cluster also were highly bootstrap supported (85%) (supplementary figure 1) . west african clade ii is larger (37 isolates) and more diverse than clade i, containing 26 isolates from 5 west african countries (burkina faso, cameroon, côte d'ivoire, ghana, and nigeria), 3 from ethiopia in east africa, and 8 from non-african countries in europe (france, italy, norway, and sweden) and the united states (minnesota). within west africa, clade ii has persisted for ≥1.9 (table 1 ). in contrast, the east african isolates were collected during 2012, and all non-african isolates were collected from november 2012 to january 2013. critically, 10 of the 11 non-west african isolates (except for a single outlier from france: a/paris/1878/2012) cluster together within a highly supported subclade (bootstrap, 100%), consistent with 1 migration event of this lineage out of west africa. although the majority of west african isolates from clade ii also cluster together on the na phylogeny ( supplementary figure 1) , the non-west african isolates from ethiopia, europe, and the united states are positioned together in a different part of the na tree, indicative of reassortment. four amino acid substitutions in the h1 sequences were observed among 37/37 clade ii viruses: a15t, n490d, t491k, and v537a ( supplementary figure 3) . the a15t, n490d, and t491k substitutions were observed globally at extremely low levels: <1% among the other 8790 pandemic h1 sequences. a central question in influenza virus epidemiology and evolution is whether viral lineages can persist at low levels of circulation on local and regional scales, or whether new viruses must be reseeded continually from a globally sustained gene pool. in the past 7 years, large-scale phylogenetic analyses of global influenza virus sequence data have provided important insights into viral migration and persistence [1] [2] [3] [4] [5] . the great intensification of iav sequencing that occurred during the 2009 h1n1 pandemic provided the best opportunity to identify viral persistence on local scales, including low-frequency variants. heightened influenza surveillance during new york state's first pandemic wave revealed that a/h3n2 seasonal influenza viruses persisted into the early summer, although this persistent lineage did not give rise to the state's subsequent fall epidemic virus [10] . in the united kingdom, baillie et al [11] detected 2 united kingdom-specific ph1n1 lineages that persisted between the first and second pandemic waves of 2009, although the close timing of the 2 waves meant that the lineages persisted for <6 months in the united kingdom. our analysis of viral evolution in west africa revealed the sustained persistence of 2 ph1n1 clades over a nearly 2-year period, although increased sampling is required to confirm that isolates from other localities are not interspersed within these clades. the intensity of global sampling of pandemic a/h1n1 influenza viruses during this time (>8000 full-length ha sequences were included in this analysis) reduces the likelihood that the persistence observed here is an artifact of unsampled diversity. in fact, the relative sparseness of sampling in west africa compared to other regions (115 of 8712 global sequences, 1.3%) means that our data are strongly biased against the detection of clades comprised solely of west african viruses. that air traffic volume within west africa is lower than in other global regions further supports the plausibility of influenza virus lineage persistence for several years without widespread dissemination to other continents. the delayed appearance of the pandemic h1n1 virus in west africa 6 months after the emergence of the virus in north america in march 2009 further supports the possibility that influenza virus evolution in west africa is less strongly linked to other continents owing to reduced international air traffic [12] . the lack of synchrony among the variable seasonal patterns of geographically linked african countries may also facilitate the persistence of influenza viruses within west africa. in contrast to temperate areas where influenza virus epidemics are strongly synchronized during winter and undergo strong bottlenecks during summertime, the virus may persist through continual migration among seasonally variable and asynchronous west african localities, similar to what is thought to occur within the southeast asian network [3] . in senegal, which has been conducting influenza surveillance for more than a decade, peaks in influenza activity coincide with the rainy season during july through september [13] . nigeria, côte d'ivoire, and cameroon exhibit more variable patterns of influenza virus seasonality, although longer time series of data are required [14, 15] . the phylogenies indicate that both west african clades, clades i and ii, evolved within multiple west african countries, with no evidence of long-term persistence within a single country, which is consistent with a role for viral migration between west african countries in the persistence of the virus. high levels of bootstrap support for a subclade within clade ii that contains isolates from ethiopia, europe, and the united states indicates that clade ii viruses may have disseminated from west africa to east africa and from east africa to europe and the united states around 2012. additionally, the detection of a singleton virus from france within both clade i (a/lyon/ chu/43.28/2010) and clade ii (a/paris/1878/2012) could relate to the frequency of air traffic between francophone west africa and france. the lack of detection of either west african clades i or ii in north or southern africa may also reflect low human mobility between these regions and west africa. further understanding of influenza virus migration within africa and between africa and other continents will require additional sequence data. although the intensity of influenza surveillance in africa still lags behind that of other continents, these findings suggest that substantial viral diversity circulates within africa, including viral lineages that are unique to the region but capable of disseminating to other continents. small sample sizes at country levels necessitate cautious inference of clade prevalence, and it remains unknown whether clades i and ii comprised substantial proportions of the h1n1 diversity in west african countries. the possibility of minor variants evolving locally within west africa undermines the assumption that a vaccine matched to globally dominant lineages will necessarily protect against these local lineages, although more data is clearly required. further knowledge of the viral lineages that circulate within africa, including antigenic characterization, is required to understand the full diversity and global ecology of influenza viruses in humans and to inform vaccination strategies within africa. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org/). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. phylogenetic analysis reveals the global migration of seasonal influenza a viruses the genomic and epidemiological dynamics of human influenza a virus the global circulation of seasonal influenza a (h3n2) viruses global migration dynamics underlie evolution and persistence of human influenza a (h3n2) temporally structured metapopulation dynamics and persistence of influenza a h3n2 virus in humans influenza in africa: uncovering the epidemiology of a long-overlooked disease influenza in africa muscle: multiple sequence alignment with high accuracy and high throughput maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models unseasonal transmission of h3n2 influenza a virus during the swine-origin h1n1 pandemic evolutionary dynamics of local pandemic h1n1/2009 influenza virus lineages revealed by whole-genome analysis delayed 2009 pandemic influenza a virus subtype h1n1 circulation in west africa sentinel surveillance for influenza in senegal influenza viruses in nigeria, 2009-2010: results from the first 17 months of a national influenza sentinel surveillance system spatiotemporal circulation of influenza viruses in 5 african countries during 2008-2009: a collaborative study of the institut pasteur international network acknowledgments. we thank dr workenesh ayele, phd at ethiopian health and nutrition research institute for providing a/h1n1 ha sequences from ethiopia, east africa, for comparison with sequences from west africa.financial support. this work was supported by the multinational influenza seasonal mortality study, an ongoing international collaborative effort to understand influenza epidemiology and evolution, led by the fogarty international center, national institutes of health, with funding from the office of global affairs at the department of health and human services (c. v. and m. i. n.). e. c. h. is supported by an national health and medical research council (nhmrc) australia fellowship.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-261241-eqf6ame6 authors: van beek, josine; veenhoven, reinier h; bruin, jacob p; van boxtel, renée a j; de lange, marit m a; meijer, adam; sanders, elisabeth a m; rots, nynke y; luytjes, willem title: influenza-like illness incidence is not reduced by influenza vaccination in a cohort of older adults, despite effectively reducing laboratory-confirmed influenza virus infections date: 2017-08-15 journal: j infect dis doi: 10.1093/infdis/jix268 sha: doc_id: 261241 cord_uid: eqf6ame6 background: data on the relative contribution of influenza virus and other respiratory pathogens to respiratory infections in community-dwelling older adults (≥60 years) are needed. methods: a prospective observational cohort study was performed in the netherlands during 2 winters. nasopharyngeal and oropharyngeal swabs were collected during influenza-like illness (ili) episodes and from controls. viruses and bacteria were identified by multiplex ligation–dependent probe amplification assay and conventional bacterial culture. results: the ili incidence in the consecutive seasons was 7.2% and 11.6%, and influenza virus caused 18.9% and 34.2% of ili episodes. potential pathogen were detected in 80% of the ili events with influenza virus, coronaviruses, rhinoviruses, human metapneumovirus, respiratory syncytial virus, parainfluenza viruses, and haemophilus influenzae being the most common. influenza vaccination reduced influenza virus infection by 73% (95% confidence interval [ci], 26%–90%) and 51% (95% ci, 7%–74%) in ili patients. however, ili incidence was similar between vaccinated (7.6% and 10.8%) and nonvaccinated (4.2% and 11.4%) participants in 2011–2012 and 2012–2013, respectively (p > .05). conclusions: influenza virus is a frequent pathogen in older adults with ili. vaccination reduces the number of influenza virus infections but not the overall number of ili episodes: other pathogens fill the gap. we suggest the existence of a pool of individuals with high susceptibility to respiratory infections. clinical trials registration: ntr3386. influenza virus causes seasonal epidemics, resulting in 3-5 million severe cases and 250 000-500 000 deaths globally each year [1] . elderly persons, individuals with certain medical conditions and children aged <2 years have the highest risk for complications. vaccination is an important tool to prevent infection and to reduce morbidity and mortality [1] . in the netherlands, individuals aged ≥60 years are offered the annual influenza vaccination. however, public acceptance of vaccination is moderate [2] . vaccine effectiveness (ve) varies per season and depends on age and health of the recipients, and the antigenic match of vaccine strains with circulating strains [3] [4] [5] . furthermore, there is scientific debate about the methodology of determining ve [5, 6] . these discussions reach the media and influence the general opinion on influenza vaccine benefit. moreover, to the public, flu as caused by influenza virus is the same as influenza-like illness (ili) caused by respiratory pathogens, against which influenza vaccination will not protect. consequently, influenza vaccination is perceived to be ineffective and vaccine uptake is reduced. to counter this trend, data on the relative contribution of influenza virus and other respiratory infections to ili in older community-dwelling adults are lacking and needed [7] [8] [9] [10] . this group is underrepresented in the dutch primary care sentinel surveillance system [10, 11] and by definition absent in the dutch sentinel nursing home surveillance network [12] . this is also the case in many other studies worldwide [7] [8] [9] . the aim of this prospective observational study was to determine the relative contribution of influenza virus and other respiratory pathogens to ili in older adults (aged ≥60 years) in 2 consecutive seasons in the netherlands. in addition, influenza ve was estimated in both seasons. upon ili, we determined the presence of potential pathogens in the pharynx of the participant within 72 hours of symptom onset. as a control, we analyzed samples of the same individuals taken after recovery (8 weeks) and in a subset of asymptomatic controls for the same potential pathogens. this prospective observational study was conducted during 2 consecutive influenza seasons (from december 2011 to april 2012 [2011] [2012] and from october 2012 to may 2013 [2012] [2013] ) in the netherlands. adults aged ≥60 years were recruited through their general practitioner or through the civil registry. for the second season, participants were reinvited and additional participants were recruited through the civil registry. we also started earlier to use the same monitoring period as in the dutch sentinel surveillance system [11] . there were no exclusion criteria for the study. influenza vaccination status was recorded (2009) (2010) (2011) (2012) . participants were part of the study for the entire duration of each season and contacted at the end of season to verify participation. written informed consent was obtained from all participants. all trial-related activities were conducted according to good clinical practice, which includes the provisions of the declaration of helsinki. the study was approved by the acknowledged ethical committee metc noord holland (http://www.trialregister.nl; ntr3386). participants were instructed about ili symptoms according to the dutch pel criteria, defined by fever (≥37.8°c) with at least 1 other symptom of headache, myalgia, sore throat, coughing, rhinitis, or chest pain [13] and to report ili as soon as possible after onset. a research nurse performed a home visit within 72 hours of fever onset. during this acute phase, nasopharyngeal and oropharyngeal swabs were obtained and additional information on demographics and comorbidities was recorded. a second visit (recovery phase) was performed 8 weeks (±1 week) later, during which the same samples were collected. if a new ili episode was reported, participants were visited again. in the second season, a control group of asymptomatic participants, equally distributed over the different age groups and season, was sampled and questioned. nasopharyngeal and oropharyngeal samples were obtained with a sterile swab with a flocked nylon tip and stored separately in 1 ml modified liquid amies transport medium (eswab, copan, brescia, italy). samples were transported at room temperature to the laboratory and processed and stored at -80°c within 8 hours after sampling. dna and rna were isolated from 200 µl of both swabs by easy-mag isolation and eluted in 25 µl of buffer (biomérieux, the netherlands). five microliters was used for the detection of a panel of respiratory viruses and bacteria by a real-time polymerase chain reaction (pcr)-based multiplex ligation-dependent probe amplification (mlpa) assay (respifinder smart 22 kit; pathofinder, the netherlands). all analyses were performed on a roche lightcycler 480. mlpa analysis was performed on both swabs separately. a participant was excluded from the analysis and considered missing if either of the swabs or data for a swab were missing. for mlpa data analysis, a participant was considered positive for a target if at least 1 swab of the participant was positive. influenza virus-positive samples were subtyped by real-time reverse-transcription pcr using the roche lightcycler 480 system with slightly modified protocols as described previously [14, 15] . conventional culture of oropharyngeal swabs was performed for streptococcus pneumoniae and haemophilus influenzae according to standard procedures [16] . discrimination of h. influenzae and haemophilus haemolyticus was performed by matrix-assisted laser desorption/ionization-time of flight (maldi-tof) [17] . participants without available oropharyngeal swabs were excluded from analysis and designated as missing. for analysis of the contribution of influenza virus, a sample size of 200 ili cases was estimated to be required. based on an expected ili incidence of 7.5% and a drop-out rate of 5%, a cohort size of 2100 participants was calculated. based on results of the first season, 2500 participants were included in the second season. pearson χ 2 testing and independent samples t test of the means was applied to analyze participant characteristics with spss 19.0 for windows software. a p value ≤.05 was considered significant. the incidence of different viruses or bacteria was calculated as the percentage of swabs positive with the potential pathogen of the number of ili events during the season. attack rates were calculated as percentage of detected pathogens per number of monitored participants. vaccine effectiveness was determined by test-negative design analysis of ili positive participants in the influenza-active period [18] . the analysis is restricted to the period that influenza virus was circulating in the netherlands for that particular season, defined by the national influenza surveillance weekly reports. participants with <14 days between the date of vaccination and the date of home visit were excluded from the ve analysis, as it is uncertain whether the vaccine already had any effect in this period. second and third ili periods were included in the analysis only if an earlier ili period was influenza virus negative. for ili participants, influenza virus positive was considered "case" and influenza negative "control. " the ve is calculated as (1 -odds ratio [or]) × 100% with 95% confidence interval (ci) and is calculated per influenza virus subtype or lineage. the following factors were regarded as potential confounders: period in the season (early and late season), sex, smoking, comorbidity, and age (natural smoothing spline, 4 degrees of freedom). the association between the potential confounders and influenza virus positivity (any subtype) was analyzed with univariate logistic regression. variables with p < .20 were considered in the multivariable analysis. variables that changed the or by at least 5% are included in the final multivariable logistic regression model for any influenza subtype (backward selection). all analyses were performed with sas version 9.4 software. sensitivity analyses were performed in the ve analyses for other control groups (supplementary materials). in this prospective study, we observed an ili incidence of 7.2% (143/1992) and 11.6% (275/2368) in 2 consecutive seasons (2011-2012 and 2012-2013, respectively) ( figure 1a ; table 1 ). the average age of vaccinated individuals was significantly higher than that of unvaccinated individuals (respectively, 70.4 vs 66.9 years in 2011-2012 and 71.9 vs 67.9 years in 2012-2013; table 2 ). the asymptomatic controls from the second season were older and more often vaccinated compared with the overall cohort ( figure 1b ; table 2 ). furthermore, participants who reported comorbidities were vaccinated significantly more often than participants who did not report comorbidities (table 3) . no significant differences were found between individuals with or without ili with respect to sex, age, and chronic illnesses (tables 1 and 3) . importantly, no differences were found in the incidence of ili episodes between vaccinated and unvaccinated participants (table 1) . in 79.1% and 78.0% of the acute ili samples from the 2 seasons, at least 1 potential pathogen could be identified by mlpa or bacterial culture ( in 16.2% and 17.0% of the samples from the acute phase, >1 potential pathogen was detected, but no specific combinations of viruses and/or bacteria were observed (data not shown). in recovery samples, 8 weeks after acute ili, potential pathogens were detected in 27.0% and 24.8% of cases in 2011-2012 and 2012-2013, respectively. in asymptomatic control samples, similar potential pathogens were observed as in recovery samples (21.5%) ( figure 2c ). in 2011-2012, influenza virus was detected in 18.9% of the acute ili samples, predominantly of the a(h3n2) subtype (96.3%) ( figure 3 ; supplementary table 1a) . influenza virus was not detected in the corresponding recovery samples in this season, suggesting that influenza virus was the actual cause of ili. in 2012-2013, influenza virus was detected in 34.2% of the acute ili samples, and all 4 circulating subtypes were detected: 43.6% a(h3n2), 25.5% a(h1n1)pdm09, 25.5% b/yamagata lineage, and 5.3% b/victoria lineage. in addition, influenza virus was detected at a very low level in both the recovery samples and in samples of asymptomatic controls (1.1% and 0.9%, respectively). we investigated which other viruses and bacteria were detectable during ili episodes. in 60.8% (2011-2012) and 44.7% (2012-2013) of ili samples, potential pathogens other than influenza virus were detected (figure 3 ; supplementary table 1 ). coronaviruses of all 4 common human subtypes (18.2% in 2011-2012 and 11.3% in 2012-2013), human metapneumovirus (hmpv) (20.3% and 3.6%), rhinoviruses (8.4% and 21.1%), respiratory syncytial virus (rsv) (4.9% and 6.5%), and parainfluenza viruses (2.8% and 5.1%) were detected in >5% of the ili samples in at least 1 season. hmpv and rhinovirus varied most between the 2 seasons. all viruses were detectable at low levels in recovery and control samples. rarely, the same virus was observed during both the ili event and the recovery sampling, except for rhinoviruses, which were detected frequently at both visits (8.3% in 2011-2012 and 17.2% in 2012-2013), but as our test only detected rhinovirus in general, we cannot exclude that these are different serotypes. the attack rate of influenza virus was significantly higher in the second season compared to the first season (p < .0001; table 4 ). interestingly, the increased attack rate could be attributed to significant increases in the attack rates of h1n1, the b/victoria-like subtype, and the b/yamagata-like subtype (p < .0001, p = .04, and p < .0001, respectively), whereas the attack rate of the h3n2 subtype was not significantly different between these seasons. for the other viruses, we observed significant increased attack rates in the second season for rhinovirus (p < .0001) and parainfluenza viruses (p = .04), whereas hmpv attack rates were significantly lower in the second season (p = .0004). the other viruses had similar attack rates during the 2 seasons. the only bacterial species detected by conventional culture in a significant number of acute ili cases was h. influenzae (15.4% in 2011-2012 and 11.3% in 2012-2013), frequently as the sole pathogen, while presence of other bacteria such as h. haemolyticus and s. pneumoniae was low. haemophilus influenzae was also detected frequently in recovery and control samples. we evaluated whether influenza vaccination reduced the overall influenza virus infection incidence and whether this influenced the incidence of ili. although influenza virus lost to follow-up n=20 no ili reported n=2240 per protocol no ili reported n=2108 a subject could have multiple ili episodes per season. an ili visit (v1) was considered "out of window" if the sample was taken >72 hours after start of fever. for the recovery visit (v2), the window was 7-9 weeks after ili onset. subjects were considered lost to follow-up if they did not respond to the end of study mailing and had no ili visit (a). after the baseline visit had been performed, a subject could have an ili event. subjects were considered lost to follow-up if they did not respond to the end of study mailing and had no ili visit (b). infection incidence was significantly lower in influenza vaccinated than in unvaccinated individuals (table 5) , the incidence of ili cases was not reduced by vaccination (table 1) . among participants with ili, we observed a high ve of 73% (95% ci, 26%-90%) in 2011-2012 and a moderate ve of 51% (95% ci, 7%-74%) in 2012-2013 against influenza virus during the influenza-active period (table 6 ). furthermore, the ve for the predominant influenza virus subtype a(h3n2) in 2011-2012 was 71% (95% ci, 19%-90%). in 2012-2013, the ve against influenza virus type a(h3n2) was 67% (95% ci, 20%-86%). additional sensitivity analyses for multiple ilis, households with ili, and the presence or absence of other virus infections did not affect this conclusion (supplementary table 2 ). in this study in a cohort of community-dwelling older adults in the netherlands, we show that influenza virus was present in 18.9% and 34.2% of ili cases in 2 consecutive seasons and that influenza vaccination significantly reduced laboratory-confirmed influenza virus infection. in 60.8% and 44.7% of the acute ili cases, potential pathogens other than influenza virus were detected. in addition, these pathogens were more often present during ili than after recovery or in asymptomatic elderly persons. in 20% of the ili cases, no potential pathogen was detected. the incidence of ili cases was expected to decrease by a reduction in influenza virus-caused ili through vaccination; however, this effect was not observed. instead, the incidence of c individuals in the asymptomatic subset were selected to be evenly distributed over the different age groups; therefore, the overall vaccination level was higher in the asymptomatic subset compared to the ili and non-ili groups. ili remained the same between the vaccinated and nonvaccinated individuals. influenza vaccination is offered to individuals 60 years and older in the netherlands, as older adults are at increased risk for morbidity and mortality from influenza virus infections due to an aging immune system and age-related chronic illnesses [19, 20] . however, this policy is based on studies mostly performed in elderly persons in nursing homes, who are substantially older and more frail compared with community-dwelling elderly individuals. studies in community-dwelling older adults are scarce [7] [8] [9] [10] , and only a restricted number of pathogens have been analyzed in these surveillance studies. to fill the gap in knowledge, we recruited participants through general practitioners and the civil registry, resulting in >99% of the participants living in the community. in line with the risk-based vaccination strategy in the western world, older participants and participants with chronic conditions were more likely to be vaccinated [2] . the age distribution in the cohort was similar to that observed for those ≥60 years in the general dutch population; only the oldest age group (>80 years) was underrepresented (table 2) . these elderly persons are often less likely to participate in studies. "healthy user effect" or "frailty selection bias" is a well-known issue in observational influenza vaccine studies, as the very frail elderly persons are difficult to reach [21, 22] . we found that influenza virus is involved in 18.9%-34.2% of ili cases in the 2 seasons studied. in addition, coronaviruses, rhinoviruses, hmpv, rsv, parainfluenza viruses, and h. influenzae were frequently observed as the sole detected pathogen in acute ili cases, whereas they were low to absent in most of the recovery samples. however, rhinoviruses and the bacterium h. influenzae were also commonly detected in asymptomatic controls. the rhinoviruses may be of different subtypes, as we did not type these viruses. viruses can often be detected in individuals without clinical manifestations [7] , but when they are found as the sole agent in the context of disease, they are commonly considered to be the cause of the disease. for bacteria, this is less clear as they are commonly carried in asymptomatic persons but may expand during respiratory viral infection or new acquisition. haemophilus influenzae may induce an enhanced inflammatory state in the presence of other pathogens, as was shown in children [23] . this may lead to ili. however, in most ili cases where we found h. influenzae, we did not find a second pathogen that could also explain the ili symptoms. influenza virus incidence in our cohort broadly matched the incidence reported in the dutch sentinel surveillance system [11] , reported a high ili incidence and mortality in the 2011-2012 season [24, 25] . this underlines a potential difference between the generally healthy community-dwelling elderly persons and that of the generally more frail institutionalized elderly persons, who may overlap in age. it also shows that data acquired in one group do not necessarily apply to the other, including exposure and susceptibility to infection, as well as influenza ve. we show that the incidence of influenza virus infection was reduced in individuals who received influenza vaccination and that ve to laboratory-confirmed influenza virus infection was high to moderate in the 2 seasons. the 95% cis of the data are wide, probably due to the relatively small sample size, a common problem in similar studies. ve estimates are notoriously variable between studies [18], but the ve data from this study are in line with those reported by van der hoek et al [18] . the most striking finding in this study was the similar incidence in ili cases observed between vaccinated and the nonvaccinated individuals. this may be explained by assuming that a pool of people exists that is highly susceptible to respiratory infections. the reduction by vaccination in the number of cases caused by influenza virus infections is offset by a rise in the number of cases caused by infections by other pathogens. cowling et al have described a similar increased risk of noninfluenza respiratory virus infection in influenza-vaccinated children [26] . however, we cannot attribute this effect to a specific pathogen. influenza virus may take preference over other viruses and prevent them from filling the niche, possibly by inducing a prolonged antiviral state, as has been described for other viruses [23, 27] . when vaccination reduces influenza virus infections, the other pathogens can fill the gap. it needs to be confirmed in other studies whether influenza virus vaccination has no effect on the total number of ili cases. it would have important consequences for decisions on implementation of vaccination for community-dwelling older adults when looking at the overall disease burden and cost-effectiveness. however, a limitation of our study is that we did not directly monitor the duration and severity of disease in the ili cases: difference in disease or hospitalization was only registered in post-ili questionnaires, and no significant events were reported. more detailed data on severity and duration of symptoms would allow assessing the relative risk posed by different pathogens, taking into account that influenza virus, unlike most other potential pathogens, can vary in pathogenicity between seasons. in combination with other medical information, it may then be possible to assemble a profile of individuals potentially at risk for ili or worse. such a profile would be of great value to public health professionals. a limitation of this study is the definition of ili used, which differs between the world health organization, the european centre for disease prevention and control, and different countries, although most include fever and cough [28] [29] [30] . in this study we used the dutch pel criteria [13] , which includes fever. it has been described that this specific ili definition can affect the number and type of pathogens detected. falsey et al showed that fever is more frequently associated with influenza virus infection in the elderly persons compared with other respiratory infections [7] . cough is not a prerequisite in the pel criteria, but as >80% of the participants with ili and >90% of the participants with influenza virus-positive ili reported coughing (data not shown), it is unlikely that cases were missed due to this difference in definition. in summary, we show that influenza virus caused between 18.9% and 34.2% of ili cases in community-dwelling older adults aged ≥60 years in 2 influenza seasons in the netherlands, leaving the remainder caused by other pathogens. we also show that influenza vaccination was effective in reducing the incidence of influenza virus infections but did not reduce the ili incidence, which may have important public health and healthcare consequences. our data will also help to better for more details, see supplementary table 3 . abbreviations: ci, confidence interval; ve, vaccine effectiveness. a corrected for the possible confounders age group, comorbidity, sex, and smoking. data were calculated for the influenza-active period in the netherlands as defined by the netherlands institute for health services research [11] . inform the public what to expect from influenza vaccination and how it will not protect against all cases of ili, popularly seen as "flu. " supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. notes influenza (seasonal) social determinants of health and seasonal influenza vaccination in adults ≥65 years: a systematic review of qualitative and quantitative data effectiveness of seasonal influenza vaccine in community-dwelling elderly people: a meta-analysis of test-negative design case-control studies the efficacy of influenza vaccination in elderly individuals. a randomized double-blind placebo-controlled trial vaccines for preventing influenza in the elderly cochrane re-arranged: support for policies to vaccinate elderly people against influenza respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden influenza vaccine effectiveness among elderly persons living in the community during the 2003-2004 season a case-control study of acute respiratory tract infection in general practice patients in the netherlands nivel influenza surveillance sentinel surveillance network on infectious diseases in nursing homes study group. absence of influenza a(h1n1) during seasonal and pandemic seasons in a sentinel nursing home surveillance network in the netherlands proefonderzoek naar de frequentie en de aetiologie van griepachtige ziekten in de winter 1963-1964 preparing the outbreak assistance laboratory network in the netherlands for the detection of the influenza virus a(h1n1) variant differentiation of influenza b virus lineages yamagata and victoria by real-time pcr effect of 7-valent pneumococcal conjugate vaccine on nasopharyngeal carriage with haemophilus influenzae and moraxella catarrhalis in a randomized controlled trial identification of haemophilus influenzae and haemophilus haemolyticus by matrix-assisted laser desorption ionization-time of flight mass spectrometry letter to the editor: influenza vaccine effectiveness: heterogeneity in estimates for the 2012/13 season frailty, inflammation, and immunity vaccines for the twenty-first century society mortality benefits of influenza vaccination in elderly people: an ongoing controversy effectiveness of influenza vaccine in aging and older adults: comprehensive analysis of the evidence dutch rsv neonatal network. respiratory syncytial virus and recurrent wheeze in healthy preterm infants excess mortality among the elderly in 12 european countries increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine does viral interference affect spread of influenza? world health organization. ili sari surveillance case definition overview of influenza surveillance in the united states european centre for disease prevention and control. influenza case definitions we gratefully acknowledge all participants for their time and commitment to the study. we thank the study staff at the spaarne hospital and the laboratory staff members at the regional laboratory kennermerland and the centre for infectious disease control at the national institute for public health and the environment.financial support. this work was supported by the dutch ministry of health, welfare and sport.potential conflicts of interest. e. a. s. has received research grants from pfizer and gsk, in addition to fees paid to wilhemina children's hospital/university medical center for advisory boards and participation in independent data monitoring committees for pfizer and gsk. r. v. has received research support from gsk and pfizer for vaccine studies and consulting fees for gsk. all other authors report no potential conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-285527-1mceq6v0 authors: kinloch, natalie n; ritchie, gordon; brumme, chanson j; dong, winnie; dong, weiyan; lawson, tanya; jones, r brad; montaner, julio s g; leung, victor; romney, marc g; stefanovic, aleksandra; matic, nancy; lowe, christopher f; brumme, zabrina l title: suboptimal biological sampling as a probable cause of false-negative covid-19 diagnostic test results date: 2020-06-28 journal: j infect dis doi: 10.1093/infdis/jiaa370 sha: doc_id: 285527 cord_uid: 1mceq6v0 false-negative sars-cov-2 test results can negatively impact the clinical and public health response to covid-19. we used droplet digital pcr (ddpcr) to demonstrate that human dna levels, a stable molecular marker of sampling quality, were significantly lower in samples from 40 confirmed or suspected covid-19 cases that yielded negative diagnostic test results (i.e. suspected false-negative test results) compared to a representative pool of 87 specimens submitted for covid-19 testing. our results support suboptimal biological sampling as a contributor to false-negative covid-19 test results and underscore the importance of proper training and technique in the collection of nasopharyngeal specimens. a c c e p t e d m a n u s c r i p t accurate covid-19 diagnosis is critical to a successful clinical and public health response. current covid-19 tests detect one or more targets in the sars-cov-2 rna genome, usually by real-time reverse transcriptase (rt)-pcr, and nasopharyngeal swabs have been the preferred sample for testing to date [1] . while pcr-based tests are highly sensitive, falsenegative covid-19 test results do occur [2, 3] , though reported rates vary. a recent large retrospective study estimated the clinical sensitivity of sars-cov-2 molecular assays to be between 58% and 96% [4] , while another reported a 67% sars-cov-2 rna detectability rate in respiratory samples taken within 7 days of hospitalization for covid-19 [5] . various factors other than molecular technology contribute to test sensitivity, including the timing of sample collection with respect to infection stage [6, 7] as well as specimen storage and transport [2] . improper specimen collection could also contribute to false-negative covid-19 test results. although nasopharyngeal swabs are routinely ordered for respiratory viruses, the collection of a high quality specimen requires training and expertise as it involves insertion of the swab to posterior nasopharynx, a depth of roughly 7cm, followed by rotation and withdrawal of the swab [8] . to investigate suboptimal sample collection as a possible cause of false-negative test results, we quantified human dna levels recovered on nasopharyngeal swabs submitted to a single laboratory for covid-19 testing, hypothesizing that human dna could serve as a stable molecular marker of specimen collection quality. the st. paul's hospital (sph) virology laboratory is one of five provincially designated sars-cov-2 diagnostic laboratories in british columbia, canada. covid-19 testing on a c c e p t e d m a n u s c r i p t nasopharyngeal swabs (copan utm® collection kit or bd universal viral transport system) was performed by total nucleic acid extraction from 500μl medium on the roche magna pure 96 followed by real-time reverse-transcriptase (rt)-pcr using the roche lightmix® 2019-ncov real-time rt-pcr assay, which uses e-sarbeco primers/probes [9] , or using the roche cobas® sars-cov-2 test. between march to may 2020 we identified 40 suspected false-negative nasopharyngeal swab test results from presumed or confirmed covid-19 cases for which >1ml medium remained for re-testing. these included 23 negative samples from individuals who recorded a positive test within ±12 days of the negative test (where the median time elapsed between negative and positive tests was 4 days, with an interquartile range of 1-6 days) and 17 samples from individuals who tested negative but for whom there was high clinical suspicion of infection by the treating physician with no alternate diagnosis established. a convenience sample of 87 consecutively submitted nasopharyngeal swabs served as a comparison dataset. remnant specimens were stored at -20°c until re-testing. to standardize nucleic acid extraction across all specimens and to maximize viral rna recovery, 1ml of medium was extracted on the biomérieux easymag and eluted in 35μl buffer. sars-cov-2 detection in suspect falsenegative samples was re-attempted using a nested rt-pcr and sequencing protocol targeting conserved regions in orf-1a and spike [10] , where the lower limit of detection of this assay was estimated in-house using serial dilutions of synthetic sars-cov-2 rna standards (exact diagnostics). human dna levels were quantified using droplet digital pcr (ddpcr), a technique where each sample is fractionated into 20,000 nanolitre-sized water-in-oil droplets prior to pcr amplification with sequence-specific primers and fluorescent probes, and where poisson detection sensitivity compared to the original real-time rt-pcr assay, we re-tested the 40 suspected false-negative specimens by nested rt-pcr. all suspect false-negative samples however again tested negative. this indicated that the original negative results were not likely attributable to suboptimal real-time rt-pcr assay performance, but rather suggested that sars-cov-2 rna was exceedingly low or absent in these samples. we then investigated whether human dna recovered on the nasopharyngeal swab could serve as a molecular marker of specimen collection quality, reasoning that dna (by virtue of its stability) would be well-preserved in remnant clinical specimens. we employed a sensitive, multiplexed ddpcr protocol for absolute human rpp30 gene copy number quantification [12] . overall, we observed significantly lower human dna levels in the suspected false-negative nasopharyngeal swab samples compared to a panel of consecutive samples submitted for testing during the same period, though overlap between groups was still substantial (figure 1, p<0.001) . specifically, suspected false negative specimens harbored a median 3,409 (interquartile range we chose human dna as a molecular marker of sampling quality because of its stability. rnasep rna-specific primer/probe set, in part to assess sample quality [11] . to investigate the relationship between human cells as measured by rpp30 gene copy number using ddpcr, and our results underscore the importance of proper training and technique in the collection of high quality nasopharyngeal specimens. they also highlight the potential utility of including a molecular marker of sampling quality in sars-cov-2 diagnostic assays that could serve as an endogenous control. while the major commercial assays (e.g. roche cobas® sars-cov-2; a c c e p t e d m a n u s c r i p t https://www.fda.gov/media/136049) include an internal rna control for nucleic acid extraction and rt-pcr amplification, these do not provide a measure of biological sampling quality. while the us-cdc 2019-ncov real-time rt-pcr diagnostic panel does feature a human rnasep rna-specific primer/probe set, in part to assess sample quality [11] , our findings suggest that the interpretation criteria for this control may be too liberal. specifically, the us-cdc's instructions for use, issued on 15-mar-2020, state that failure to detect rnasep within 40 pcr cycles can indicate insufficient biological material in the sample or other assay problems. in our retrospective test panel of 91 remnant nasopharyngeal nucleic acid extracts however, the 90th percentile ct value for rnasep was 25.9 (range 19.65 to 27.77; see results). the observation that even the lowest decile of samples in terms of rnasep rna levels (possibly representing those for which sampling was the least robust) still amplified well before ct<40 suggests that this threshold may be insufficient to identify suboptimally-collected samples. some limitations of our study merit mention. our use of a convenience sample of 87 consecutively submitted nasopharyngeal swabs may not represent an ideal control group, as there is no guarantee that these samples were collected using appropriate or consistent technique. indeed, the wide range of human dna levels observed in this group, and the substantial overlap with the suspected false negative group, corroborate this notion. this limitation however should only serve to reduce our study's statistical power. moreover, approximately 40% of our suspected false negative tests derived from patients with high clinical suspicion of sars-cov-2 infection but whose diagnosis was never confirmed. as diagnoses may not have been made in a consistent manner across treating physicians, these samples may be less likely to represent false negative results. however, our observation that human dna levels in both subcategories of the false-negative group were significantly lower than in the comparison group suggests that this a c c e p t e d m a n u s c r i p t limitation may be minimal. it is also important to note that our study was not designed to identify a threshold of human dna (or rna) that could define a properly-collected sars-cov-2 nasopharyngeal swab, and that future studies attempting to do so would need to consider that recovery efficiency (and thus total yield) of different types of nucleic acid may differ by extraction platform (e.g. human dna levels recovered in the present study differed between biomérieux and magna pure platforms, see results), and possibly by swab type. it is also important to note that, although human dna levels can serve as a surrogate marker of the amount of biological material collected, sampling the correct anatomical location is also critical, particularly for nasopharyngeal swabs. our observations strongly support suboptimal biological sampling, but not pcr sensitivity for sars-cov-2 rna detection, as a contributing cause of false-negative covid-19 test results. m a n u s c r i p t a c c e p t e d m a n u s c r i p t human dna levels (rpp30 gene target) were measured using ddpcr in nasopharyngeal extracts as a molecular marker of biological sampling quality. "suspect false-negatives" included 23 negative samples from individuals who recorded a positive test within ±12 days of the negative test (grey) and 17 samples from individuals with high clinical suspicion of being infected but never molecularly confirmed (white). the comparison dataset was a consecutive set of 87 samples submitted for testing in april 2020 to the same laboratory (black). p-values report the significance level between the comparison dataset and the "suspect false-negative" group as a whole (black), between the comparison dataset and the negative samples from individuals who reported a positive test within ±12 days (grey) and between the comparison dataset and the negative samples from individuals with high clinical suspicion (white). a c c e p t e d m a n u s c r i p t figure 1 interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease false negatives and reinfections: the challenges of sars-cov-2 rt-pcr testing false negative tests for sars-cov-2 infection -challenges and implications clinical performance of sars-cov-2 molecular testing antibody responses to sars-cov-2 in patients of novel coronavirus disease virological assessment of hospitalized patients with covid-2019 variation in false-negative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure how to obtain a nasopharyngeal swab specimen detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr detection of second case of 2019-ncov infection in japan cdc 2019-novel coronavirus (2019-ncov) real-time rt-pcr diagnostic panel instructions for use a quantitative approach for measuring the reservoir of latent hiv-1 proviruses we thank dr. christopher sherlock for helpful discussions. we thank the laboratory teams at the a c c e p t e d m a n u s c r i p t key: cord-007288-lzxi6q1p authors: pazin, george j.; harger, james h.; armstrong, john a.; breinig, mary k.; caplan, richard j.; cantell, karl; ho, monto title: leukocyte interferon for treating first episodes of genital herpes in women date: 1987-12-17 journal: j infect dis doi: 10.1093/infdis/156.6.891 sha: doc_id: 7288 cord_uid: lzxi6q1p women experiencing their first episodes of genital herpes were treated, beginning within three days of the onset of lesions, with 5 × 10(4) units of human leukocyte interferon/kg of body weight for 12 doses over 14 days (total, ∼3.6 × 10(7) units) or with placebo in equivalent volumes. life-table analysis revealed quicker healing and significant reductions in the duration of shedding of virus in interferon-treated patients. maximum daily geometric mean titers of virus and total area of unhealed lesions also decreased more quickly. no statistically significant difference in resolution of pain was seen between the two groups. interferon had no effect on onset or frequency of subsequent recurrences recorded over one year of follow-up. moderate, transient neutropenia occurred in 13 of 34 interferon-treated patients. a therapeutic effect of human leukocyte interferon on initial genital herpes was documented, but the clinical usefulness of interferon treatment of genital herpes is limited at this time. since its discovery in 1957, human leukocyte interferon has been shown to have potent antiviral, immunomodulating, and antiproliferative effects. the biologic significance and therapeutic potential of interferon, however, remain uncertain despite demonstration of clinical benefits in controlled studies of the common cold [1] [2] [3] [4] , herpes zoster [5] , and condyloma acuminatum [6, 7] . in particular, the role of interferon in relation to herpesvirus infections continues to interest researchers and clinicians [8, 9] . interferon has significantly increased survival of experimental animals infected with herpes simplex virus type 2 (hsv-2), a result suggesting that interferon may have an effect on the virus's invading the cns and on its establishing latency [10] . in humans, interferon administered before and after microvascular decompression of the trigeminal sensory root has reduced the frequency and severity of postsurgical reactivations of herpes simplex virus type 1 (hsv-1} [11] , but administration either before or after the procedure had no beneficial effect [12] . on the basis of these studies, we hypothesized that early treatment with interferon during the initial episode of genital herpes might ameliorate the severity of the initial episode and might also prevent the establishment or affect the extent of latent infection with herpes simplex virus (hsv) in sacral ganglia. we undertook this placebo-controlled, double-blind study to test these hypotheses. we clinically and virologically assessed the effect of early treatmentwith leukocyte interferon (cantellvariety) [13] on the initial episode of' genital herpes.weindirectly evaluated the effect of .interferon on latency by determining the incidence and frequency of both asymptomatic reactivations and symptomatic recurrences during an intensive one-year follow-up period. recruitment and selection ofpatients. women experiencing their first episodes of genital herpes were referred to us by physicians and clinics informed about the study. eligible patients had had lesions for less than 72 hr; no prior history of genital herpes; a negative pregnancy test (urine chorionic gonadotropin test and confirmatory serum assay); no major cardiac, renal, or pulmonary disease; no personal, emotional, or professional factors that could be expected to interfere with the course of treatment or follow-up; no psychiatric or addictive disorders that could preclude informed consent; a leukocyte count of~5,000/mm3,a platelet count of 100,000/mm3,and a hemoglobin level of~12 g/dl. treatment and follow-up. patients were randomly assigned to receive interferon or placebo. neither the clinical personnel nor the patient knew which group she was in. on the day of enrollment, the patient received two doses of interferon (5 x 10 4 u/kg im) or an equivalent volume of 4.5 mg of human serum albumin/ml. on the second through the eighth day and on days 10, 12, and 14, single doses were given. the total amount of interferon received over 14 days was 6 x 10 5 u/kg. this was equivalent to 3 x 10 6 u/dose or a total of 3.6 x 10 7 u for a 60-kg subject. patients were examined daily for eight days then every other day until the lesions were healed. at these visits, specimens for cultures of virus were obtained during speculum examination of the cervix and vagina and from least-healed vulvar lesions. complete blood counts were performed on days 1, 4, 7, 10, and 14, and tests of liver and renal function were done on days 1, 7, and 14. blood was drawn 1 hr after treatment on days 2, 7, 10, and 14 to determine interferon levels. samples for hsv serology were obtained at enrollment and on day 28 or 35. to document the frequency and severity of recurrences or to detect asymptomatic reactivations, one of the study staff examined and collected specimens for culture of hsv from each patient every two weeks for one year. when this was not possible, we maintained contact with the patients by telephone, letter, or both. culture and typing of virus. specimens for isolation of virus were taken with sterile cotton swabs, placed in 1.5 ml of hbss with 0.5070 gelatin, and stored frozen at -70 c. throat-wash specimens were obtained by having patients gargle with 10 ml of hbss and gelatin. gentamicin sulfate (100 ug/ml) and amphotericin b (2.5 ug/ml) were added to throat washes before they were stored at -70 c. swab cultures were taken from suspected lesions and from pazin et at. the vulva, vagina, and cervix. a "sweep" culture was also made by sweeping the swab outward from the cervix, to contact both the vagina and vulva. cultures of secondary baby-rabbit kidney cells in sixwell plastic dishes were used to isolate hsv. undiluted swab specimens (0.2 ml in each of two wells) and throat-wash specimens (0.3 ml in each of two wells) and 10-fold dilutions through 10-3 (0.1 ml/well) of these specimens were inoculated into the wells of these dishes. cultures were examined daily for cpe and were held for seven days. at first, isolates of virus were typed by plaque formation on cultures of chick embryo cells; more recently, commercially available type-specific monoclonal antibodies (syva, palo alto, calif) were used. in our hands, this method correlated precisely with determination of hsv type by restriction endonuclease analysis of viral dna. serology. antibody to hsv was measured by using a plaque-reduction assay. the neutralizing titer was defined as the dilution of the test serum reducing plaque number by 50070. the assay was carried out in monolayers of vero cells. hsv strains f and g, provided by dr. bernard roizman (university of chicago, chicago) were used as the standard strains of hsv-1 and hsv-2, respectively. because hsvneutralizing antibody is not type specific, the higher titer, usually that obtained with hsv-1 (strain f), was accepted as the titer of the tested specimen. any titer~1:4 was considered positive. interferon. partially purified human leukocyte interferon (ifn-a) was prepared in helsinki as previously described [13] and was transported and stored frozen at -70 c. the interferon preparation contained 4.5 mg of protein/ml, and its specific activity was 1.33 x 10 6 u/mg of protein. interferon was titrated by using a semi-micro method [14] with human foreskin fibroblasts and vesicular stomatitis virus to assay its antiviral activity. titers are expressed as 10g1o u/unit volume, estimated by comparison with a laboratory standard that in turn was calibrated with respect to the ifn-a international standard 69/19. distribution and characteristics of the subjects. sixty-nine women were enrolled, but five subjects were excluded from analysis because of lack of virological confirmation of diagnosis. one subject was seronegative throughout follow-up, and 365 146 219 figure 1 . duration of follow-up of the study population. the number of days of follow-up was determined by using the last day of clinically useful patient contact; 365 days was the number used for patients followed up for more than a year. tions accounted for 37.50/0 of the cases. one subject who initially shed hsv-l also shed hsv-2 beginning 10 days after enrollment. two others who initially shed hsv-l had subsequent episodes in which hsv-2 was isolated (see below). there was a slight imbalance in number between the experimental group (34 patients) and the placebo group (30 patients). all of this imbalance was accounted for in the hsv-l subgroup, in which 14 subjects received interferon and 10 received placebo. the duration of follow-upis shown in figure 1 . although a few subjects dropped out during or immediately after the treatment period,800j0 were followed up for atleast 230 days, and 500/0 were followed up for at least 360 days. the area under the curve in figure 1 represents the fraction ( rv800/0) of the total theoretical follow-up time achieved. interferon levels. patients who received interferon showed a rise in antiviral activity in serum equivalent to 85 vlml of serum in the specimen taken 1 hr after the dose was given on day 2. the levels fluctuated between 60 and 70 vlml during daily therapy and decreased to 30-50u/ml during alternateday therapy. the minor baseline levels «10 v) of antiviral activity present before treatment in patients receiving placebo remained unchanged throughout the course of treatment. adverse effects. total leukocyte counts were similar and were within normal limits at enrollment for both experimental and control groups. absolute lymphocyte counts in the two groups varied little. counts of segmented and banded pmnls did not change significantly in the placebo group, but we observed a progressive decrease in these values in the interferon-treated patients during the daily treatment phase; a modest recovery occurred during the period no. of patients receiving hsv was never isolated. hsv was not isolated at any time from four other subjects who were seropositive for hsv on enrollment. sixty-two of the qualified subjects were enrolled in pittsburgh and the remaining two in rochester, ny. the mean ages for the experimental group and the placebo group were 25.8 years (range, 18-40 years) and 24.9 years (range, 17-38 years), respectively. the group receiving interferon included two black subjects; there was one black patient in the placebo group. all others were white. the subjects were classified by disease type, infecting virus, and treatment (table 1). disease was classified as "primary" or as "initial, not primary" (np). primary disease was defined as disease occurring in a subject who had neutralizing antibody titers to hsv <1 :4 on the day of enrollment. they represented 750/0 of our study group (table 1).the remaining 250/0 (16 subjects) had titers of neutralizing anti-body~1:4 to at least one hsv type and were classified as having np disease. twoof these patients who had titers of antibody to hsv-l of only 1:4 and no detectable antibody to hsv-2 may also have had true primary disease produced by hsv-2. although np disease was less frequent in patients from whom hsv-l was isolated (170/0) than in those with hsv-2 (300/0), this difference was not statistically significant. the distribution of patients by type of virus isolated is also shown in of alternate-day therapy. these decreases were reflected in corresponding decreases in totalleukocyte counts. thirteen interferon-treated patients had transient reductions in pmnl counts to 1,000imm 3 ; one patient had a single count <500imm 3 • treatment was cautiously continued with carefulobservation of all patients, with their awareness and consent. no patient receiving placebo had a pmnl count <1,000imm 3 • interferon-treated patients also showed a reduction in platelet counts during daily treatment and normalization during alternate-day therapy. no platelet count <100,000/mm 3 was observed, and only six patients had counts transiently <150,000imm 3 • platelet counts increased progressively throughout the treatment period for placebo-treated patients; no counts <150,000 were observed. no significant changes were detected in tests of hepatic or renal function, except for a minor increase in serum aspartate aminotransferase levels (soot) among interferon-treated patients at day 7; levels returned to normal by day 14, the last day of treatment. the initial episode. the effects of interferon treatment on the initial episode of genital herpes are shown in figures 2-5. our previous study [11] on the effect of interferon on the reactivation of oral herpes virus showed that of the disease parameters studied, shedding of virus was the one most sensitive to interferon. the duration of shedding of virus in the present study is shown in figure 2 . patients whose lesions healed before they stopped shedding virus were not followed up to the cessation of shedding. therefore, we used life-table analysis to make the best use of our censored observations (le., data from patients still shedding hsv at the last visit associated with the initial episode of herpes). half of the group receiving interferon had stopped shedding hsv from a genital site (vulva, cervix, vagina, or any combination of the three) by day 10 (median duration of shedding); half of the group receivingplacebo was still shedding virus on day 14. from day 6 of treatment until day 16, the day after the end of treatment, the cumulative p value associated with the mantel-haenzel statistics is <.05, a result confirming the impression given by figure 2 that the duration of shedding was shortened while interferon was being given. thereafter, the rates are no longer different; after the last observation, on day 32, the adjusted global mantel-haenzel statistic is not significant. by that time, however, the percentage of subjects shedding virus was <20070 in both groups. in 770/0 of subjects, the vulva was the last site from which virus was isolated. because cultures were taken on each day at different sites, some of which had no lesions, we decided to calculate geometric mean titers for the groups from the maximum titers obtained on each day for individual patients. as shown in figure 3 , the mean of the maximum hsv titer was reduced by f\j10-fold (90070) in the group receiving interferon during the latter part of the treatment period. this inhibition is highly significant (p< .001). after day 14,the end of the treatment period, no further inhibition was seen. the mean of the titers of virus was, however, by that time low in both groups. to assess the effect of treatment on lesions, we estimated the area of each lesion of the vulva and vestibular mucosa at each examination until the scab came off or until complete re-epithelialization occurred. although cervical lesions were often present, their area could not be reliably measured. the sum of the areas of the measurable lesions was used to calculate the mean of the areas for the two groups ( figure 4 ). the striking difference (which is not, however,statistically significant) seen between treatment groups is partly due to two subjects in the placebo group who had very extensive lesions. after treatment ended on day 14, the group receiving interferon actually had a slightly larger mean lesion area than did the placebo group; however, only small lesions were still present at this time in most subjects. life-table analysis of the duration of lesions, or time to healing, is shown in figure 5 . lesions were considered healed when the scab was gone or when an ulcer re-epithelialized, even if some residual erythema remained. half of the interferon-treated patients were healed by 16 days (median time to healing); it took 22 days for half of the subjects receiving placebo to heal. because we observed much variation in time to healing, the difference in the survival curves was statistically significant only in the period from 18 to 20 days (p < .05). pain during the episode was similarly examined by life-table analysis (data not shown). the mean of the last days on which patients reported pain was plotted for each group. although the mean duration of paininthe group receiving interferon was consistentlytwodays shorter than that for the placebo group.the difference was not significant, pain was quite a variable parameter: some subjects never ex-perienceddiscbffif()rt severe enough for them to describe it as pain, whereas others suffered pain for more than four weeks. the mean duration of pain for all patients with hsv-l infection was 11.5 days and was similar (12.1 days) for patients with hsv-2. systemic manifestations were not reduced by interferon treatment. in fact myalgia, malaise, and fever were slightly more frequent in the group treated with interferon. subjects receiving placebo had myalgia for an average of 4.3 days, malaise for an average of 4.6 days, and fever for an average of 0.9 days; the group receiving interferon averaged 4.8, 5.3, and 1.2 days for the three symptoms, respectively. these differences were not statistically significant. most, but not all, subjects took an analgesic/antipyretic, usually acetaminophen, as desired; therefore no conclusions should be drawn from these data. overall, interferon treatment at rv3 x 10 6 u/day had an ameliorative effect on both shedding of virus and the time to healing of initial episodes of genital herpes, but had no significant effect on the associated pain. recurrences. two aspects of the effect of treatment on the frequency of subsequent recurrences have been analyzed. the first of these was the time to the first recurrence after initial disease. this parameter was examined by life-table methods, as shown in figure 6 . the small differences in time to recurrence were not statistically significant. it was assumed in these analyses that all episodes of hsv disease seen during the follow-up period were recurrences (reactivations) of the initial infection, rather than reinfections. in fact, two subjects who first presented with hsv-l infections later had episodes of disease during which hsv-2 was isolated. we considered these subjects to be lost to followup, for the purpose of measuring recurrence rates, at the time of the new infection. we assumed that their episodes of hsv-2 disease were due to reinfection and were not recurrences of the initial infection. one other subject was not analyzed for recurrences sponse to interferon. the overall frequency of recurrences is low in subjects infected with hsv-l (table 2) , and there were only four np episodes of genital herpes among our subjects (table 1) . therefore, only subjects with hsv-2 infection can be analyzed. the mean time to first recurrence was 80 days for primary disease and 72 days for np disease. this difference is not significant; interferon treatment did not result in significant changes. the mean rate of recurrences was 5.7/year for all primary hsv-2 infections and 4.6/year for the np episodes. no treatment effects were seen. because, after presenting with hsv-l, she started shedding both hsv-l and hsv-2later in the course of her initial disease. the second parameter used was the frequency of recurrence during the year of follow-up. because not all subjects could be followed up for a full year (see figure 1 ), a rate (number of recurrences/duration of follow-up in years) was calculated for the subjects followed up for at least four weeks (table 2) . ouf data are consistent with the well-known fact that hsv-2 genital infections reactivate more frequently than do hsv-l infections [15] . because virus type is the major source of variability in frequency of recurrence, the data were analyzed by two-way analysis of variance using the method of rao [16] for groups of unequal size. as expected, the analysis showed that hsv-l and hsv-2 differ significantly in reactivation rates (p < .025), but the small reductions in rates seen for the groups treated with interferon were not significant. the frequency of recurrence may partly depend on whether the first episode of disease was primary; it is possible that this parameter might affect rethis double-blind, placebo-controlled study was undertaken to clarify the therapeutic and preventive potential of treating first episodes of genital herpes with interferon. besides assessing the effects of interferon treatment on the clinical and virological aspects of the initial episode, we evaluated the effect of interferon on preventing or reducing subsequent asymptomatic reactivations or symptomatic recurrences. these issues are important both biologically and clinically. our patients were heterogeneous in that, as a group, they had both primary and initial, nonprimary episodes of disease caused by either type 1 or 2 of hsv. these different entities were, however, fairly well distributed in the treatment and placebo groups. without stratification into the above-mentioned disease categories, leukocyte interferon, totaling~3.6 x 10 7 u over 14 days, exerted a moderately beneficial effect upon the natural course of initial genitalherpes. survival-curve analysis revealed statistically significant decreases in duration of positive cultures of virus and in time to healing for the group receiving interferon. at the respective midpoints of survival-curve analysis, duration of positive cultures was reduced by four days and time to healing was reduced by six days. although titers of virus were nearly identical for the two groups at enrollment, they decreased more rapidly in patients receiving interferon. progression of disease, as measured by total area of unhealed lesions at each visit, was substantially reduced in interferon-treated patients. no significant reduction in pain was observed, however. preventing or reducing the latent activity of the virus was another objective of this study. we hypothesized that interferon might prevent or affect the extent of latency if it were administered early. we therefore set stringent requirements for admission to this study that made it difficult to complete. enrollment within three days of the development of lesions was a strict requirement. as a result, two patients, approximately, were excluded for each patient enrolled. willingness and availability to be followed up by visits at two-week intervals for one year was an additional criterion for admission. the adequacy of our follow-up is shown in figure 1 . ourdata clearly indicate that interferon did not prevent establishment of latency. most patients infected with hsv-2 experienced recurrences regardless of their treatment; neither time to first recurrence nor number of recurrences was reduced. markedly fewer recurrences due to hsv-l were, however, observed in both treatment groups, an observation agreeing with previously reported studies of the natural history of genital herpes [15] , but recurrences of hsv-2 were also not significantly reduced. apparently, the incubation period of genital herpes is long enough that the latent state is firmly established by hsv before it can be appreciably affected by interferon. careful monitoring for toxicity revealed that 13 interferon-treated patients developed transient reductions «1,000/mm 3 ) in levels ofneutrophilic granulocytes during treatment. no patient experienced untoward effects during the transient neutropenia, and treatment was not discontinued in anyone. modest reductions in platelet counts to <150,000/mm 3 were observed during daily treatment with interferon in six patients, but counts returned to normal during alternate-day therapy. except for minor elevations of soot on day 7 of therapy, interferon-treated patients did not show abnormalities of hepatic or renal function. thus, the dose of interferon was reasonably well tolerated and was suitable for ambulatory therapy. recombinant interferon has been used to treat initial and recurrent episodes of genital herpes [9] and as suppressive therapy in patients with frequent recurrences [8] . in the former study, no significant benefits were shown during treatment of initial episodes, but this result could be due to the fact that treatment was instituted later than in our study, that treatment groups were smaller, or that men and women were analyzed separately. an initial five-day treatment period was followed by "maintenance" treatment three times per week for three months to prevent or treat recurrences. during maintenance prevention of natural colds by contact prophylaxis with intranasal alpha--interferon intranasal interferon as protection against experimental respiratory coronavirus infections in volunteers intranasal interferon-a prophylaxis of natural respiratory virus infection intranasal interferon-a2b for seasonal prophylaxis of respiratory infection human leukocyte interferon for the treatment of herpes zoster in patients with cancer intralesional recombinant alpha-2 intereron for the treatment of patients with condyloma acuminatum or verruca plantaris interferon therapy for condylomata acuminata suppression of recurrent genital herpes simplex virus infection with recombinant a2 interferon effect of recombinant interferon a2 on clinical course of first episode genital herpes infection and subsequent recurrences effect of treatment with exogenous interferon, polyriboinosinicpolyribocytidylic acid or polyriboinosinic-polyribocytidylic acid-poly-t-lysine complex on herpesvirus hominis infections in mice prevention of reactivated herpes simplex infection by human leukocyte interferon after operation on the trigeminal root dummer ls, 1annetta pl. paradoxical effects of interferon on reactivation of oral infection with herpes simplex virus after microvascular decompression for trigeminal neuralgia preparation of human leukocyte interferon for clinical use semi-micro, dye binding assay for rabbit interferon risk of recurrence after first episodes of genital herpes. relation to hsv type and antibody response linear statisticalinference and its applications intravenous acyclovir for the treatment of primary genital herpes treatment of first episodes of genital herpes simplex virus infection with oral acyclovir: a randomized double-blind controlled trial in normal subjects double-blind placebo-controlled trial of oral acyclovir in first-episode genital herpes simplex virus infection key: cord-007255-jmjolo9p authors: pulliam, juliet r. c.; dushoff, jonathan title: ability to replicate in the cytoplasm predicts zoonotic transmission of livestock viruses date: 2009-02-15 journal: j infect dis doi: 10.1086/596510 sha: doc_id: 7255 cord_uid: jmjolo9p understanding viral factors that promote cross-species transmission is important for evaluating the risk of zoonotic emergence. weconstructed a database of viruses of domestic artiodactyls and examined the correlation between traits linked in the literature to cross-species transmission and the ability of viruses to infect humans. among these traits-genomic material, genome segmentation, and replication without nuclear entry-the last is the strongest predictor of cross-species transmission. this finding highlights nuclear entry as a barrier to transmission and suggests that the ability to complete replication in the cytoplasm may prove to be a useful indicator of the threat of cross-species transmission. previous studies have compared emerging human pathogens to nonemerging human pathogens and looked for characteristics typical of those considered to be emerging [1] [2] [3] . to ask which characteristics predict host jumps requires a different approach. specifically, we must examine the pool of other hosts' pathogens that a target species regularly encounters. from this pool we can compare the characteristics of microbes that are able to infect the target host versus those that manifest no evidence of an ability to infect the target host. molecular characteristics that facilitate cross-species transmission are likely to be substantially different between viruses, bacteria, and protozoa, because of large differences in the pathobiology of these different taxa. here, we focus on cross-species transmission of viral infections and examine the effects of 3 characteristics that are described in the literature as expected to affect the ability of a viral group to infect a novel host species: genome segmentation, genomic material, and site of replication. the ability to rapidly explore genetic state space is expected to increase the probability of a host jump, so we expect that viruses with rna genomes will have a higher probability of jumping than viruses with dna genomes [1, 4] and that viruses with segmented genomes will have a higher probability of jumping than viruses with nonsegmented genomes [4] . complex interactions with a host's cellular machinery, on the other hand, are expected to decrease the probability of a host jump, so we expect that viruses that are able to complete replication in the cytoplasm will have a higher probability of jumping than viruses requiring nuclear entry [5] . to examine the effects of these characteristics, we should choose a target species that will maximize the chance that viral infection due to cross-species transmission events will have been detected; the obvious choice is humans. likewise, we should minimize differences in exposure of the target host to infectious virions produced by the source hosts. humans have regular contact with all potentially infectious bodily fluids of domestic food animals; we thus ensure that the target species has contact with all viral groups infecting the source hosts by analyzing the pool of viral species known to infect sheep, goats, cattle, and pigs. methods. we constructed a database containing taxonomic and molecular data on known viruses of domestic artiodactyls. to determine which viruses to include in the database, we searched the primary literature for references documenting infection of these species with all recognized species in all viral genera known to infect mammals. for each viral species infecting sheep, goats, pigs, or cattle, we then searched the literature to determine whether human infections have been documented (see table a1 in appendix a, which appears only in the electronic edition of the journal). viruses dependent on coinfection with other viral species, known to be maintained through continuous transmission within humans (see appendix a), or for which documented instances of artiodactyl infection resulted from human-to-animal transmission or experimental infection were excluded from the database. all literature searches were performed between 10 january 2007 and 15 february 2007 using web of science. the database contains information on the 3 molecular characteristics hypothesized to influence the potential of a virus to cross host species: site of replication (x sr ; whether replication is completed in the cytoplasm or requires nuclear entry), genomic material (x gm ; rna or dna), and segmentation of the viral genome (x seg ; segmented or nonsegmented). these characteristics are conserved at the family level, and classifications were made on the basis of standard reference books [6, 7] . we used a combination of hypothesis testing and modelbased prediction to analyze the database. hypothesis testing allowed us to determine how likely it was that the observed patterns were due to chance, whereas model-based prediction allowed us to determine what trait or set of traits was the best predictor of a livestock virus's ability to infect humans and to estimate the probability that a particular virus species would be able to jump host species, given knowledge of the traits of interest. computer code and data are available at http://lalashan .mcmaster.ca/hostjumps/ or from the authors. to determine the statistical significance of the effect of each trait on zoonotic transmission independent of the 2 other traits of interest, we performed a series of randomization tests. for a particular trait, we held the values of the other 2 traits and the ability of the viral species in the database to infect humans constant and permuted the values of the trait of interest within each subset defined by the other 2 traits (thereby preserving the full cross-correlational structure of the data with regard to the 3 viral traits) 100,000 times. the p value was given by the proportion of permutations that allowed the model to predict outcomes as well as or better than the model that was constructed using the observed data, and an ␣ level of 5% was used to determine the statistical significance of results. we compared model fit by use of a logistic regression model that predicted the ability to infect humans as a function of replication site, genomic material, and segmentation. the logistic model was fit in the r statistics package [8] , and fits were compared on the basis of likelihood. because the 3 traits examined are conserved at the family level for all species in our database, treating species as independent may bias our results. we therefore repeated our analysis at the genus and family levels. permutations of the data set were constructed by permuting the values of the trait under consideration at the taxonomic level examined and assigning species within a genus (or family) the corresponding trait value after permutation. p values were calculated as in the species-level analysis. to examine the magnitude and relative importance of the effects that the 3 molecular characteristics of interest have on the ability of the viral species in the database to infect humans, we developed a set of logistic regression models. each model included some combination of viral traits as independent variables and the ability to infect humans as the dependent variable. traits not having a statistically significant effect on the ability of livestock viruses to infect humans were still considered for modelbased predictions, because sample sizes were limited and small-but real-effects may not be detected via hypothesis testing. we estimated parameter values for each model in r and compared models using akaike's information criterion adjusted for small sample size (aic c ) [9] . results. a total of 146 viral species were found to infect the livestock species of interest and meet other criteria for inclusion in the database. of these, 141 species (representing 59 genera in 22 families) fulfilled the criteria for inclusion in the analysis. the effect of site of replication was significant at all 3 taxonomic levels examined (p ͻ .001, p ϭ .018, and p ϭ .014 for the species, genus, and family levels, respectively), with nearly half of the virus species completing replication in the cytoplasm able to infect humans. neither genomic material nor segmentation showed a significant effect on the ability of livestock viruses to infect humans at any taxonomic level. logistic regression model comparisons are summarized in table 1. the models are given in order as ranked by aic c . figure 1 compares the observed data with the results of the best model. the best model included site of replication as the only variable (odds ratio, 17.4 [95% confidence interval, 3.98 -75.8), and the top 4 models were the 4 that included site of replication. each of these models showed a positive correlation between replication in the cytoplasm and the ability to infect humans, as expected. segmentation appears in models 2, 4, 6 and 7, and all 4 models showed a positive correlation between having a segmented genome and the ability to infect humans. genomic material appears in models 3, 4, 5, and 6. again, all 4 models showed a correlation in the expected direction. it is interesting to note that both of the viral species that caused major pandemics in humans in the 20th century (hiv and influenza virus a) require nuclear entry for replication. because influenza virus a infects domestic artiodactyls but was excluded from our database because it is maintained through continuous transmission in humans, we confirmed the robustness of our results to this exclusion; we also confirmed that our findings were robust to the inclusion of viral species for which human infection data were based solely on serology (see table b1 in appendix b, which appears only in the electronic edition of the journal). discussion. our analyses indicate that viral species infecting domestic artiodactyls are more likely to infect humans if they complete replication in the cytoplasm without nuclear entry. the observed effect of cytoplasmic replication on host-jumping ability is not surprising given the complex molecular pathways regulating nuclear entry. viral species that are unable to complete replication in the cytoplasm require intracellular transport from the site of penetration, targeting of the nucleus through nuclear localization signals, and importation of genetic material, proteins, and/or whole virions through the nuclear pore complex [10] . the combination of molecular mechanisms governing this chain of events is likely to be highly host specific, because of strong selective pressure against admission of foreign particles into the nucleus. to date, discussion of barriers to viral replica-tion has largely focused on receptors for cellular entry. the concentration on this aspect of the viral life cycle exists for 2 substantive reasons. first, the inability to enter a cell obviously precludes viral replication; second, several well-documented viral host jumps have been shown to occur after point mutations that modify interactions between viral particles and cellular receptors [11] [12] [13] . the effect of nuclear entry seen in our data set emphasizes that cellular entry, while a necessary step, is insufficient for completion of the viral life cycle. the ability to produce genetic diversity is the factor most widely discussed as expected to increase viral host-jumping ability [1, [3] [4] [5] 14] . although the observed effects of genomic matenote. x sr , x gm , and x seg are variables indicating the molecular characteristics of a viral species (see methods). ln(ᐉ ) is the log likelihood of the best-fit parameter combination for a given model. k is the no. of model parameters for a given model. aic c is the value of akaike's information criterion with small sample size correction for each model; thus, ⌬aic c is the difference in aic c value between a given model and the best model (i.e., the model with the lowest aic c value). w i is the akaike weight of the model. ␤ seg , ␤ gm , and ␤ sr are regression coefficients for genome segmentation, genomic material, and site of replication, respectively. ␤ i represents the estimated intercept for the best-fit parameter combination for each model. rial and segmentation were not statistically significant, our data do not necessarily contradict this expectation. the hypothesized effect of segmentation, in particular, may be obscured in our data set by a combination of the small number of viral species with segmented genomes and the absence of segmented dna viruses. on the other hand, the lack of predictive power associated with genomic material and segmentation in our data set may indicate that consideration of these traits alone is insufficient to capture the potential to generate useful genetic diversity. the degree to which the pool of viruses infecting domestic artiodactyls is typical of all potentially zoonotic viral species is uncertain. other pools of viral species should be examined to determine the generality of our results. similarly, further studies should examine whether the observed patterns hold for crossspecies transmission of viruses to other target host species, including wildlife and domestic animals. given the rapid rates at which ecological relationships between species are changing as a result of anthropogenic landscape changes, global warming, and globalization of both human and animal populations, the development of indicators of the risk of cross-species pathogen transmission is an increasingly important goal. as humans, domestic animals, and wildlife are brought into contact with species from which they were formerly isolated, they inevitably encounter the pathogens that these species carry. the finding that the ability to complete replication in the cytoplasm is the best predictor of zoonotic transmission and that nearly half of domestic artiodactyl viruses that are able to complete replication in the cytoplasm can infect humans suggests that cytoplasmic replication will be a useful indicator of the ability of a newly encountered virus species to jump hosts, an essential prerequisite to epidemic or pandemic emergence [15] . it should be noted, however, that the present analysis focused exclusively on the ability to infect the target host, and the viral traits influencing this step in the emergence process may differ from those that predispose a virus to cause severe disease in a novel host as well as from those that facilitate transmission within a novel host species. diseases of humans and their domestic mammals: pathogen characteristics, host range, and the risk of emergence risk factors for human disease emergence host range and emerging and reemerging infectious diseases evolvability of emerging viruses viral host jumps: moving toward a predictive framework virus taxonomy: eighth report of the international committee on taxonomy of viruses the springer index of viruses r: a language and environment for statistical computing. vienna: r foundation for statistical computing in: model selection and multimodel inference: a practical information-theoretic approach viral entry into the nucleus the natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor structure of sars coronavirus spike receptor-binding domain complexed with receptor a single amino acid in the pb2 gene of influenza a virus is a determinant of host range molecular constraints to interspecies transmission of viral pathogens origins of major human infectious diseases key: cord-007237-8y7218oj authors: manning, ashleigh; willey, samantha j.; bell, jeanne e.; simmonds, peter title: comparison of tissue distribution, persistence, and molecular epidemiology of parvovirus b19 and novel human parvoviruses parv4 and human bocavirus date: 2007-05-01 journal: j infect dis doi: 10.1086/513280 sha: doc_id: 7237 cord_uid: 8y7218oj background. parv4 and human bocavirus (hbov) are newly discovered human parvoviruses with poorly understood epidemiologies and disease associations. we investigated the frequencies of persistence, tissue distribution, and influence of immunosuppression on replication of these viruses. methods. at autopsy, bone marrow, lymphoid tissue, and brain tissue from human immunodeficiency virus (hiv)—infected individuals with acquired immunodeficiency syndrome (aids) and those without aids and from hiv-uninfected individuals were screened for parvovirus b19, parv4, and hbov dna by means of quantitative polymerase chain reaction analyses. results. b19 dna was detected both in hiv-infected study subjects (13 of 24) and in hiv-uninfected study subjects (8 of 8), whereas parv4 dna was detected only in hiv-infected study subjects (17 of 24). hbov dna was not detected in any study subjects. the degree of immunosuppression with hiv infection did not influence b19 or parv4 viral loads. b19 or parv4 plasma viremia was not detected in any study subjects (n = 76; viral load <25 dna copies/ml). a significantly older age distribution was found for study subjects infected with b19 genotype 2, compared with those infected with b19 genotype 1. two genotypes of parv4 were detected; study subjects carrying prototype parv4 (genotype 1) were younger (all born after 1958) than those infected with genotype 2 (parv5; study subjects born between 1949 and 1956). conclusions. tight immune control of replication of b19 and parv4 was retained despite profound immunosuppression. recent genotype replacement of parv4, combined with absent sequence diversity among genotype 1 sequences, suggests a recent, epidemic spread in the united kingdom, potentially through transmission routes shared by hiv. ruses (aavs) in the dependovirus genus. recently, 2 additional human parvoviruses have been discovered [1, 2] . parv4 was detected in a large-scale screening of exogenous dna and rna sequences in a panel of blood samples from individuals with acute infections of undiagnosed etiology [1] . parv4 does not resemble any other known mammalian parvoviruses and probably will be classified as the sole member of a new parvovirus genus. at the same time, a second novel human parvovirus was discovered in pooled respiratory samples [2] . the virus has been named "human bocavirus" (hbov), since it is most closely related to 2 other parvoviruses, bovine parvovirus 1 and a minute canine virus in the bocavirus genus (i.e., bovine/canine) of parvoviridae. hbov infections have been found to be widely distributed among young children worldwide [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] , with growing evidence for an etiological role in severe lower respiratory tract infections. in contrast, detection of parv4 infections to date has been confined to the original infected study subject [1] and to occasional pooled plasma samples used for blood-product manufacturing [13] . thus, the prevalence, epidemiology, transmission routes, and clinical associations of parv4 remain undetermined. on the basis of the observation of relatively short-lived viremia and the development of a strong immune response that conferred protection from reinfection, b19 infections have been considered to be acute and spontaneously resolving. however, more recent evidence from tissue biopsy specimens and autopsy samples has indicated frequent persistent infection in previously exposed individuals [14] [15] [16] [17] [18] [19] , even though infection is controlled effectively by the immune system to the extent that persistent viremia is infrequent and has an invariably low titer among seropositive individuals [20] [21] [22] . the combination of long-term persistence and immunity from reinfection has been shown to create a so-called bioportfolio in which b19 and possibly other persistent dna viruses, such as aavs, that are encountered early in life may be preserved lifelong, thus constituting a record of virus variants that had circulated several decades previously [19] . in the current study, we examined a series of samples of different tissues, collected at autopsy, for the presence of hbov and parv4 dna sequences and compared the frequencies of detection, viral loads, and genetic variability with those for b19. samples from hiv-infected individuals who did not have aids (hereafter referred to as "pre-aids study subjects") and those with terminal aids at time of death were compared with corresponding samples from hiv-uninfected individuals, to investigate the relationship between viral load and immunosuppression and, thus, the role of the immune system in controlling virus replication. bone marrow, lymphoid tissue, and brain tissue were examined as part of an initial investigation of the cellular tropism of these different parvoviruses in vivo. the autopsy tissue samples used in this project were obtained from the edinburgh medical research council hiv brain and tissue bank at western general hospital, edinburgh. consent for use of postmortem tissue was obtained from the lothian research ethics committee. for the studies of viral load in autopsy tissue, lymphoid (lymph node or spleen), brain (frontal or occipital lobe), and bone marrow tissue samples were obtained from 13 hiv-infected individuals with aids at time of death, 11 hiv-infected individuals without aids, and 8 hiv-uninfected individuals (table 1). samples were stored at ϫ80њc for use in subsequent polymerase chain reaction (pcr) analyses. all but 2 of the study subjects with aids were untreated with antiretroviral drugs before death. all pre-aids study subjects had a cd4 count of у200 cells/ml at the time of death, except for 1 study subject with a cd4 count of 167 cells/ml. all presymptomatic study subjects died from aids-unrelated causes [23] . plasma samples were obtained from a total of 36 previously untreated hiv-1-infected individuals at varying stages of disease progression, which was reflected in their cd4 lymphocyte counts (table 1). plasma samples were obtained from 40 low-risk hiv-negative individuals who were approximately age matched to the hiv-positive individuals; samples were collected over the same period of time (2003) (2004) (2005) . dna extraction. tissue samples were extracted as described elsewhere [24] . extracted dna was quantified by spectrophotometry at 260/280 nm. a maximum of 0.5 mg dna was assayed by pcr. dna was extracted from 0.4-ml volumes of plasma by use of the qiaamp dna blood minikit (qiagen). extracted dna was resuspended in 50 ml elution buffer, and 5 ml was assayed by pcr. amplification of parvovirus b19, hbov, and parv4 dna sequences. nested-pcr methods were used for amplification of b19, hbov, and parv4 dna. b19 primers were based on a highly conserved region in the ns gene, with the following sequences (position of the 5 base in b19-au sequence m13178 in parentheses): outer sense, gtgtatcagcaatttgtrs-aatt (2478); inner sense, tytgtttgacttagttgctcg (2867); inner antisense, actggrtgataaggcatgggg (2984); and outer antisense, taagtcttcactagataatac (3038). for screening hbov and parv4 dna, we used previously described primer sequences from the ns gene [12] . a shared amplification protocol for each target was used [12] . nested pcrs for the 3 virus targets were sensitive for single copies of the target sequence [12] , enabling use of limiting dilution [24] for quantitation of positive samples. for the autopsy samples, initial screening was performed by use of 0.5 mg dna from each sample, and positive samples were titrated further in 5-fold dilution steps until limiting dilution was observed in 4 or 8 replicate reactions; viral loads then were calculated from the frequencies of positive reactions at limiting dilution [25] and were expressed as copies per million cells. plasma samples were screened for b19 and parv4 sequences by means of pcr using 5 ml of the extracted dna (40 ml of the original sample). based on single-copy detection [12] , the assay had a theoretical sensitivity of 25 dna copies/ml of plasma. to rule out the presence of impurities in extracted dna that could reduce pcr sensitivity, 1-mg quantities of extracted dna from 15 different autopsy tissues and 3-5-ml volumes of 8 different plasma samples were amplified in reactions spiked with 10 copies of b19 or parv4 dna. no inhibition of amplification was detected in any sample, when compared with the 10-copy amplification controls. nucleotide sequencing. parv4-positive samples were sequenced in the region amplified by the screening primers, by use of the big dye terminator kit (applied biosystems). because the b19 amplicon obtained from screening was short, sequencing used the following alternative set of primers in the ns gene: outer sense, gugguaaraaaaayacmcugugg (1638); inner sense, suguuccaguruauggcaugg (1716); inner antisense, aaguaaugucaccauugcugg (1943); and outer antisense, guaascccaugucagggcugcauc (2048). all samples positive with the screening primers were able to be amplified with the sequencing primers. accession numbers. the nucleotide sequences of b19 and parv4 obtained in this study have been submitted to gen-bank and have been assigned accession numbers ef371379-ef371417. detection of parvoviruses in autopsy tissue. dna samples extracted from lymphoid tissue (lymph node or spleen, depending on availability), bone marrow, and samples of brain tissue (cerebral cortex) were screened for parvovirus dna by means of nested pcr using sets of b19-, hbov-, and parv4specific primers (table 2) . b19 dna was frequently detected in study subjects from all 3 categories (hiv negative, pre-aids, and aids), whereas parv4 dna was detected only in those study subjects with hiv coinfection (7 of 11 pre-aids study subjects and 10 of 13 study subjects with aids). in contrast to the frequent detection of b19 and parv sequences, hbov dna was not detected in any study subjects. because of the previously described association of hbov with respiratory infections, we also assayed autopsy lung samples from 3 study subjects with aids, but pcr analysis showed that all samples were negative for hbov dna (data not shown). samples positive for b19 and parv4 dna were quantified by limiting-dilution pcr (figure 1). viral loads varied widely within each tissue-sample type and subject category, ranging from undetectable (!6 dna copies/10 6 cells) to several samples with 11000 copies of b19 or parv4/10 6 cells. when data from all 3 subject categories were combined, the highest b19 viral loads were found in bone marrow samples, with a median level of 582 dna copies/10 6 cells versus 92 dna copies/10 6 cells in lymphoid tissue ( , mann-whitney nonparametric u p p .07 test) and 69 dna copies/10 6 cells in brain tissue ( ) p p .006 ( figure 1a ). among the hiv-infected study subjects, a similar difference was found in parv4 viral loads in bone marrow versus lymphoid tissue (median values of 220 and 46 dna copies/10 6 cells, respectively; ) (figure 1b). p p .0005 neither b19 nor parv4 showed an association between increased viral load and immunosuppression (figure 1). for b19, median viral loads for hiv-uninfected individuals had a range of values similar to that for the pre-aids and aids subject categories (e.g., median viral load in lymphoid tissue from hivuninfected individuals was 203 dna copies/10 6 cells, compared with 120 and 46 dna copies/10 6 cells for pre-aids study subjects and those with aids, respectively [ ]). among hiv-p 1 .05 infected study subjects, a correlation between cd4 lymphocyte count and b19 viral loads was not found in any of the tissues examined, as determined by means of the nonparametric spearman's rank correlation test ( to .04). using previously r p ϫ.01 determined hiv viral loads for the subjects in this study group [23, 26] , we also found no association between the extent of b19 infection and the extent of hiv infection in any of the tissues examined ( to .20). r p ϫ. 22 although parv4 was found only in the lymphoid tissue and bone marrow of hiv-infected individuals, there was no correlation between immunosuppression and parv4 viral load. viral loads were similar in pre-aids study subjects and those with aids ( figure 1b) (table 2) . the individuals in these study groups were demographically similar to the hiv-positive study subjects from whom autopsy samples were obtained and also showed a range of cd4 lymphocyte counts that substantially overlapped with that of the latter group (table 1). given the likelihood that a substantial proportion of the tested individuals were persistently infected with b19 and parv4, negative findings from analysis of their plasma samples indicate that persistent infection with either virus is not associated with frequent viremia. b19 and parv4 genotypes. amplified dna from samples positive for both b19 and parv4 was sequenced to identify which genotypes infected the study subjects (figure 2). among hiv-infected and -uninfected study subjects, sequences of b19 genotypes 1 and 2 were detected (12 [60%] and 8 [40%] study subjects, respectively), with a significantly different age distribution between genotypes (figure 2a). the median year of birth for genotype 1-infected study subjects was significantly later than that for genotype 2-infected study subjects (1963-1964 vs. 1955, respectively; ). genotype 2 sequences differed p p .004 from genotype 1 sequences by 9.5% in the region sequenced (positions 1737-1922 in the ns region of the b19-au prototype sequence) and showed a highly constrained pattern of variability; all substitutions between and within genotypes were confined to synonymous sites (i.e., non-amino acid changing). the pattern of sequence variability among parv4 sequences was remarkably similar to that of the b19 sequences. two variants of parv4 were detected and differed from each other by 13% in the region sequenced (positions 1455-1569 in the complete parv genome sequence nc_007018); all substitutions again were confined to synonymous sites. fourteen of the sequences were identical to nc_007018 (referred to as "genotype 1"), and the remaining 4 (provisionally termed "genotype 2") showed greater intragroup variability (mean pairwise distance of 3.1%). genotype 2 infections were found only in those born before 1956 (median year of birth was 1953, compared with 1964 for those with genotype 1 infections [ ; figure 2b] ). p p .003 as indicated by their frequent detection in autopsy tissue, both b19 and parv4 showed evidence for the lifelong persistence of infection, which contrasted markedly with the findings for hbov. the high frequency of detection of b19 in this study is consistent with previous pcr-based evidence for frequent, potentially lifelong persistence of infection [19, 27] -that is, strong anti-b19 cytotoxic t cell reactivity among seropositive individuals [28] and the appearance of the igg4 subclass of antibody to b19, which usually is associated with chronic viral infection [29] . indeed, ongoing immunoreactivity to b19 at sites of replication has a potential etiological role in several chronic inflammatory diseases, such as arthritis [22, [30] [31] [32] [33] [34] [35] , although high frequencies of b19 detection also have been reported in a variety of tissues (synovium, liver, bone marrow, myocardium, and lymphoid tissue) in study subjects without inflammatory disease [14] [15] [16] [17] [18] [19] . as indicated by the results of the current study, persistence may be the usual outcome of b19 infection [14] , and the detection of b19 may be incidental to inflammatory diseases [15, 36] . since a high frequency of b19 infection was detected in lymphoid cells in this and a previous study (figure 1a) [19] , the higher viral loads in inflamed tissues, such as synovial tissue in subjects with arthritis, may have arisen through the migration of b19-infected inflammatory cells into affected tissues. although the highest b19 and parv4 viral loads were detected in bone marrow (which is consistent with the propensity of parvoviruses to target dividing cells) and both were found in lymphoid tissue, only b19 was detected in the brain autopsy samples. the spread of b19 into the central nervous system (cns) occurred in all study subjects and was not related to immunosuppression (figure 1a); these findings are consistent with the frequent detection of b19 dna sequences in prefrontal cortex samples in a recent study [37] . the detection of b19 dna sequences in autopsy brain samples is unlikely to be simply a spillover effect associated with the entry of lymphoid or stem cells into the cns; parv4 and b19 viral loads were similar in lymphoid and bone marrow-derived cells, but parv4 was consistently absent from the brain samples (table 2) . a surprising finding in the current study was the similarity in b19 viral loads between immunocompetent and immunosuppressed individuals ( figure 1a ). despite the destruction of the immune system in patients with terminal aids, b19 replication remained under tight control, with no evidence of increased viral loads in autopsy tissue from the study subjects with aids, compared with pre-aids and hiv-uninfected individuals; b19 or parv4 viremia was undetectable in a larger study population of 76 individuals that included several with low cd4 lymphocyte counts. the behavior of b19 and parv4 contrasted markedly with other persistent dna viruses, such as human cytomegalovirus, for which reactivation leading to clinically significant disease frequently develops during aids. as another example, the ubiquitously distributed and persistent small dna virus torquetenovirus (ttv) also shows evidence of increased viral replication in immunosuppressed individuals [38] [39] [40] . using the same autopsy samples that were analyzed in the current study, an ∼1000-fold increase in ttv viral load was found in the bone marrow, lymphoid tissue, and brain tissue samples from the study subjects with aids, compared with the samples from pre-aids and hiv-uninfected study subjects (s. j. willey, g. j. hughes, s. lucas, j. e. bell, and p. simmonds, unpublished data). while b19 dna sequences were detected in study subjects in all 3 categories, detection of parv4 dna was confined to hiv-infected individuals (table 2) . three hypotheses might explain this restricted distribution. it is possible that, in immunocompetent individuals, the immune system controls parv4 replication to such an extent that parv4 dna becomes undetectable in autopsy samples, whereas reactivation of parv4 occurs in immunosuppressed individuals through hiv infection. alternatively, parv4 infection may be nonpersistent in immunocompetent individuals, but the pre-aids study subjects and those with aids in the current study may have developed a persistent infection after reexposure to parv4 while infected with hiv. finally, it is possible that exposure and infection with parv4 is restricted to the hiv-infected group because of shared routes of transmission, such as sexual contact or parenteral exposure. the latter possibility is supported by the finding of a history of injection-drug use in each of the parv4-infected study subjects. without an antibody assay for parv4, it is currently difficult to distinguish between these hypotheses. however, if the replication of parv4 is controlled by the immune system in way that is similar way b19 replication, then several observations in the current study point toward the third hypothesis. first, evidence from the measurement of parv4 viral loads in pre-aids study subjects or those with aids does not indicate that the degree of immunosuppression had any effect on viral replication ( figure 1b) . if the lack of parv4 detection in hivuninfected individuals was the result of immune control, then viral loads would be expected to be much lower in the pre-aids group, since they retain a modestly functioning immune system, compared with the study subjects with aids. second, there was clear differentiation in parv4 infection status, with concordant results obtained from lymphoid and bone marrow samples from all but 1 individual. there was similarly a distinct distribution of parv4 viral loads among infected individuals from negative samples (figure 1b). if parv4 infection was under varying degrees of immune control at different stages of hiv disease progression, such a clear differentiation between hiv-infected and -uninfected individuals would be unlikely. future investigations of autopsy samples from hiv-infected individuals, non-injection-drug users (idus), and hiv-uninfected idus will help resolve the underlying reasons for the association of parv4 infection with hiv infection in the current study. understanding of the epidemiology and transmission patterns of b19 has advanced greatly, owing to the discovery of a temporal succession of infections with genotypes 1 and 2 over the past 60-70 years [19] , as well as recent evidence of much higher rates of sequence change in parvoviruses than had been thought previously [41, 42] . our finding of a significantly older age distribution among study subjects infected with b19 genotype 2 closely reproduced findings from a finnish study that demonstrated that b19 variants detected in biopsy samples corresponded to the original infecting strains [19] . the observed disappearance of genotype 2 infections in all but 1 individual born after 1965 closely matches data in the current study, in which the last genotype 2 infection was detected in an individual born in 1962 (figure 2a); this similarity provides some evidence for long-range b19 transmission networks between quite distantly separated countries in northern europe. evidence was obtained for an equivalent temporal succession of infection with 2 different parv4 genotypes in the study group. infection with divergent variants of parv4, provisionally referred to as genotype 2 in the current study and corresponding to the previously described parv5 variants [13] , was restricted to study subjects born in 1956 or earlier; this age range was even more sharply differentiated from that for genotype 1, compared with the difference observed between the b19 genotypes. if parv4 infection is associated with needle sharing or hiv infection (as discussed in the previous subsection), then the time scale for this succession would be substantially more recent than that for b19. thus, parv4 infection might have occurred only after 1982, when the first cases of hiv infection occurred in edinburgh. this hypothesis of a very recent source of parv4 infection in this risk group is supported by the nucleotide-sequence diversity of parv4. no sequence variation was observed in the 13 genotype 1 sequences of 114 bp. the recently described high rates of sequence change in b19 and canine parvovirus [41, 42] can be used to infer maximum time since divergence for these variants, based on the assumption that substitution rates at synonymous sites in the viral nonstructural regions are similar between different genera of parvoviridae. by application of the observed mean substitution rate of substitutions ϫ4 1.8 ϫ 10 per site per year (95th percentile range, to 3.0 ϫ 10 ϫ4 ϫ5 7.5 ϫ 10 substitutions) [41] , the parv4 variants that infected the study subjects, as well as the individual in whom parv4 was originally discovered, would have diverged within the last 14 years (range, 8-34 years) before death. this prediction indicates a very recent introduction and rapid dissemination of parv4 infection in the hiv/idu risk groups in edinburgh in the 1980s-1990s. in contrast, the same calculation applied to b19 genotype 1 variants from the study subjects (mean pairwise distance, 0.47%) predicted an earlier average divergence time of 52 years, which is remarkably consistent with the observed replacement of b19 genotype 2 by genotype 1 in the 1960s (figure 2) [25] . rapid turnover and changes in transmission dynamics of genetic variants of b19 and parv4 revealed by these new models of parvovirus evolution may apply more broadly to other members of parvoviridae. the combined set of 125 published sequences from the np1 gene of hbov sequences, including those from geographically disparate sources such as sweden [2] , canada [8] , japan [5] , and australia [3] , showed a mean pairwise distance of 0.003, which corresponds to a predicted time of origin of 34 years (range, 21-83 years). as an intriguing alternative to the hypothesis that hbov was not detected in autopsy samples because infections were nonpersistent (see above), the genetic data support the possibility that hbov also might have spread to scotland very recently and was not circulating during the childhood years of the study subjects (1930s-1970s) , when hbov infection usually seems to be acquired. further studies, including the development of serological assays for antibody to hbov, are clearly required in order to understand more fully the transmission dynamics and persistence of hbov, parv4, and b19 infections. new dna viruses identified in patients with acute viral infection syndrome cloning of a human parvovirus by molecular screening of respiratory tract samples evidence of human coronavirus hku1 and human bocavirus in australian children frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections detection of human bocavirus in japanese children with lower respiratory tract infections bocavirus infection in hospitalized children the association of newly identified respiratory viruses with lower respiratory tract infections in korean children human bocavirus infection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus in french children simmonds p. epidemiological profile and clinical associations of human bocavirus and other human parvoviruses novel parvovirus and related variant in human plasma persistence of parvovirus b19 dna in synovial membranes of young patients with and without chronic arthropathy a study of the role of parvovirus b19 in rheumatoid arthritis integrity and full coding sequence of b19 virus dna persisting in human synovial tissue evidence for persistence of parvovirus b19 dna in livers of adults high prevalence of viral genomes and multiple viral infections in the myocardium of adults with "idiopathic" left ventricular dysfunction bioportfolio: lifelong persistence of variant and prototypic erythrovirus dna genomes in human tissue persistent b19 infection in immunocompetent individuals: implications for transfusion safety persistent parvovirus b19 infection without the development of chronic anemia in hiv-infected and -uninfected children: the women and infants transmission study parvovirus b19 infection-persistence and genetic variation an immune control model for viral replication in the cns during presymptomatic hiv infection human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers limiting dilution assays for the determination of immunocompetent cell frequencies. i. data analysis identification of shared populations of human immunodeficiency virus type 1 infecting microglia and tissue macrophages outside the central nervous system hedman k. persistence of human parvovirus b19 in human tissues prolonged activation of virusspecific cd8 + t cells after acute b19 infection parvovirus b19-specific dna in bone marrow from b19 arthropathy patients: evidence for b19 virus persistence clinical and laboratory findings in immunocompetent patients with persistent parvovirus b19 dna in bone marrow presence and significance of human parvovirus b19 dna in synovial membranes and bone marrow from patients with arthritis of unknown origin persistence of b19 parvovirus in synovial membranes of patients with rheumatoid arthritis chronic human parvovirus b19 infection in rheumatic disease of childhood and adolescence persistence of parvovirus b19 dna in synovium of patients with haemophilic arthritis parvovirus b19 and chronic arthritis-causal or casual association? detection of adeno-associated virus 2 and parvovirus b19 in the human dorsolateral prefrontal cortex ttv viral load as a marker for immune reconstitution after initiation of haart in hiv-infected patients effect of immune modulation on tt virus (ttv) and ttv-like-mini-virus (tlmv) viremia tt virus infection: prevalence of elevated viraemia and arguments for the immune control of viral load phylogenetic evidence for the rapid evolution of human b19 erythrovirus high rate of viral evolution associated with the emergence of carnivore parvovirus we are grateful to frances carnie for technical assistance with the brain and other autopsy tissue samples and to gareth hughes and jill douglas for providing plasma samples from hiv-infected and -uninfected individuals. key: cord-007176-61e9obb3 authors: jackson, george gee; muldoon, robert lee title: viroses causing common respiratory infections in man. iii. respiratory syncytial viroses and coronavimses date: 1973-11-17 journal: j infect dis doi: 10.1093/infdis/128.5.674 sha: doc_id: 7176 cord_uid: 61e9obb3 nan 1. size. rs virus was estimated, from sucrose density gradient centrifugation studies, to be 90-120 nm in diameter [2] ; viral particles in infected cells measured 65 nm by electron microscopy. particles negatively stained with phosphotungstic acid measured 120-300 nm [23] . some viral particles were as large as 860 nm [25] . structure. in ultrathin sections of infected tissue culture, electron microscopy revealed viruslike particles in vacuoles or invaginations within the cytoplasm [14] , but complete particles were not found in the cell cytoplasm [21] . the enveloping membrane has been described as fringed and the nucleoprotein strand as having a herring-bone appearance, with a mean diameter of 13 cf antibody formed by infants during an rs infection has a higher antigen requirement than antibody present in sera of adults or than that found as maternal antibody [113] . neutralizing secretory antibody occurs in one-half or more of patients with lower respiratory disease and in 10%-20 % of those with milder infections [97, 116] . b. secondary. a rise in cf and neutralizing antibody was observed after reinfection in the presence of pre-existing serum antibody [18] . the cf antibody response in adults was always weak compared with that in children [65] . a. infection of tissue culture. isolates were made from 0.1-0.2 ml of specimen inoculated onto a monolayer consisting of 100,000-300,000 hep-2 cells. eagle's basal medium, containing 5 %inactivated chicken serum, is a suitable maintenance medium that should be changed every three to four days. stationary incubation at 36 c is satisfactory. inoculation of a culture of chang liver cells, four to six days old, maintained with a medium of eight parts eagle's basal medium, two parts inactivated horse serum, and 0.2 parts l-glutamine is satisfactory. the medium is changed every four days. virus can be propagated in kb cells in a medium consisting of eagle's basal medium with 2 %chicken serum. preferred cell line. the most sensitive cell cultures are hep-2, monkey kidney, human amnion, and human kidney, in decreasing order [5, 44] . i. the cytopathic effect begins with small syncytial areas randomly distributed early in infection. within one to four days, the entire cell sheet may be involved, with syncytial areas enlarging and becoming more numerous [2] . the time of appearance of cpe depends, within limits, on the number of serial passages of the virus [2] but is nearly always demonstrable within five days. ii. cytology. eosinophilic cytoplasmic inclusions are commonly found in infected cells, especially in the syncytia [15] . these inclusions are devoid of dna, rna, virions, and demonstrated specific antigens. chromosomal abnormalities are not observed. in infected vero cells, dense intracytoplasmic inclusions have diameters ranging from 90 to 130 nm [115] . iii. plaque formation. small macroscopic plaques developed after incubation for seven to nine days in hep-2 cells overlayed with agar [30] . four conditional-lethal, temperature-sensitive mutants of rs have been isolated and shown to be genetically stable. one of the mutants produced a typical, nonsyncytial plaques [93]. iv. hemadsorption. none demonstrated. no data available. one-day-old mice inoculated intracerebrally or intraperitoneally [1, 2] , weanling hamsters, rabbits, guinea pigs, mice, weighing 10 g, and young adult rats inoculated via the previously mentioned routes were refractory to infection [1] . a. natural infection 1. clinical. infection with rs virus has been observed every year since its recognition. it occurs in rather sharp epidemics, recurring at intervals of nine to 14 months, usually in the fall or spring. illness may be gradual or abrupt after an incubation period of three to five days. the symptoms associated with infection in children are cough (97 %), fever (93 %), rhinitis (57 %), pharyngitis (47%), malaise (38 %), vomiting (30 %), anorexia (27%), lymphadenopathy (22%), otitis media (17%), conjunctivitis (13 %), and abdominal pain (7 %) [13, 29, 49] . in an analysis of symptoms produced by infections with rs and by influenza a viruses, the two diseases could not be differentiated on a clinical basis [112] . in infants, as many as 60% the first isolations of virus were made one to two days before the onset of symptoms. isolates were more frequent and positive over a longer period from subjects with illness than from those who were infected but without symptoms. 3. immunity. all adults tested possessed detectable levels of neutralizing antibody to rs virus before challenge, but the titer of naturally acquired antibody had no significant effect on subsequent rs infection of volunteers and was poorly correlated with development of mild clinical illnesses. resistance to infection and illness appeared to be related to the level of nasal antibody but not to the level of serum antibody [121] . infection elicited an increase in the titer of serum antibody by cf and neutralization. immunity upon rechallenge was not tested. illnesses from infection are more severe in children than in adults [8, 10] . the cf test, with 8 units of antigen, will detect 90 %of infections among individuals older than six months and is as sensitive as neutralization. below six months, the cf test detects only 20 %of infections. the neutralization test is more sensitive than cf when serum from infants is used, but rises in neutralizing antibody have been detected in only half of the virus-positive infections in this age group. the use of unheated serum or the addition of antibody-free fresh serum increases the sensitivity of tests for neutralizing antibody [42, 60]. antiserum was produced in guinea pigs by three weekly ip inoculations of 1 ml of infected tissue culture harvest [2] . rabbits given three weekly iv inoculations of 1 ml each followed by two weekly im inoculations of 1 ml of infected tissue fluid combined with 2 ml of a mixture of mycobacterium butyricum, paraffin oil, and arlacel, produced cf and neutralizing antibody [2] . in adult rabbits, an alternative procedure is to give three injections at two-week intervals, the first two consisting of 8 ml of virus and adjuvant given im, and the third injection of virus alone administered iv [5] . respiratory syncytial virus is considered to be a paramyxovirus on the basis of its size, appearance by electron microscopy, and sensitivity to ether; it differs from other paramyxoviruses in that it has no known hemagglutinin. although minor antigenic variants have been found, they are not well discriminated by antibody in human sera. because there has been no sequential drift in the antigenic character of the prevalent strain, rs virus is considered to be a single type. epidemiologically, respiratory syncytial virus is very important, because it causes annual epidemics of acute respiratory diseases affecting infants, children, and adults. infection spreads rapidly from person to person and characteristically occurs as a discrete outbreak of acute respiratory illness in the winter or early spring. in infants and children, especially during the first six months of life, respiratory syncytial virus is the most important cause of bronchiolitis and a major cause of pneumonia. serum antibody acquired by transplacental passage does not provide immunity against infection and might possibly augment the local respiratory disease by an immunopathologic process. the virus may replicate in the middle ear, but its role in otitis is unproven. croup is an infrequent manifestation. pneumonia, as determined radiographically, is frequent, usually bilateral, and multilobar; it may be lobar with secondary bacterial infection. the pathologic lesion is one of necrosis of the epithelial mucosa of the trachea and bronchi and an interstitial inflammation. in adults, infection with rs virus usually causes upper respiratory symptoms; however, because of the prevalence of infection, it is an important cause of exacerbations of bronchitis, pneumonia, and "flu," requiring the hospitalization of adults. in some years, virus infection has given rise to an increase in secondary pneumonia due to diplococcus pneumoniae. infection is followed by an increase in serum cf and neutralizing antibody; also by secretory neutralizing antibody in the nasopharyngeal and tracheal secretions. primary infection does not establish complete immunity, and reinfection is common at all ages. inactivated vaccines of the type and potency previously produced have been detrimental because they have failed to prevent infections and they have induced a more severe disease with exaggerated pneumonia. attenuated live virus vaccines have not yet been successful, nor has any effective chemotherapy been developed. takano a virus isolated from wild cottontail rabbits was shown to possess chemical, physical, and biologic characteristics of the paramyxovirus family. although formation of syncytia was characteristic of its growth in tissue cultures, no antigenic relationship was detected by cf or neutralization tests with any known member of the paramyxovirus family [1] . isolates shown to be antigenically related to human rs virus were recovered from cattle with bronchopneumonia [2] . cytologic examination of bhk2 1 cells infected with bovine rs virus revealed intranuclear and intracytoplasmic viral components [4] . an a type particle with a diameter of 65 nm has been described in the cytoplasm of such cells [3] . the viral envelope was added as the virion passed through the cytoplasmic membrane in a budding process [4] . a. physical properties characterization 1. size. by gradocol filtration, the size of the virion was calculated to be 89 nm [2] . by electron microscopy, the usual diameter was measured as 80-160 nm [5] . 2. structure. the virions are pleomorphic. most particles are covered with projections (spikes) more densely packed than those seen on influenza viruses. these spikes are attached to the virion-by narrow stalks with a thickening (90-110 a) at the distal end [5, 11] . the internal component is a hollow, threadlike structure with a diameter of 70 nm and a definite structural pattern [21] . 3. heat stability. infectivity was destroyed at 56 c within 10 min. there was no loss of titer after 2 hr at 37 c or 10 days at 4 c. rates of thermal inactivation are dependent on the amount of particle aggregation. aggregation is dependent on the concentration of serum in the medium [ a. group antigen common cf antigens have been observed in known members of the coronavirus family, except in avian bronchitis virus. serologic data are still incomplete, but some observed interrelations are shown in table 1. table 1 . antigenic mosaic of coronavirus as determined by neutralization (n) and cf tests with animal serum [20, 25] . antigens produced in tissue culture or mouse brain cf antigens have been prepared from harvests of infected tissue culture and mouse brain, but attempts to prepare antigens from organ cultures have not been successful [2] . strains oc38 and oc43 cross-react, as shown by neutralization tests in mice or monkey-kidney cell culture. strains 229e and lp cross-react in neutralization tests but not to identical titers, indicating a close but not completely similar antigenic mosaic. a one-way cross exists between 229£ and oc32; antisera against the latter and b814 do not neutralize other coronaviruses. avian bronchitis virus reacts only with homologous virus (see table i ). in immunodiffusion tests, the number of detectable lines of precipitation varies from one (strain b814) to four (strain oc43). this may be a result of the procedure used for production of antibody [25] . c. antigenicity i. animals. in animals, specific antibody is elicited by the initial series of inoculations of cell-culture harvests. in neutralization tests with animal serum, no antigenic relationship has been detected between strains 229e and oc38-43 [20] . a. primary response. initial infection results in an increase of specific neutralizing antibody. about 50% of volunteers challenged with the oc strains of coronaviruses developed cf rises to mhv [20] . b. secondary response. the antigenic primacy of the initially infecting strain, heterologous interrelationships, and anamnestic responses are still to be worked out. [16] . coronaviruses of avian origin have phenotypic differences in the susceptible avian host cell range [4] and grow to a limited degree in nonavian tissues [32] . murine coronavirus has grown adequately only in tissues from mice [20] . coronavirus of rats has isolation propagation grown in only one of several cell lines studied [24] . coronaviruses of swine grow only in tissues of porcine origin [37] . a. infection of tissue culture. for growth of explants, a medium of 2 ml of eagle's medium with 0.2 % (wt/vol) bovine plasma albumin and incubation at 33 c in a humidified atmosphere containing 5 %(vol/vol) co 2 in air is satisfactory [5] . a ph of 7.0 is required for growth since inactivation is accelerated at ph 7.7 and 6.7 [25] . i. preferred cell line. human tracheal organ cultures. ii. growth cycle. after 1 hr at 33 c, only 18%of l132 cells wereinfected with strains 229e. virus structures were detected 6-8 hr later [17] .· infection of wi-38 cells with strain 229e resulted in a reorganization of the cytoplasm, as determined by electron microscopy. new viral structures were observed 6-12 hr after infection. clusters of virus were observed in intracytoplasmic vacuoles, called cisternae, 24-36 hr after challenge. strain oc43 in wi-38 matures in intracytoplasmic vesicles similar to those observed with strain 229e.. budding, such as that described for strain 229e was not observed [30] , and the budding process described for coronavirus is into cytoplasmic vesicles rather than from the plasma membrane, as has been observed with myxoviruses [6, 8, 28] . iii i. cpe. the cpe that gradually developed in human diploid cells gave them a stringy appearance. some intracytoplasmic vacuoles were observed [2] . strain b814 caused no cpe and could be detected only by electron microscopy or by interference with echovirus type 11 [1] . ii. cytology. no inclusions have been observed [2] . fluorescent antibody has been used for identification of viral antigen [15] . morphology, as observed by electron microscopy, is characteristic [5, 37] . the use of specific antisera, in combination with electron microscopy, can facilitate recognition of the virus in culture harvests [38] . iii. plaques. at 33 c, with a methyl cellulose overlay, plaques were formed in wi-38 cell cultures [8] . plaques can be produced in l132 cells infected with the 229e strain [17] . [4, 12, 24] . after four to five passages in tissue culture, strains oc38 and oc43 were administered intracranially to suckling mice. on the first passage in mice, illness, characterized by tremors, rigidity, and lethargy, was observed on days 11-15 after challenge. by the fourth passage in mice, these viruses were lethal for mice within 48-60 hr after challenge [9] . there is marked host specificity of different strains. a. natural infection 1. clinical. the use of explants of human embryonic nasal epithelium or trachea has resulted in the isolation of coronaviruses. serology has shown them to be associated with acute respiratory diseases of man. the exact importance of these viruses with accompanying epidemiologic data is unavailable because of the variability of strains and the difficult techniques required to establish diagnosis of the infection. in most studies, coronaviruses usually caused infections in the period from january through march [18, 33, 34] . only about one-half of naturally occurring infections cause clinical illness [26] . spread appears to be preferentially within families. in one study, secondary cases occurred in 17 of 26 families [22] . serum neutralizing antibody does not influence the occurrence of reinfection [33] . acquisition of infection with avian bronchitis virus from chickens is suggested by studies in poultry handlers [13] . nonsusceptible cells infection in man sponses occurred in 10%-20 % of these and other volunteers infected with coronaviruses [29] . strain 229e was recovered from sick as well as healthy volunteers in each of the four challenge passages [7] . volunteers challenged with the oc or b816 strain often showed cf rises to strains 229e and lp [20] . 3. immunity. incomplete. c. prevention 1. vaccine. none. none. diagnosis is based on electron microscopic examination of cell explants or, with some strains, detection of cpe in monolayers after serial blind passage. b. serology rises in the titer of cf antibody against strain 229e and hal antibody against strain oc43 are the most practical tests. neutralization is the serologic test of choice for specific identification. commercially unavailable. prevention laboratory diagnosis coronaviruses are a distinct group of viruses with common morphology and various degrees of antigenic similarity. the number of different coronaviruses that infect man and their exact interrelations are still unknown. their etiologic role in respiratory diseases has been established. also, they are associated with hepatitis in mice and avian bronchitis. data suggest that coronaviruses cause comment 3 %-4 % of acute respiratory illnesses in humans. the clinical syndrome is usually that of a common cold. asymptomatic infections also occur, possibly because of partial immunity to reinfection. coronavirus infections are most prevalent in the winter months and may occur in epidemic fashion, with the same strain being geographically widespread. recurrent epidemics of the same type do not seem to occur in sequential years. preliminary serologic investigations indicate that certain strains may infect children preferentially, whereas others are prevalent in adults. an alternative explanation of the findings is the emergence of new epidemic strains with disappearance of older strains for a period. transmission of the virus within families is frequent, and acquisition may be possible from poultry and other sources. no effective vaccines or chemotherapy have been developed. recovery of cytopathogenic agent from chimpanzees with coryza recovery from infants with respiratory illnesses of a virus related to chimpanzee coryza agent (cca). i. isolation, properties,and characterization recovery from infants with respiratory illness of virus related to chimpanzee coryza agent (cca). ii. epidemiologic aspects of infiltration in infants and young children association of the chimpanzee coryza agent with acute respiratory disease in children growth characteristic of chimpanzee coryza agent in tissue cultures role of respiratory syncytial virus in bronchiolitis, pneumonia, and minor respiratory disease. i. virus recovery and other observations during 1960 outbreak studies of acute respiratory illness caused by respiratory syncytial virus. i. laboratory findings in 109 cases respiratory syncytial virus infection in adult volunteers. ii. correlation of illness and clinical observations an outbreak of febrile illness and pneumonia associated with respiratory syncytial virus infection respiratory syncytial virus infection in adult volunteers. iii. prediction of illness and clinical observations studies on acute respiratory syncytial virus. 2. epidemiology and assessment of importance role of respiratory syncytial virus in bronchiolitis, pneumonia, and pharyngitis with bronchitis in children. ii. serologic studies over a 34 month period studies on acute respiratory illnesses caused by respiratory syncytial virus. 3. clinical and laboratory findings morphology and development of respiratory syncytial virus in cell culture growth and serologic characteristics of respiratory syncytial virus respiratory syncytial virus experimental cytial virus antigens by agar gel diffusion and immunoelectrophoresis interferon and respiratory syncytial virus speculation on pathogenesis in death from respiratory syncytial virus infection the late detection of respiratory syncytial virus in cells of respiratory tract by immunofluorescence double infection with rs virus and influenza virus infections in children. clinical comparison of overlapping outbreaks of influenza a2-hong kong-68 and respiratory syncytial virus infections differentiation of actively and passively acquired complementfixing antibodies in infants with respiratory syncytial virus infection the use of cough-nasal swabs in the rapid diagnosis of respiratory syncytial virus infection by the fluorescent antibody technique morphogenesis of respiratory syncytial virus in a green monkey kidney cell line (vero) respiratory syncytial virus neutralizing activity in nasopharyngeal secretions rsv infections and infant deaths respiratory syncytial virus tissue culture immunofluorescence as a laboratory aid rapid diagnosis of respiratory syncytial virus infection in children by the immunofluorescent technique experimental respiratory syncytial virus infection of adults. possible mechanisms of resistance to infection and illness recovery of a new syncytium virus from a cottontail rabbit a respiratory syncytial virus of bovine origin cultivation of a novel type of common cold virus in organ culture a new virus isolated from the human respiratory tract a new virus cultivated only in organ culture of human ciliated epithelium immunofluorescence of avian infectious bronchitis virus in primary chicken embryo kidney, liver, lung, and fibroblast cell cultures the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) effects of a "new" human respiratory virus in volunteers growth and intracellular development of a new respiratory virus growth in suckling mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease recovery in tracheal organ cultures of novel viruses from patients with respiratory disease direct electron microscopy of organ culture for detection and characterization of viruses neutralization of infectious bronchitis virus by human serum cultivation of "difficult" viruses from patients with common colds intracellular avian infectious bronchitis virus: detection by fluorescent antibody techniques in nonavian kidney cell cultures sensitivity of l132 cells to some "new" respiratory viruses the propagation of "coronaviruses" in tissue culture isolation from man of "avian infectious bronchitis virus-like" viruses (coronaviruses) similar to 229e virus with some epidemiological observations some characteristics of hemagglutinin of certain strains of "ibv-like" virus antigenic relationships among the coronaviruses of man and between human and animal coronaviruses symposium on the biology of large rna viruses community-wide outbreak of infection with 229e-like coronavirus in tecumseh, michigan seroepidemiologic studies of coronavirus infection in adults and children rat coronavirus (rcv); a prevalent naturally occurring pneumotropic virus of rats coronaviruses of man seroepidemiologic survey of coronavirus (strain 0c43) related infections in a children's population presence of neutralizing antibody against the 229e strain of coronavirus in the sera of residents of sendai electron microscopic studies of coronavirus coronavirus antibody titres in sera of healthy adults and experimentally infected volunteers intracellular development and mechanism of hemadsorption of a human coronavirus oc43 studies with human coronaviruses. ii. some properties of strains 229e and jackson and muldoon oc43 replication of avian infectious bronchitis virus in african green monkey kidney cell line vero virologic studies of acute respiratory disease in young adults. v. coronavirus 229e infections during six years of surveillance coronavirus infections in working adults: eight year study with 229e and oc43 protein composition of coronavirus oc43 hemadsorption by coronavirus strain oc43 characteristics of a coronavirus (strain 67n) of pigs detection of coronavirus strain 692 by immune electron microscopy of five subjects from whom coronaviruses were isolated, all developed a serologic response [3] .b. serologic. measurement of the cf antibody response was found to be twice as sensitive an index of infection as virus isolation. the cf antibody tends to be transitory, whereas titers of neutralizing antibody remain elevated for a longer period of time [13] . the observed prevalence of infection varies widely in different years [29, 33] .during a spring outbreak of respiratory disease in 1967, the infection rate in a community was 34 % [22] . from observations in a children's home, it was estimated (based on serology) that 19% of respiratory illness in a single season was caused by coronavirus strain oc43 [26] . among groups of adults during two of four winters when there were high rates of respiratory disease and infrequent virus recovery, infection with 229b occurred in 10%-24% of those with upper respiratory tract disease [18] .in three separate studies, each covering a seven-or eight-year period, coronavirus oc43 accounted for 3 % of 1,328 illnesses observed in children [26] ; coronaviruses 229b and oc43 accounted for 4 % of colds in an 'adult industrial population [34] , and 15%-35 % of students were infected during three seasons of high prevalence [33] .a serologic survey of adults and children showed that infection in infants below one year of age was infrequent. infection with oc38 and/ or oc43 occurred principally in the preschool years, whereas infection with 229b occurred later [23] . data from sera collected in 1966indicate that 30 % of adults and 15%-20 % of children had neutralizing antibody specific for strain 229e [7] . in a study of sera collected since 1967, 50 %-80 % of the population were found to have neutralizing antibody against 229e at a dilution of 1:20, as measured by plaque reduction in l132 cells. in cf tests (serum diluted 1: 20) between 50 %and 98 %of those tested had antibody [25] . sera collected from 139 adults between january 1969 and october 1970 showed that 8.6% had a neutralizing antibody titer 2:: 1: 8 against strain 229b [27] . 1. challenge. in an early study, harvest medium from human embryonic trachea containing strain b814 was used as inoculum. illness was observed in five of 11 volunteers [1] . after tissue and organ-culture passage, strain 229e was passed four times in volunteers and produced illness in each passage [7] . later, six coronaviruses isolated from persons with illness were given to volunteers. all six produced colds. heterologous antibody rekey: cord-007190-x1v4jpl4 authors: hardison, jenny l.; kuziel, william a.; manning, jerry e.; lane, thomas e. title: chemokine cc receptor 2 is important for acute control of cardiac parasitism but does not contribute to cardiac inflammation after infection with trypanosoma cruzi date: 2006-06-01 journal: j infect dis doi: 10.1086/503812 sha: doc_id: 7190 cord_uid: x1v4jpl4 the cc chemokine ligand 2 (ccl2) and cc chemokine receptor 2 (ccr2) are expressed in the heart after infection with trypanosoma cruzi suggesting that they play an important role in host defense. infection of ccr2-deficient (ccr2(−/−)) mice with t. cruzi resulted in increased cardiac parasitism, yet the severity of cardiac inflammation was not affected. in addition, expression of interferon-γ and inducible no synthase in the heart, which are associated with effective killing of trypomastigotes, was not affected in ccr2(−/−) mice. these observations reveal that ccr2 signaling plays a distinct role that is separate from that of influencing either chemotaxis or previously defined anti-trypomastigote mechanisms for the control of t. cruzi’s replication in the heart infiltrates are composed primarily of cd8 + t cells but also contain cd4 + t cells and macrophages [2] . as the disease progresses, the heart becomes enlarged, and, over time, this condition can result in congestive heart failure and sudden death [1, 2] . therefore, an understanding of the mechanisms participating in promoting and maintaining inflammation in the heart may foster identification of new therapeutic targets in the treatment of chronic chagas disease. during the past several years, there has been increased interest in characterizing the expression of proinflammatory chemokine genes in the hearts of t. cruzi-infected mice [3] [4] [5] . chemokines and their cognate receptors direct the extravasation of leukocytes and monocytes from the bloodstream into tissues and also regulate the positional migration of these cells in tissues. in addition, in numerous disease models, chemokines have been shown to play a significant role in the influx of cells, where they participate in the control of pathogens and/or contribute to chronic inflammation. chemokine genes are expressed in the hearts of t. cruzi-infected mice, suggesting that chemokines may play a role in host defense and/or disease. in support of this idea, we and others have determined that cxc chemokine ligand 9 (cxcl9) and cxcl10, as well as cc chemokine ligand 5 (ccl5), are detectable in the heart during both acute and chronic cardiomyopathy [3, 5] . when mice are treated with neutralizing antibodies specific for cxcl9 and cxcl10, the result is increased parasitemia, indicating that these chemokines play an important role in generating a protective immune response [5] ; however, blocking these chemokines does not ultimately alter the severity of chronic cardiomyopathy, as characterized by both the parasitic burden and chronic inflammation in the heart. one factor controlling the response to chemokine-ligand expression is the corresponding expression of the appropriate chemokine receptor(s). we have recently shown that t. cruzi-infected mice lacking cc chemokine receptor 5 (ccr5), which recognizes ccl5, results in an increase in both parasitemia and parasitic burden in the heart, an increase that correlates with a paucity of infiltration of t cells and macrophages [4] . in addition to inducing the directional migration of targeted populations of leukocytes during periods of inflammation in response to infection or injury, chemokines are now recognized to be important in numerous biological processes, one of which is to exert antimicrobial effects against various types of pathogens, including parasites [6] . indeed, after a series of in vitro studies, aliberti et al. [7] reported that ccl2 signaling both increased the macrophage uptake of t. cruzi and enhanced the production of no, which resulted in the killing of this parasite. these observations suggest that, by controlling this parasite's replication, ccl2 may play a role in resistance to infection with t. cruzi. previous studies in our own and other laboratories have demonstrated that, in response to infection with t. cruzi, ccl2 is expressed in the heart [3, 5] . in addition, the ccl2 receptor, ccr2, also is expressed in the heart after infection with t. cruzi, which suggests that it plays an important role in the ccl2:ccr2 signaling axis, thereby promoting host defense. to characterize the in vivo role that ccl2 signaling plays in leukocyte trafficking, ccr2-deficient (ccr2 ϫ/ϫ ) mice were infected with t. cruzi, and both the ability to control this parasite's replication in the heart and the severity of cardiac inflammation were evaluated. materials and methods. female ccr2 wild-type (ccr2 +/+ ) mice (b6129f2/j, obtained from the jackson laboratory in bar harbor, me), and female ccr2 ϫ/ϫ mice (129b6f2-cmkbr2 tm1kuz ), all of which were 6-8 weeks old, were bred and housed in pathogen-free conditions, in enclosed filter-top cages. mice were infected subcutaneously with 50 blood-derived colombiana-strain trypomastigotes, and parasitemia was monitored weekly, as described elsewhere [5] . mice were killed at days 0, 15, 30, 60, 120, and 200 after infection, and their hearts were collected for histological and rna analysis by rnase protection assay. the hearts were fixed in 10% formalin and were embedded in paraffin, and 5-mm sections were cut in 100-mm increments, for staining with hematoxylin-eosin. each section was scored for inflammation, by an investigator blinded as to its source; and infected cardiomyocytes were counted according to a method described elsewhere [5] . significant differences in parasitism were determined by mann-whitney u test; p р was considered to be significant. .05 total rna was extracted from hearts by use of trizol reagent (invitrogen). a minimum of 3 mice were evaluated at each examination time. chemokine, chemokine-receptor, cytokine, and cell-surface marker transcripts were analyzed by use of custom multitemplate probe sets (bd pharmingen), and individual transcripts were normalized to housekeeping gene l32 [4, 5] . normalized units are obtained by dividing the band intensity of each individual transcript by the band intensity of l32 and are expressed as percentages. data are expressed as means ‫ע‬ sd, and significant differences between ccr2 +/+ mice and ccr2 ϫ/ϫ mice, at each examination time, were determined by student's t test; was considered to be significant. p р .05 results and discussion. at days 30-60 after infection, the hearts of t. cruzi-infected ccr2 +/+ mice had detectable mrna transcripts for both ccl2 and ccr2, suggesting that ccl2 and ccr2 potentially play a role in the regulation of host defense and/or inflammation in the heart (figure 1a). evaluation of parasitemia revealed no difference between ccr2 ϫ/ϫ mice ( ) and ccr2 +/+ mice ( ), and all mice in n p 11 n p 12 both groups survived to day 200 after infection, the last point at which survival was measured (data not shown). collectively, these data indicate that (1) ccr2 signaling is not required for control of t. cruzi's replication in the blood and (2) ccr2 is not an important influence in survival. however, at day 30 after infection, cardiac parasitism in ccr2 ϫ/ϫ mice was ∼6-fold higher than that in ccr2 +/+ mice (table 1 and figure 1b) . infected cardiomyocytes in ccr2 ϫ/ϫ mice were often found in close proximity and were frequently quite large, a result that contrasts with what was observed in ccr2 +/+ mice ( figure 1b) . however, the deficiency in the ccr2 ϫ/ϫ mice's control of this parasite's replication in the heart was eventually overcome, and, at days 60-200 after infection, there were no differences between the 2 groups of mice (table 1) . to determine whether the increase in cardiac parasitism in ccr2 ϫ/ϫ mice correlated with a muted inflammatory response, the severity of cardiac inflammation was determined by scoring of the hematoxylin-eosin-stained tissues and, by use of the rnase protection assay, examination of the mrna transcripts for the presence of infiltrating immune cells. as shown in table 1, at no examination time after infection was there any difference between the severity of inflammation in ccr2 ϫ/ϫ mice and that in ccr2 +/+ mice. consistent with these results is the finding that the mrna transcript levels for cd4, cd8, and f4/80 detected in the hearts of ccr2 +/+ mice were similar to those detected in the hearts of ccr2 ϫ/ϫ mice (figure 1c and d). both interferon (ifn)-g and inducible no synthase (inos) have been shown to be important in reducing the parasitic burden in infected tissues [2] ; in the present study, however, at no examination time after infection were the transcript levels for either ifn-g or inos in ccr2 ϫ/ϫ mice different from those in ccr2 +/+ mice ( figure 1c and d) . similarly, there were no deficiencies in expression of chemokine transcripts in the hearts of ccr2 ϫ/ϫ mice, compared with that in the hearts of ccr2 +/+ mice ( figure 1e) ; rather, at days 60 and 120 after infection, expression of ccl5 was increased in ccr2 ϫ/ϫ mice during chronic infection. expression of some chemokine receptors was intermittently reduced in ccr2 ϫ/ϫ mice after infection (figure 1f); for example, at day 30 after infection, expression of cxcr3 in ccr2 ϫ/ϫ mice was ∼3-fold lower than that in wild-type control mice, and, at day 60 after infection, expression of ccr5 in these mice was ∼5-fold lower than that in wild-type control mice. evaluation either by histological analysis or on the basis of expression of markers for t cells and macrophages showed that at no examination time after infection was the infiltration of leukocytes into the heart decreased in ccr2 ϫ/ϫ mice, a finding that implies that signaling through ccr2 may play a role in both cell activation and expression of other chemokine receptors. the immune response in peripheral tissue also did not appear to be decreased in ccr2 ϫ/ϫ mice: at days 30 and 60 after infection, expression of chemokines in the spleen of ccr2 ϫ/ϫ mice was increased, compared with that in the spleen of ccr2 +/+ mice; however, at days 15, 30, 60, and 120 after infection, expression of chemokine receptors was similar in both groups of mice (data not shown). collectively, these data (1) demonstrate that ccr2 neither enhances immune-cell infiltration into the heart nor influences antiparasitic effector responses such as expression of either ifn-g or inos and (2) imply that ccr2 is important in the control of t. cruzi's replication in the heart. therefore, the mechanism by which ccr2 controls this parasite's replication is not dependent on the regulation of cardiac inflammation. ccr2 is the primary receptor for ccl2 and is found on monocytes, activated t cells, b cells, and nk cells [8] . recent studies have indicated that, in response to stimuli or infection, ccr2 ϫ/ϫ mice display deficiencies in trafficking of t cells and macrophages [9, 10] . previous studies have also demonstrated that ccl2 can activate t cells and macrophages, presumably through binding to ccr2 expressed on the surface of these cells [10, 11] . indeed, leukocyte populations from ccr2 ϫ/ϫ mice do not bind ccl2 and exhibit defects both in activation and in recruitment to sites of inflammation [9] . infection of ccr2 ϫ/ϫ mice with various microbial pathogens has increased our understanding of how this receptor contributes to immunological events involved in host protection; for example, ccr2 ϫ/ϫ mice with lungs infected with cryptococcus neoformans exhibit a decreased protective th1 response accompanied by a switch to a strong th2 response [12] . this switch in th responses correlates with both prolonged infection and the infecting agent's greater dissemination throughout the body. analysis of ccr2 ϫ/ϫ mice either infected with leishmania donovani or immunized with shistosomal antigen has revealed an impaired cellular immune response characterized both by reduced t cell and macrophage recruitment to sites of antigen localization and by decreased expression of ifn-g [11, 13] . collectively, these studies strongly suggest that expression of ccr2 is required for the development of protective th1 responses after microbial challenge. the findings of the present study indicate that, in t. cruzi-infected mice, the absence of ccr2 does not decrease expression of ifn-g (figures 1c and d); nor were there increased numbers of transcripts for the th2associated cytokines il-4 and il-10 (data not shown). therefore, it is unlikely that the increased parasitic burden in the hearts of ccr2 ϫ/ϫ mice is a result of a muted th1 response accompanied by increased expression of th2-associated cytokines. alternately, differential expression of chemokine receptors in ccr2 ϫ/ϫ mice may contribute to the inability to control t. cruzi's replication in the heart during acute infection, in light of in vitro studies that have shown that chemokines play a role in promoting the control of this parasite's replication [7, 14] ( figure 1f ). chemokines such as ccl2 and cxcl10 increase nitrite production by infected cardiomyocytes to a level that is sufficient for control of this parasite's replication [14] . moreover, by virtue of their enhancement of both production of no and phagocytosis of trypomastigotes, ccl2, ccl3, ccl4, and ccl5 have been implicated in promoting the trypanocidal activities of macrophages, with ccl2 being the most potent chemokine promoting these activities [7] . in addition to promoting these trypanocidal activities, ccl2 may also be important in promoting the effective survival responses of cardiomyocytes. indeed, in a model of myocardial trauma, ccl2 signaling through ccr2 has been found to be associated with increased cardiomyocyte survival [15] . these findings highlight the possibility that ccr2 plays a previously unappreciated role in host defense after infection with t. cruzi. the results of the present study show that the contribution that ccr2 makes to host defense is transient and is limited to the acute stages of infection, which coincide both with increased expression of ccl2 and with cardiac parasitism (table 1 and figure 1a ). this finding suggests that, during this time, ccr2 is used to inhibit the spread of parasites and to promote the killing of these parasites in the heart. potential mechanisms by which ccr2 signaling enhances tissue-specific host defense may include regulation of phagocytosis of trypomastigotes and/ or control of t. cruzi's intracellular replication, possibly through the sequestering of required growth factors. ongoing studies are under way to further characterize the mechanisms by which ccr2 promotes the killing of this parasite. chagas' disease immunological control of trypanosoma cruzi infection and pathogenesis of chagas' disease kinetics of cytokine gene expression in experimental chagasic cardiomyopathy: tissue parasitism and endogenous ifn-gamma as important determinants of chemokine mrna expression during infection with trypanosoma cruzi the cc chemokine receptor 5 (ccr5) is important in control of parasite replication and acute cardiac inflammation following infection with trypanosoma cruzi the chemokines cxcl9 and cxcl10 promote a protective immune response but do not contribute to cardiac inflammation following infection with trypanosoma cruzi ip-10 is critical for effector t cell trafficking and host survival in toxoplasma gondii infection beta-chemokines enhance parasite uptake and promote nitric oxide-dependent microbiostatic activity in murine inflammatory macrophages infected with trypanosoma cruzi expression and characterization of the chemokine receptors ccr2 and ccr5 in mice severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in cc chemokine receptor 2 lack of ccr2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus defects in the generation of ifng are overcome to control infection with leishmania donovani in cc chemokine receptor (ccr) 5-, macrophage inflammatory protein-1-a-, or ccr2-deficient mice ccr2 expression determines t1 versus t2 polarization during pulmonary cryptococcus neoformans infection effect of c-c chemokine receptor 2 (ccr2) knockout on type-2 (schistosomal antigen-elicited) pulmonary granuloma formation: analysis of cellular recruitment and cytokine responses trypanosoma cruzi-infected cardiomyocytes produce chemokines and cytokines that trigger potent nitric oxide-dependent trypanocidal activity chemokine expression in myocardial ischemia: mip-2 dependent mcp-1 expression protects cardiomyocytes from cell death key: cord-003115-y40knklf authors: amlabu, emmanuel; mensah-brown, henrietta; nyarko, prince b; akuh, ojo-ajogu; opoku, grace; ilani, philip; oyagbenro, richard; asiedu, kwame; aniweh, yaw; awandare, gordon a title: functional characterization of plasmodium falciparum surface-related antigen as a potential blood-stage vaccine target date: 2018-09-01 journal: j infect dis doi: 10.1093/infdis/jiy222 sha: doc_id: 3115 cord_uid: y40knklf plasmodium falciparum erythrocyte invasion is a multistep process that involves a spectrum of interactions that are not well characterized. we have characterized a 113-kda immunogenic protein, pf3d7_1431400 (pf14_0293), that possesses coiled-coil structures. the protein is localized on the surfaces of both merozoites and gametocytes, hence the name plasmodium falciparum surface-related antigen (pfsra). the processed 32-kda fragment of pfsra binds normal human erythrocytes with different sensitivities to enzyme treatments. temporal imaging from initial attachment to internalization of viable merozoites revealed that a fragment of pfsra, along with pfmsp1(19,) is internalized after invasion. moreover, parasite growth inhibition assays showed that pfsra p1 antibodies potently inhibited erythrocyte invasion of both sialic acid–dependent and –independent parasite strains. also, immunoepidemiological studies show that malaria-infected populations have naturally acquired antibodies against pfsra. overall, the results demonstrate that pfsra has the structural and functional characteristics of a very promising target for vaccine development. malaria is a deadly infectious disease that affects inhabitants of the tropics and subtropical regions of the world and accounts for approximately 212 million cases and 429 000 deaths annually [1] . the clinical manifestation of the disease begins from the blood stage of the infection, during which the parasite invades human erythrocytes [2] . plasmodium falciparum erythrocyte invasion is a complicated process that involves an array of receptor-ligand interactions [3] and/or protein-protein interactions [4] [5] [6] [7] [8] that occur at the parasite-host cell interface and facilitate the recruitment of the parasite's invasion machinery. thus, recent identification of p. falciparum surface proteins that are accessible to both humoral and cellular immune systems is a major advancement toward vaccine development against malaria [9] [10] [11] [12] . this has given great impetus to the idea of a multiantigen vaccine as an intervention strategy against blood-stage malaria. therefore, the selection and prioritization of candidate antigens are critical aspects of this strategy. although several blood-stage antigens have been extensively studied, few have demonstrated the desired qualities for a vaccine candidate. one of the remarkable observations from the p. falciparum genome sequencing project was that nearly 60% of the parasite' s genes lacked sequence similarity to genes from other known organisms, and thus these genes have remained hypothetical with no defined functional roles [13] . subsequently, the availability of more comprehensive genomic, proteomic, and transcriptomic datasets from both humans and plasmodium has paved the way for further characterization of these hypothetical proteins using informatics-based approaches. this is required for successful identification of a repertoire of novel p. falciparum merozoite antigens that could be explored as targets for a rational vaccine design [14] . a detailed understanding of the functional roles of p. falciparum novel merozoite antigens, their localization, and their fate during invasion is critical to the identification of targets of host immunity and prioritization of merozoite antigens for inclusion in blood-stage malaria vaccines. herein, we have identified a novel p. falciparum protein (plasmodb id: pf3d7_1431400/pf14_0293) that we have named p. falciparum surface-related antigen (pfsra), based on its dual subcellular localization on both merozoites and gametocytes. native pfsra is proteolytically processed into multiple fragments in parasite culture supernatant, and the 32-kda fragment of pfsra exhibits erythrocyte-binding activity. more important, antibodies against pfsra potently inhibited merozoite invasion of erythrocytes by both sialic acid-dependent and sialic acid-independent parasite strains. the data also demonstrated that pfsra is a target for naturally acquired immune responses in humans. to identify new blood-stage proteins as potential vaccine candidates, a systematic screening procedure was implemented. this included analysis of temporal gene expression relative to other well-characterized invasion-type genes and in silico interrogation of protein structural features. once all of these selection criteria were ascertained, sequence alignment analysis was done to evaluate the conservation level of the target gene across the different plasmodium species orthologs. finally, we scanned the entire protein sequence using a current state-of-the-art online threading program to identify coiled-coil regions [15] . three synthetic peptides corresponding to the immunogenic epitopes were synthesized by genescript on the basis that they harbor coiled-coil signatures corresponding to the conserved regions in pfsra orthologs. ethical approval was obtained from the ethics committees as documented previously [16, 17] . immunoreactivity screenings of plasma samples were performed by enzyme-linked immunosorbent assay (elisa) [12] and immuno-dot blotting [18] using the synthetic peptides pfsra p1 (nnkdnhnkkdtnenc); pfsra p2 (cendndeygnknkns); and pfsra p3 (csnnkkkkk ndkkkk). r1 peptide (lfskfgsrmhilkc) was used as control (r1 peptide blocks ama 1-ron 4 interaction), and naive plasma was used as negative control. the c-terminal α-pfsra human antibodies (α-pfsra p3) were affinity-purified from plasma samples of malaria-exposed children as described previously [19] . plasmodium falciparum strains 3d7 and w2mef were maintained in culture as described previously [16] . schizonts were purified using percoll-alanine gradient centrifugation [20] , followed by saponin lysis, and the recovered parasite pellets were further lysed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer. parasite culture supernatant or ringstage invasion supernatant were used as the source for native pfsra, which was detected by immunoblotting using primary α-rabbit antibodies to the 3 peptides (α-pfsra p1, α-pfsra p2, and α-pfsra p3). the immunoblots were developed using goat α-rabbit horseradish peroxidase-conjugated, secondary antibody, and enhanced chemiluminescence reagents (thermo scientific). erythrocyte-binding assay (eba) was performed using parasite culture supernatant as described previously [21] . immunofluorescence microscopy was carried out using p. falciparum (3d7)-infected erythrocytes smeared onto glass slides and fixed in prechilled methanol for 30 minutes. fixed erythrocytes were permeabilized using 0.1 % triton x-100 formulated in phosphate-buffered saline. nonpermeabilized, liquid immunofluorescence assay (ifa) was carried out as described previously [22] . after the washing step for both ifa conditions, the slides were blocked for 1 hour in pbs containing 3% bovine serum albumin. slides were probed with primary and secondary antibodies for the respective antigens and mounted in vectashield (vector laboratories inc) supplemented with 4′,6-diamidino-2-phenylindole for staining the nucleus. fluorescence microscopy was performed on an olympus fluorescence microscope (bx41). images captured were processed using the open access fiji-image j software (national institutes of health). to test for the internalization of α-pfsra p3 antibodies, α-pfsra p3 and α-pfmsp-1 19 polyclonal antibodies were incubated with tightly synchronized segmenting schizonts that were allowed to rupture and release merozoites. the viability of the released merozoites was assessed at the different stages (early, mid, and late) of an invading merozoite. for each stage, smears were prepared, fixed, and examined by immunofluorescence microscopy as described above. growth inhibition assays (gias) were performed as described previously [23] , and parasitemia levels were determined using flow cytometry on a bd fortessa x-20 with flo j software. erythrocyte invasion inhibitory effects of the α-pfsra antibodies were estimated by comparison of percentage of invasion of controls with test assay. in a systematic screen of uncharacterized p. falciparum proteins for potential blood-stage vaccine candidates, we performed data-mining analysis for genes with peak mrna expression levels in late schizogony using data from p. falciparum transcriptome studies [24, 25] and another study on the prediction of pfsub-1 protease specificity [26] . the details of all analyses have been described in the supplementary material (supplementary figure 1a and 1b) . overall, pfsra emerged as the top hit with both signal peptide and a predicted glycosylphosphatidylinositol attachment site. furthermore, we generated sequence alignments with other orthologs of pfsra and showed that the c-terminus of pfsra had 5 positionally conserved cysteine residues across the different plasmodium species orthologs (supplementary figure 2) . additional predictive analysis from psi-pred revealed that the pfsra protein sequence harbored coiled-coil signatures (supplementary figure 3) . this signature forms stable structures that elicit functional antibodies and was considered as a basis for the design of 3 pfsra chemically synthesized peptides used for antibody generation. despite several optimization procedures for expression in escherichia coli, attempts to express recombinant pfsra protein were unsuccessful. however, we designed 3 peptides for synthesis that corresponded to the conserved regions of pfsra in other plasmodium species orthologs ( figure 1a ). these pfsra-derived peptides (pfsra p1, pfsra p2, and pfsra p3) include coiled-coil signatures (supplementary figure 3) . immunization of rabbits with the 3 synthetic peptides (pfsra p1, pfsra p2, and pfsra p3) by genescript resulted in high titers of pfsra peptide-specific antibodies, which were used in subsequent experiments. preimmune sera were used for all immunoblotting as a negative control that did not detect native pfsra. as expected, α-eba-175 (r217) antibodies did not detect native eba-175 under reduced condition ( figure 1b , i). the antibodies (α-pfsra p1, α-pfsra p2, and α-pfsra p3) consistently detected multiple processed fragments (17, 32 , and 58 kda) of the native pfsra in ring-stage invasion supernatants ( figure 1b , ii-v) or parasite culture supernatants ( figure 1c , i-iv) and schizont lysates ( figure 1d , i-iv). to identify the fragment(s) of pfsra that possess erythrocyte-binding activity, we performed erythrocyte binding assays. the 32-kda fragment of pfsra was clearly detected in the eluate, suggesting that it bound erythrocytes. under higher exposure, the 113-kda full-length pfsra and the 58-kda processed fragment were also detectable (supplementary figure 3) , suggesting a weaker binding relative to the 32-kda fragment. of interest, the binding was sensitive to neuraminidase and trypsin treatments but resistant to chymotrypsin treatment ( figure 1f ). as a control, recombinant eba-175 (r217) bound erythrocytes with the same sensitivity to enzyme treatments ( figure 1f ). stage-specific expression analysis in p. falciparum asexual stages by ifas showed that all α-pfsra peptide antibodies labeled the surface membranes of merozoites in intact schizonts and released merozoites, respectively (figure 2a -c). however, α-pfsra antibodies did not label developing ring-stage parasites, which served as a useful internal control for subsequent experiments in asexual stages. colabeling of segmenting or rupturing schizonts and released merozoites with α-pfsra p1 antibodies and the micronemal marker α-pfama-1 showed no colocalization ( figure 2d ), even though reports exist about the circumferential staining pattern of pfama-1 [27] . however, α-pfsra p1 antibodies and α-pfmsp1 19 antibodies colocalized at the parasitophorous vacuole in late trophozoites, segmenting schizonts and released merozoites ( figure 2e ). in segmenting schizonts and released merozoites, both proteins colocalized on the merozoite surface ( figures 2e and 3a) . liquid ifa shows that both α-pfsra p1 antibodies and α-pfama-1 antibodies labeled the surface of nonpermeabilized merozoites ( figure 3b ). a control panel shows α-pfsra p1 labeling of an invading merozoite under permeabilized condition, but no labeling was observed for α-pfs48/45 antibodies ( figure 3b ). to determine the fate of native pfsra during invasion, invading merozoites at different time-points (early, mid, late, and postinvasion) were colabeled with α-pfsra p3 and α-pfmsp1 19 antibodies. we observed labeling of both α-pfsra p3 and α-pfmsp1 19 antibodies in all of the time points of invasion ( figure 3c and 3d) suggesting that a fragment or the unprocessed forms of pfsra are carried into erythrocytes during invasion. as a control, late-stage parasites and internalized merozoites were colabeled with α-pfsra p3 and the gametocyte surface marker α-pfs48/45 antibodies. whereas α-pfsra p3 antibody labeled internalized merozoite, no labeling was observed with α-pfs48/45 antibody ( figure 3b ). a panel of plasma samples from malaria-infected children resident in 3 malaria-endemic sites in ghana (accra, navrongo, and kintampo) was evaluated for reactivity to pfsra synthetic peptides by elisa. plasma antibodies from children in accra and navrongo showed no reactivity to the r1 peptide beyond background (normal human serum [nhs]) levels, whereas those from kintampo were only slightly above background; however, this difference was statistically significant (p = .04) ( figure 4a ). plasma samples from all 3 sites appeared to recognize all 3 pfsra peptides, and all groups showed reactivity above background levels ( figure 4b -d). statistically, these differences in reactivity against pfsra p1 were significant compared with nhs for plasma from navrongo (p < .0001) and accra (p < .0001) but not kintampo ( figure 4b ). reactivity for pfsra p2 was significant compared with nhs for plasma from kintampo (p < .0001) and accra (p = .004) but not navrongo ( figure 4c ). however, the reactivity against pfsra p3 was significant for all 3 sites (kintampo, p < .0001; navrongo, p = .02; and accra, p < .0001) ( figure 4d) . pre-immune a, pfsra possesses a signal peptide and a predicted glycosylphosphatidylinositol (gpi) anchor. cut-1 and -2 represents pfsub-1 cleavage sites analyzed by the prediction of protease specificity tool. cut-3 represents the gpi-transamidase cleavage site for the predicted gpi attachment signal. pfsra p1, pfsra p2, and pfsra p3 designate conserved regions of pfsra in different orthologs that possesses coiled-coil signatures for which chemically synthesized peptides were designed. c designates the carboxyl terminus, and n designates the amino terminus. b, α-pfsra antibodies detect multiple processed fragments of native pfsra during immunoblotting of 3d7 ring-stage invasion supernatants (iss) that were probed with mouse α-eba-175 (r217) antibody (i), rabbit α-pfsra p1 antibody (lot no.: a417040387) (ii), α-pfsra p2 antibody (lot no.: a417040332) (iii), and α-pfsra p3 antibody (lot no.: a417040334) (iv). the colored bars at the far right represent the predicted processed fragments (70, 58, 32, and 17 kda) (v). c, anti-pfsra antibodies detect multiple processed fragments of native pfsra during immunoblotting of 3d7 parasite culture supernatants (css) that were probed with preimmune sera (i), α-pfsra p1 antibody (ii), α-pfsra p2 antibody (iii), and pfsra p3 antibody (iv). d, anti-pfsra antibodies detect multiple processed fragments of native pfsra during immunoblotting of 3d7 schizont lysates (sls) that were probed with preimmune sera (i), α-pfsra-p1 antibody (ii), α-pfsra-p2 antibody (iii), and pfsra-p3 antibody (iv). e, the colored bars at the far right represent the predicted processed fragments (70, 58, 32, and 17 kda) (v). f, recombinant eba-175 was used as a control, and it bound erythrocytes in a neuraminidase-sensitive, trypsin-sensitive, and chymotrypsin-resistant manner. similarly, the 32-kda processed fragment of native pfsra bound erythrocytes in a neuraminidase-sensitive, trypsin-sensitive, and chymotrypsin-resistant manner. abbreviations: ct, chymotrypsin; gh, normal human erythrocyte ghost; nt, neuraminidase; tt, trypsin; ut, untreated control. consistent with the elisa data, we also established by immuno-dot blotting that plasma samples from malaria-infected children recognized pfsra p1 ( figure 5a ). thus, α-pfsra p3specific human antibodies against the c-terminus of the protein were purified from a pooled sample of plasma from kintampo children that showed immunoreactivity ( figure 4d ). consistent with the data from rabbit α-pfsra antibodies ( figures 2e and 3a) , human α-pfsra p3 immuno-affinity purified antibodies stained schizonts and released merozoites that colocalized with α-pfmsp1 19 antibodies on the merozoite surface ( figure 5b ). we performed stage-specific expression analysis in gametocytes (stage iii-v) by microscopy and observed that α-pfsra p3 antibodies specifically labeled the membranes of gametocytes, whereas the control antibody (pfmsp1 19 ) did not label gametocytes ( figure 6a) (stage ii-v) with the respective α-pfsra peptide antibodies and the gametocyte surface marker α-pfs48/45 antibody and showed close colocalization that appeared to be stage-dependent ( figure 6b-d) . furthermore, pfsra expression in gametocytes appeared not to be sex-specific, with α-pfsra p1 antibodies labeling both male and female gametocytes and α-tubulin antibodies labeling only male gametocyte (supplementary figure 5) . we evaluated the invasion inhibitory activity of α-pfsra antibodies against p. falciparum 3d7 and w2mef and showed that all pfsra peptide antibodies exhibited 70%-80% inhibition at 750 μg/ml, whereas the preimmune control did not show any inhibition (supplementary figure 6a and 6b ). because all 3 pfsra peptide antibodies were inhibitory at higher concentrations (100-750 μg/ml) (supplementary figure 6a and 6b) , we have shown that, whereas the preimmune sera and a control immunoglobulin g (igg) did not inhibit parasite invasion at lower concentrations (25-75 μg/ml), α-pfsra antibodies exhibited a concentration-dependent inhibition of parasite invasion ( figure 7a ). of all 3 of the pfsra peptide antibodies tested, only α-pfsra p1 antibodies showed 60% inhibition of parasite invasion at 75 μg/ml. as a control, anti-basigin antibodies showed 75% inhibition of parasite invasion at 10 μg/ml ( figure 7a ). to exclude the possibility that serum contaminants might be interfering with parasite invasion, we generated antibodies against a shorter construct of r1-peptide using the same purification procedures for α-pfsra peptide-specific antibodies, and no inhibition of parasite invasion was observed ( figure 7a ). because all 3 α-pfsra antibodies were inhibitory at 100-750 μg/ml, we tested combinations of α-pfsra peptide antibodies against 3d7 and observed that this strategy did not greatly impact on parasite invasion inhibition ( figure 7b ). furthermore, we performed invasion inhibition assays with 2 ghanaian clinical isolates (misa010 and misa011) and observed similar patterns of parasite invasion inhibition for α-pfsra peptide antibodies ( figure 7c and 7d) . the focus of this work was the identification and functional characterization of a potential blood-stage malaria vaccine candidate (pfsra). using bioinformatics and data-mining analysis of published p. falciparum transcriptomes and proteomes [24, 25] , we identified pfsra to be reminiscent of a surface protein, defined by the presence of a signal peptide and a predicted glycosylphosphatidylinositol attachment signal [28, 29] . indeed, pfsra has been shown to have pfsub-1 cleavage sites that were assigned using the prediction of protease specificity analysis platform [25] . the presence of these proteolytic cleavage sites in pfsra may allude to processing events occurring prior to invasion of erythrocytes. besides, proteolytic processing in the malaria parasite has been shown to be relevant for cascades of interaction occurring at the parasite-host cell interface [21, 30] . furthermore, because orthologs are good candidates for multispecies vaccines, we generated sequence alignments with other pfsra orthologs, which showed that the c-terminus of pfsra has 5 positionally conserved cysteine residues across the different species orthologs. our attempt to express the recombinant pfsra that possesses 67% unstructured regions was unsuccessful. similarly, others have attempted the expression of the mature recombinant pfsra in hek293e cells using a codon-optimized gene (geneart) and have also been unsuccessful [28] . considering that pfsra is an asparagine-rich protein with numerous potential n-linked glycosylation sites, we did not attempt expression in insect cells because the system produces proteins with more complex n-linked glycosylation [31] that may not present the relevant sugar epitopes of native glycosylation [32] . therefore, the challenges associated with the expression of recombinant pfsra necessitated the design of chemically synthetized peptides for an epitope-based vaccine strategy. plasmodium falciparum surface-related antigen harbors coiled-coil domains that are known to be less polymorphic [33, 34] , and they form stable structures that elicit functional antibodies that block relevant domains in many organisms [35] [36] [37] . interestingly, these domains have been evaluated as potential targets for peptide-based vaccines [38] [39] [40] . the 3 pfsra peptide antibodies from rabbits specifically detected breakdown products of native pfsra in ring-stage invasion supernatant or parasite culture supernatant and schizont lysates. this indicated that pfsra synthetic peptides are antigenic mimics of the native parasite protein. our data showed that pfsra is postsynthetically processed by cleavage into parasite culture supernatant, and this is consistent with the pfsub-1 cleavage sites it harbors. the merozoite surface is remodeled by a series of proteolytic processing events; the physiological relevance of these events in the malaria parasite remains poorly described. however, there are reports suggesting that proteolytic processing may result in activation, structural rearrangement, or acquisition of other new functional properties of native parasite proteins [41] . we have described the fate and shedding pattern of native pfsra by temporal immunofluorescence imaging using α-pfsra and α-pfmsp1 19 antibodies that bound the merozoite surface and were internalized during erythrocyte invasion. consistent with this observation is a previous report that α-pfmsp1 19 antibodies were carried into invaded erythrocytes, disrupted intra-erythrocytic development, and inhibited erythrocyte invasion [42] [43] [44] . although the molecular mechanism underlying the internalization of antibodies remains debatable, it was suggested that the tight junction between the merozoite and the erythrocyte might consist of transient interactions that allow the passage of antibodies or surface proteins [19] . because all rabb3it α-pfsra peptide antibodies recognized different pfsra polypeptide fragments in ring-stage invasion supernatant or parasite culture supernatant and schizont lysates at varying thresholds, it was expedient to investigate whether processing could influence differential subcellular localization of pfsra in the parasite. interestingly, all 3 rabbit α-pfsra peptide antibodies showed circumferential association on the merozoite surface at the timing of schizont rupture and merozoite release. similarly, we performed colabeling in ifas with gametocytes (stage ii-v) using all 3 rabbit α-pfsra peptide antibodies with the gametocyte surface marker pfs48/45. a clear, punctate rim-fluorescence pattern was observed for all 3 rabbit α-pfsra peptide antibodies that appeared to colocalize with pfs48/45 in a stage-dependent manner based on the colocalization coefficient. therefore, the consistency in the staining patterns of all 3 rabbit α-pfsra peptide antibodies in both asexual and gametocyte stages suggested that proteolytic processing of pfsra does not cause changes in the subcellular localization of the protein. although the distribution of proteins in male or female gametocytes could be linked to functional divergence between the sexes [45] , we observed the expression of pfsra in both male and female gametocytes. consistent with this observation is an existing report on plasmodium berghei gametocyte egress and sporozoite traversal protein (pbgest) expression in both sexes [46] . also, it was imperative to determine whether pfsra in released merozoites was accessible to humoral immune surveillance during the short period of erythrocyte invasion. our serological screens, buttressed by immuno-dot blot assays, with plasma samples from malaria-infected children residing at different endemic sites showed differences in total igg recognition frequencies for pfsra peptides. this could be linked to varying transmission intensity rates as reported for samples collected from different endemic sites in previous studies [33, 47] . in most cases, the reactivity of all 3 pfsra synthetic peptides was low, and the likely explanation for this could be the hindered accessibility of pfsra in the native context. however, our data from ifas showed that the immuno-affinity purified, human α-pfsra peptide antibodies labeled native pfsra, which suggests that malaria-infected populations have naturally acquired antibodies against pfsra. generally, several receptors have been characterized based on their sensitivities to different enzyme treatments. notably, neuraminidase removes sialic acids from glycophorins, trypsin cleaves peptide backbones of several receptors (glycophorin a, glycophorin c, and complement receptor 1), and chymotrypsin cleaves glycophorin b and complement receptor 1, among others [48] . we have shown that the 32-kda-processed fragment of native pfsra binds normal human erythrocytes, but the molecular identity of the receptor remains unknown. however, we have classified the putative receptor for pfsra as sialic acid-dependent on the basis of its binding specificity, which is sensitive to treatments with both neuraminidase and trypsin but resistant to chymotrypsin, a binding phenotype that fits the description of the receptor glycophorin c. the enzyme sensitivity profile of pfsra binding to erythrocytes is similar to that observed for pfeba-140 (region ii), the parasite ligand for glycophorin c [49] . additional investigations are required to determine whether pfsra also interacts with glycophorin c, possibly via a different binding site. our data revealed that pfsra peptides induce functional antibodies that inhibited p. falciparum erythrocyte invasion of both laboratory strains and clinical isolates. the observed invasion inhibitory activity of rabbit α-pfsra peptide antibodies could be attributed to indirect effects of antibody binding to the merozoite surface or direct inhibition of proteolytic processing events. the demonstration that pfsra synthetic peptides induced erythrocyte invasion inhibitory antibodies and the successful purification of a limited amount of pfsra-specific human antibodies from patient plasma suggested that the synthetic peptides possessed structural integrity or conformation that mimics the native pfsra. in summary, this study has provided relevant new information regarding the proteolytic processing of pfsra that supports the idea of targeting these cleavage events for development of antimalarial therapies. the expression of pfsra in late stages of gametocytes is an unprecedented opportunity that should be explored for potential transmission-blocking vaccines. also, pfsra-specific immune responses triggered in natural infections may inform the inclusion of pfsra as a candidate for epitope-based, blood-stage malaria vaccine development. invasion of red blood cells by malaria parasites plasmodium falciparum is able to invade erythrocytes through a trypsin-resistant pathway independent of glycophorin b complement receptor 1 is a sialic acid-independent erythrocyte receptor of plasmodium falciparum an egf-like protein forms a complex with pfrh5 and is required for invasion of human erythrocytes by plasmodium falciparum multiprotein complex between the gpi-anchored cyrpa with pfrh5 and pfripr is crucial for plasmodium falciparum erythrocyte invasion essential role of the pfrh5/pfripr/cyrpa complex during plasmodium falciparum invasion of erythrocytes p113 is a merozoite surface protein that binds the n terminus of plasmodium falciparum rh5 the future for blood-stage vaccines against malaria malaria vaccine development based on merozoite surface proteins of plasmodium falciparum phase i clinical trial of a recombinant blood stage vaccine candidate for plasmodium falciparum malaria based on msp1 and eba175 new antigens for a multicomponent blood-stage malaria vaccine genome sequence of the human malaria parasite plasmodium falciparum immunoinformatics: an integrated scenario the phyre2 web portal for protein modeling, prediction and analysis analysis of erythrocyte invasion mechanisms of plasmodium falciparum clinical isolates across 3 malaria-endemic areas in ghana assessing the impact of differences in malaria transmission intensity on clinical and haematological indices in children with malaria mpp1 directly interacts with flotillins in erythrocyte membrane-possible mechanism of raft domain formation sequential processing of merozoite surface proteins during and after erythrocyte invasion by plasmodium falciparum separation of viable schizont-infected red cells of plasmodium falciparum from human blood multiple plasmodium falciparum merozoite surface protein 1 complexes mediate merozoite binding to human erythrocytes members of the low-molecular-mass rhoptry protein complex of plasmodium falciparum bind to the surface of normal erythrocytes current molecular and emerging nanobiotechnology approaches for the detection of microbial pathogens the transcriptome of the intraerythrocytic developmental cycle of plasmodium falciparum discovery of gene function by expression profiling of the malaria parasite life cycle identification and stoichiometry of glycosylphosphatidylinositol-anchored membrane proteins of the human malaria parasite plasmodium falciparum neutralization of plasmodium falciparum merozoites by antibodies against pfrh5 a library of functional recombinant cell-surface and secreted p. falciparum merozoite proteins global identification of multiple substrates for plasmodium falciparum sub1, an essential malarial processing protease processing of plasmodium falciparum merozoite surface protein msp1 activates a spectrin-binding function enabling parasite egress from rbcs plasmodium falciparum merozoite surface antigen, pfrh5, elicits detectable levels of invasion-inhibiting antibodies in humans suggestive evidence for darwinian selection against asparagine-linked glycans of plasmodium falciparum and toxoplasma gondii rapid identification of malaria vaccine candidates based on alpha-helical coiled coil protein motif sequence conservation in plasmodium falciparum alpha-helical coiled coil domains proposed for vaccine development complexes of neutralizing and non-neutralizing affinity matured fabs with a mimetic of the internal trimeric coiled-coil of hiv-1 gp41 platform technology to generate broadly cross-reactive antibodies to α-helical epitopes in hemagglutinin proteins from influenza a viruses templatebased coiled-coil antigens elicit neutralizing antibodies to the sars-coronavirus isolation of peptides that mimic epitopes on a malarial antigen from random peptide libraries displayed on phage phage-displayed mimotopes elicit monoclonal antibodies specific for a malaria vaccine candidate induction of humoral immune response against plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library extensive proteolytic processing of the malaria parasite merozoite surface protein 7 during biosynthesis and parasite release from erythrocytes formation of the food vacuole in plasmodium falciparum: a potential role for the 19 kda fragment of merozoite surface protein 1 (msp1(19)) antibodies inhibit the protease-mediated processing of a malaria merozoite surface protein analysis of antibodies directed against merozoite surface protein 1 of the human malaria parasite plasmodium falciparum proteome analysis of separated male and female gametocytes reveals novel sex-specific plasmodium biology pbgest mediates malaria transmission to both mosquito and vertebrate host plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif variation in plasmodium falciparum erythrocyte invasion phenotypes and merozoite ligand gene expression across different populations in areas of malaria endemicity characterization of a plasmodium falciparum erythrocyte-binding protein paralogous to eba-175 we thank dr anthony holder for providing msp1 19 antibody and the west african centre for cell biology of infectious pathogens (waccbip) for providing anonymized archived plasma sample from children resident at kintampo, navrongo, and accra. anti-plasmodium falciparum 48/45-kda gamete surface protein (pfs48/45) monoclonal antibody was contributed by louis h. miller and alan saul through the national institute for allergy and infectious diseases (niaid) bei resources (product number mra-316a). erythrocyte binding antigen, region ii was also contributed by niaid bei resources (product number mra-1162).disclaimer.the views expressed in this publication are those of the author(s) and not necessarily those of the supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.notes key: cord-007220-nlsduenh authors: black, r. e.; merson, m. h.; rahman, a. s. m. m.; yunus, m.; alim, a. r. m. a.; huq, i.; yolken, r. h.; curlin, g. t. title: a two-year study of bacterial, viral, and parasitic agents associated with diarrhea in rural bangladesh date: 1980-11-17 journal: j infect dis doi: 10.1093/infdis/142.5.660 sha: doc_id: 7220 cord_uid: nlsduenh enteric pathogens associated with diarrhea were studied for two years at a diarrhea treatment center in rural bangladesh. enterotoxigenic escherichia coli (etec) was the most frequently identified pathogen for patients of all ages. rotavirus and etec were isolated from ∼50% and ∼25%, respectively, of patients less than two years of age. a bacterial or viral pathogen was identified for 70% of these young children and for 56% of all patients with diarrhea. most etec isolates were obtained in the hot dry months of march and april and the hot wet months of august and september. rotavirus identification peaked in the cool dry months of december and january, but infected patients were found year-round. the low case-fatality rates for patients with watery diarrhea and substantial dehydration further document the usefulness of treating patients with diarrhea with either a glucoseor sucrose-base electrolyte solution such as those used in this treatment center. diarrhea, especially in children, is a major cause of morbidity and mortality in developing countries [1] . although classic enteric bacterial pathogens are not isolated from most patients with diarrhea, recent studies indicate that enterotoxigenic escherichia coli (etec) and rotaviruses may frequently cause diarrhea [2] [3] [4] . although these agents have been isolated from patients in developing countries, the studies have been brief, and the results do not permit an accurate assessment of the relative importance of the various causative agents. we therefore studied patients during a two-year period at a treatment center in rural bangladesh to determine the frequency, severity, and seasonality of diarrhea associated with etec, rotavirus, and other enteric agents. population studied. the matlab field research area of the international centre for diarrhoeal received for publication april 18, 1980 , and in revised form july 28, 1980. we thank g. kibriya, s. huda, and s. rahman for technical assistance and the staff of the matlab treatment center for patient care. please address requests for reprints to dr. r. e. black at his present address: center for vaccine development, university of maryland school of medicine, 29 south greene street, baltimore, maryland 21201. 660 diseases research, bangladesh (formerly the cholera research laboratory) is located in a riverine, rural area. a central treatment facility, staffed by physicians and paraprofessionals, provides free therapy for patients with diarrhea who come directly or are brought by speedboat or jeep ambulances stationed in the field area. for a twoyear period data for all patients who lived in the research area and were treated for diarrhea were gathered for a study of enteric pathogens. between february 1977 and january 1978 (year 1), 8, 139 patients were included in the study, and from february 1978 to january 1979 (year 2), 6,352 patients were enrolled. in years 1 and 2, respectively, 43070 and 41 % of all patients were children less than two years of age; 22 % each year were children two through nine years of age, and 35% and 37070 were~10 years of age. when the patient visited the treatment center, a physician or nurse did a standard clinical evaluation of the degree of dehydration on the basis of signs such as skin elasticity, sunken eyes or fontanelle, pulse,. respirations, volume of urine, and level of consciousness. dehydration was classified as none, mild (corresponding to <5 % loss of body weight), moderate (5%-10%), or severe (>10%). patients judged to have no or mild dehydration were treated with oral electrolyte solution (composition in milliequivalents/liter: na+, 90; k+, 20; ci-, 80; hc0 3 -, 30) containing 20 g of glucose/liter (year 1) or 40 g of sucrose/liter (year 2) [5] . patients with moderate or severe dehydration were given a solution (composition in milliequivalents/ liter: na+, 134; k+, 13; cl-, 86; hc0 3 -, 48) iv to replace their estimated fluid deficit and were also given the oral solution for maintenance therapy. because laboratory findings were not available at the time of admission, the assessment of dehydration and the choice of treatment were not influenced by the identification of an enteropathogen. only persons whose histories indicated a dysenteric illness were given ampicillin or trimethoprimsulfamethoxazole on admission. laboratory studies. on admission rectal swabs were taken from all patients and plated directly on salmonella-shigella, trypticase-tellurite-gelatin, and macconkey's agars. part of each specimen was enriched overnight in bile peptone and then plated on trypticase-tellurite-gelatin agar. the plates were examined for salmonellae, shigellae, and vibrios [6] by standard methods. vibriolike colonies identified on trypticase-tellurite-gelatin plates were further characterized in terms of biochemical, serotypic, and salt tolerance properties and classified as vibrio cholerae group 0: 1, non-group 0: 1 vibrios, vibrio parahaemolyticus, or group f vibriolike organisms [6, 7] . a sample of patients, stratified by age group, was further studied for etec infection. during year 1, studies were performed with 4,498 patients (52010 of the patients less than two years of age, 67010 of the patients two through 19 years of age, and 79010 of the patients~20 years of age). during year 2 the sample was modified because of the results from year 1, and 2,042 patients (19010 of the patients less than two years of age, 66010 of the patients two through 19 years of age, and 40010 of the patients~20 years of age) were studied. from each culture selected for toxin studies, 10 lactosepositive colonies with typical e. coli morphology were removed from macconkey's agar plates and pooled on nutrient agar slants. these pools were tested with the chinese hamster ovary cell assay for heat-labile toxin (lt) and with the infant mouse assay for heat-stable toxin (st) [8] . during year 1, fresh stool specimens from 40010 of the patients were treated with saline and iodine preparations and examined for intestinal parasites. in this study only stools containing vegetative-stage giardia lamblia or entamoeba histolytica were considered positive. beginning in december 1977, a second rectal 661 swab was taken from each patient and refrigerated in phosphate-buffered saline for less than one month (and generally less than one week) before being tested by enzyme-linked immunosorbent assay for rotavirus antigen [9, 10] . positive results were confirmed by testing about 30 positive specimens per month with an enzyme-linked immunosorbent assay involving wells coated with immune and nonimmune sera [11] . of 404 specimens retested, 380 (94010) were confirmed as positive. analysis. the frequency with which patients were infected with the pathogens was calculated directly; however, since only subgroups of patients were tested for etec and for parasites, those results had to be extrapolated for the entire group of patients. because rotavirus was tested for only the last two months of year 1, we included these data in the analysis of seasonality, degree of dehydration, and hospital death rates but did not try to determine the overall frequency of infection with rotavirus in year 1. for both year 1 and year 2, etec was the pathogen most frequently isolated from all patients and adults; however, it was the second most often isolated (after rotavirus) from children less than two years of age and the second most often isolated (after v. cholerae) from children two through nine years of age. most of the etec produced only st, fewer produced both st and lt, and still fewer produced only lt (table 1) . rotavirus was found in the stools of 46010 of the children less than two years of age and in the stools of 12010 and 9010, respectively, of older children and adults. v. cholerae group 0: 1 was rarely identified for children less than two years of age but was an important pathogen in older children and adults, while non-group 0: 1 vibrios were found in the stools of 4010-11 010 of the patients of each age group for both years of the study. group f vibriolike organisms were associated with diarrhea for 3010 of all patients in year 1 but rarely in year 2. v. parahaemolyticus and salmonella were rarely isolated during the study, but shigella was isolated from 5010-6010 of all of the patients treated at the center for diarrhea. vegetative g. lamblia was identified from 4010 of the older children and adults, and vegetative e. histolytica was identified from 13010 of the older children and 8010 of the adults. for 2,039 patients tested for group f vibriolike organisms note. this study was conducted at a diarrhea treatment center (matlab) in rural bangladesh. * st = strain producing heat-stable enterotoxin; lt = strain producing heat-labile enterotoxin. bacterial pathogens, including etec, and for rotavirus (but not for parasites) in year 2, an enteropathogen was identified for 70% of the children less than two years of age, 47070 of the children two through nine years of age, and 56% of the adults. infection with more than one pathogen was found for about 20% of all patients. the seasonal patterns of occurrence of the three most common enteric pathogens are illustrated in figure 1 . infections with v. cholerae were decidedly seasonal, with a peak occurrence during the hot monsoon period. in contrast, infections with etec had two seasonal peaks, one in the hot dry months of march and april and the other in august through september. although the highest numbers of patients with diarrhea associated with rotavirus were seen in the cool dry months of december 1977 and january 1978, there was no comparable peak in year 2 of the study. the number of infections with non-group 0: 1 vibrios and group f vibriolike organisms peaked in april and may, whereas the incidence of shigella infections peaked between june and august. no seasonal pattern could be determined for infections with g. lamblia or e. histolytica. the degree of dehydration at the time that patients visited the treatment center during year 1 was tabulated for those infected with v. cholerae, etec, or rotavirus after data for patients with known mixed infections were excluded. among children less than two years of age, moderate or severe dehydration requiring inpatient therapy occurred in 24 (40%) of 60 patients with cholera; this proportion was significantly higher than the 68 (20%) of 340 and 76 (16%) of 473 patients with etec and rotavirus diarrhea, respectively (both p < 0.001, x 2 ) . among adults moderate or severe dehydration was found in 307 (77%) of 398 patients with cholera and 269 (43%) of 624 with etec diarrhea (p< 0.001). there were no significant differences in the levels of dehydration accompanying diarrhea associated with etec of different toxin types in children or adults. in spite of substantial dehydration in patients of all ages, the hospital case-fatality rate for this twoyear period was very low (table 2) . furthermore, there were no significant differences in fatality rate between year 1, when glucose-electrolyte oral therapy solution was used, and year 2, when a sucrose-base solution was used. the fatality rate for patients with diarrhea associated with shigella was higher than those for patients infected with v. cholerae, both group 0: 1 (fisher's exact test, p < 0.01) and non-group 0: 1 (p < 0.02), rotavirus (p < 0.01), or etec (p < 0.001). that relatively few young children had cholera whereas many had diarrhea caused by other pathogens suggests that v. cholerae may have a pattern of transmission different from that of other agents or, less likely, that infants have substantial immunity to v. cholerae but not to other organisms. a bacterial or viral pathogen could be identified for 70070 of the children less than two years of age and for a majority of all patients. these proportions are substantially higher than those observed before the recognition of etec and rotavirus [14, 15] . furthermore, g. lamblia and e. histolytica, which were identified from 6070 of the patients in year 1, probably caused some of the episodes in year 2. the failure to find a pathogen for a minority of patients may be due in part to the relative insensitivity of some of the techniques used, such as testing a pool of 10 colonies of e. coli for toxin production rather than testing multiple individual isolates [8] . also, the assays we used could not detect some agents of diarrhea, including bacterial pathogens such as invasive or enteropathogenic e. coli, campylobacter fetus, and yersinia enterocolitica and viruses such as parvovirus-like agents and other recently reported particles (adenoviruses, caliciviruses, coronaviruslike agents) that may be associated with diarrhea [16] [17] [18] [19] . patients with cholera were generally more severely dehydrated than patients with diarrhea associated with other pathogens, including ltand st/lt-producing e. coli, which produce an enterotoxin similar to that of v. cholerae. this finding suggests that these organisms differ in the amount of toxin released or in other properties of virulence such as the ability to adhere to the mucosal surface and to colonize the small bowel. the low case-fatality rates for patients who had watery diarrhea and substantial dehydration at the time that they visited the treatment center further document the effectiveness of oral rehydration therapy in conjunction with sufficient iv fluid to correct shock. recent comparative studies have demonstrated that a sucrose-base electrolyte formula is nearly as good as a glucose-base formula for oral fluid replacement [5, 20] . our experience with the two solutions, each used for one year of this study in a center annually treating 6,000-8,000 patients with diarrhea, supports this conclusion in that the case-fatality rates for the two years of study were comparable. etec organisms were the pathogens most frequently isolated from patients of all ages and were the second most frequently isolated (after rotavirus) from young children coming to the treatment center in rural bangladesh. our study indicates that rotavirus is the most common pathogen for children less than two years of age visiting a treatment center for diarrhea; this finding is in agreement with other studies in both developed and developing countries [3, 4, 12, 13] . older children and adults may also have had symptomatic infections caused by rotavirus, but because of frequent concomitant infections with other pathogens it is difficult to determine whether rotavirus caused diarrhea for the infected adults in this study. in this highly endemic area cholera remains an important cause of life-threatening diarrhea. although few children less than two years of age had cholera, v. cholerae was the most frequently isolated pathogen from children two through nine years of age and was the second most frequently isolated (after etec) in adults. the observation selective primary health care. an interim strategy for disease control in developing countries enterotoxigenic escherichia coli and diarrheal disease in mexican children enterotoxigenic escherichia coli and reovirus-like agent in rural bangladesh comparison of glucose with sucrose in oral rehydration therapy of rotavirus diarrhea in infants and young children polyphasic taxonomy of the genus vibrio: numerical taxonomy of vibrio cholerae, vibrio parahaemolyticus, and related vibrio species the use of colony pools for diagnosis of enterotoxigenic escherichia coli diarrhea enzyme-linked immunoassay (elisa) for detection of human reovirus-like agent of infantile gastroenteritis elisa for rotavirus analysis of nonspecific reactions in enzyme-linked immunosorbent assay testing for human rotavirus human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children comparison of human rotavirus disease in tropical and temperate settings infection and disease in a group of south indian families. iv. bacteriologic methods and a report of the frequency of enteric bacterial infection in preschool children acute diarrheal disease in less developed countries. i. an epidemiological basis for control epidemic yersinia enterocolitica infection due to contaminated chocolate milk related vibrio in stools recent advances in viral gastroenteritis characteristics of noncultivable adenoviruses associated with diarrhea in infants: a new subgroup of human adenoviruses oral rehydration in rotavirus diarrhoea: a double blind comparison of sucrose with glucose electrolyte solution key: cord-275108-snqbrxgr authors: daverio, marco; amigoni, angela; cavicchiolo, maria elena title: testing for novel coronavirus antibodies: a necessary adjunct date: 2020-05-22 journal: j infect dis doi: 10.1093/infdis/jiaa283 sha: doc_id: 275108 cord_uid: snqbrxgr nan we read with interest the article by cowling and aiello [1] about the use of proactive public health measures to help slow the spread of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (sars-cov-2) over the world. the size of this pandemic and the exorbitant increase in number of patients seems to have become unstoppable. more than 200 countries are now involved in this emergency, and, as of 30th may 2020, about 6.1 million persons have tested positive and > 360 000 patients have died [2] . it now seems clear that the implementation of such measures has unfortunately not been sufficient to contain the outbreak, considering that this dramatic increase in numbers occurred within only a few months after the first case in wuhan, china [3] . this new endemic disease has proved itself not only a worldwide clinical disaster but also an economic disaster. it caused the lockdown of economic activities and the collapse of worldwide markets [4] . furthermore, the numbers of individuals infected are difficult to estimate, owing to the presence of both sars-cov-2-positive asymptomatic individuals and symptomatic, self-isolating individuals in whom nasopharyngeal swab samples were not obtained. many experts estimate that the real number of persons positive for sars-cov-2 is underreported by up to 10-fold [5, 6] . the problem of asymptomatic individuals spreading sars-cov-2 is critical. knowing the number of truly infected people is important not only for epidemiological reasons but also in order to restart the world economy, which would otherwise be blocked until an uncertain date in the future. the most feasible solution to knowing how many people have actually been infected lies in the possibility of carrying out large-scale serosurveys, evaluating antibody titers in individuals who have not undergone nasopharyngeal swab viral rna testing, particularly in heavily infected areas. it may seem a waste of resources but knowing people's serological status regarding sars-cov-2 could allow those who were previously infected to return to work and restart the world economy before the entire pandemic is over. the revenues obtained from a recovery of the economic system would far exceed the expenses needed to support the "antibody search" policy. we therefore suggest, along with all the necessary public preventive measures, performing target testing for sars-cov-2 antibodies in particular subpopulations, for example, young and healthy persons who can actively work. this strategy could accurately identify previously infected individuals who could return to work. in this way the economy could be relaunched while minimizing the risk of worsening the epidemic. public health measures to slow community spread of covid-19 covid-19 coronavirus pandemic a pneumonia outbreak associated with a new coronavirus of probable bat origin economic impacts of wuhan 2019-ncov on china and the world estimation of covid-19 outbreak size in italy correcting under-reported covid-19 case numbers key: cord-278260-3o91v72a authors: halstead, scott b; katzelnick, leah title: covid 19 vaccines: should we fear ade? date: 2020-08-12 journal: j infect dis doi: 10.1093/infdis/jiaa518 sha: doc_id: 278260 cord_uid: 3o91v72a might covid 19 vaccines sensitize humans to antibody dependent enhanced (ade) breakthrough infections? this outcome is unlikely because coronavirus diseases in humans lack the clinical, epidemiological, biological or pathological attributes of ade disease exemplified by the dengue viruses (denv). in contrast to denv, sars and mers covs predominantly infect respiratory epithelium, not macrophages. severe disease centers on older persons with pre-existing conditions and not young infants or individuals with previous coronavirus infections. live virus challenge of animals given sars or mers vaccines has resulted in vaccine hypersensitivity reactions (vah), similar to those in humans given inactivated measles or respiratory syncytial virus vaccines. safe and effective covid 19 vaccines must avoid vah. a c c e p t e d m a n u s c r i p t 3 introduction. not since pandemic smallpox or the 1918 influenza have humans confronted a epidemic viral pathogen as successful as sars cov-2, a member of a family of viruses that cause serious diseases in many vertebrates. [1] it has proved difficult to achieve robust vaccine protection against avian, bovine, porcine, canine and feline coronaviruses, failures sometimes attributed to "antibody dependent enhancement (ade)." [2] the possibility that a sars cov-2 vaccine may sensitize recipients to ade has received considerable scrutiny. [3] on inspection, ade is not one but two vaccine-related immunopathological phenomena: intrinsic ade (iade) and vaccine hypersensitivity (vah). iade describes interactions between igg antibody and microbial pathogen immune complexes that attach to fc receptors to initiate infection but also enhance replication of the microbe by suppressing innate cellular immune defenses. [4, 5] vah was first described in humans in the early 1960s, after formalin-inactivated measles vaccines were introduced in the us and europe. within months large numbers of vaccinated children developed a severe breakthrough disease, called "atypical measles." [6] a similar outcome, "vaccine associated enhanced respiratory disease (vaerd)," was observed in infants, 4 -12 months of age, who were given formalininactivated respiratory syncytial virus (rsv) and a few months later infected by rsv. [7] the outcomes observed were attributed to delayed type hypersensitivity and/or an arthus reaction. [8] lung lesions revealed damage to parenchymal tissue, a pulmonary neutrophilia with abundant macrophages and lymphocytes and excess eosinophils. from studies in laboratory animals, it is thought that formalin de-conformed viral antigens raised nonprotective antibodies that led to a th2 polarization of the immune response and a deficit of cytotoxic t cells. it was also the case that mice immunized with rsv inactivated with uv radiation, a purified fusion (f) protein, or a vaccinia-rsv replicative construct experienced similar pathology following challenge with wild-type virus. a similar pathological response has repeatedly accompanied live virus challenge in several species of laboratory animals a c c e p t e d m a n u s c r i p t 4 vaccinated with sars and mers cov constructs, with and without adjuvants. [9, 10] vah may best be defined as a coombs type iii antigen hypersensitivity. it should be emphasized there is no formal proof that vaerd is antibody mediated. the mechanism(s) of the postmeasles vaccine disease enhancement and its similarity to vaerd are not known. the biological behavior of some coronaviruses in non-human species together with evidence that human coronavirus antibodies enhanced infection of sars or mers covs in fc receptor-bearing cells, in vitro, have led to speculations that ade contributes to disease severity in humans. [11] it has been reported that high levels of sars cov-1 igg antibodies circulated in severe sars cases and that anti-s igg neutralizing antibody (nab) responses developed significantly faster after the onset of clinical symptoms in fatal compared with recovered cases leading some to attribute enhanced tissue damage to ade. [12] because sera from sars or mers vaccinated animals sera enhanced cov infections, in vitro, it was assumed that post-vaccination pathologies, too, were ade responses. [13] dengue ade if sars or mers infection outcomes are affected by iade they should have epidemiological and disease features in common with denv. these are compared in table 1 . in vivo, iade requires an initial immunological event, termed "sensitization." in dengue, this occurs in three settings: 1) first infections, [14] 2) multitypic dengue antibodies passively transferred to infants (high antibody levels protect, low levels enhance), [15] and 3) vaccination resulting in incomplete protective immunity. [16, 17] crucial to the occurrence of iade is the circulation of four antigenically related denvs. after a first infection, there is a 1 -2 year period of relative cross protection after which heterotypic denv infection may cause severe disease. [18] third or fourth sequential infections are not pathogenic. pre-outbreak age-specific distributions of dengue antibodies control age-specific ade disease attack rates. during heterotypic infections, viremias may be enhanced early but as illness progresses the titers and duration of viremias are shortened. [19] a c c e p t e d m a n u s c r i p t 5 dengue disease is a serious and widespread global health problem. in many dengue endemic countries there is an estimated 2% lifetime risk of hospitalization for enhanced dengue disease. [20] severe denv iade infections are short duration illnesses that elicit a stereotypical clinical course: an abrupt onset of fever and generalized symptoms followed around the time of defervescence by a rapid loss of fluid from the vascular compartment and, in turn, anoxia, shock and gastrointestinal hemorrhage. [21] the first suggestion of an immunopathology was finding that denv infected peripheral blood leukocytes (pbl) from dengue-immune monkeys and humans but not pbls from non-immune donors. [22] severe denv infections in infants implied that antibodies were etiological factors, a hypothesis confirmed when antibody mediated enhanced denv infection was produced in monkeys. [23] peak viremia titers observed early in illness are predictive of severe dengue in humans. [19] careful pathologic studies identified splenic and lymph node monocytes, macrophages and dendritic cells as major targets of denv infection. [24] fluid loss from the vascular compartment is attributed to capillary damage caused by circulating toxic viral protein (nonstructural protein 1 -ns1). [25] in humans, disruption of endothelial glycocalyx components by ns1 correlates with plasma leakage during severe denv infection. [26] denv ns1-induced endothelial cell intrinsic pathway vascular leakage is related to loss of integrity of endothelial glycocalyx components both in vitro and in vivo and is independent of inflammatory cytokines. [27] two corollary iade phenomena have been described: 1) passively acquired dengue antibodies efficiently enhance infection/disease and 2) disease severity rates may increase rapidly during epidemics. severe dengue accompanies first infections in infants circulating dengue antibodies acquired from multi-immune mothers. [28] during the course of secondary denv 2 epidemics in cuba in 1981 and 1997, disease severity increased month to month. it has been suggested that a single amino acid mutation in non-structural protein 1 (ns1) may observed as early as 4 days after onset of illness. it is thought that competent t cell immunity is essential for recovery. [34] while many clinical and pathological features are shared by sars, mers and covid 19, lungs from patients with covid-19 show distinctive severe endothelial injury associated with the presence of intracellular virus and disrupted cell membranes. histologic analysis of pulmonary vessels in patients with covid-19 showed widespread thrombosis, microangiopathy and a reactive angiogenesis. [35] indeed, there is growing evidence of thromboembolic phenomena in covid 19. [36] in severe and fatal sars and mers the dominance of the inflammatory response gave rise to the concept that cellular damage was due to a "cytokine storm." [37] because cytokines are stimulated by viral infection itself, it is difficult to distinguish between cytokines as cause or effect of infection. "cytokine storm" has also been invoked as a pathogenic mechanism in dengue, instead, capillary damage results from a circulating viral toxin. [25] concluding remarks: with others, we conclude that the differences in clinical, epidemiological and pathological features of sars and denv diseases suggest that iade does not contribute to the severity of natural human coronavirus infections. [39] a question asked frequently is whether sars or mers cov infections convey solid protective immunity. viral respiratory infections often fail to protect the respiratory tract from reinfection by the same organism. among immune individuals, respiratory tract superinfections occur frequently, but, usually without systemic disease. [40] for example, natural and vaccine immunes were re-infected with measles or rubella viruses and these infections may contribute to the spread of virus. [41] vah is a post-vaccination outcome that may be associated with non-protective antibodies. vah is a complex and poorly defined immunopathology. several different sars and mers vaccines have been shown to elicit a post-challenge vah in laboratory animals. ominously, when sars-cov-1-immune monkeys were challenged with homologous virus most animals had evidence of lung inflammation. [40] it is important to note that inactivated measles vaccine and dengvaxia exhibited short-term protection. [6, 16] a central challenge to sars cov-2 vaccine development will be differentiating early from sustained protection and will be greatly aided by a sars cov-2 model of vah in laboratory animal models. recognition of vaccine constructs that achieve solid protection in humans might be controlled by prevalence of 1 st denv infection antibodies. [47] no antibody effect observed ade with passive antibody 5 -11 month-old infants born to denvimmune mothers [47] not observed viral pathogenicity increases month to month; single mutation controls [49] not observed vaccine ade dengvaxia raises non-protective (ade) antibodies, sensitizing non-immunes [16, 17] challenge virus produces vah in vaccinated animals [9, 10, 50] animal coronavirus vaccines: lessons for sars an update on feline infectious peritonitis: virology and immunopathogenesis a perspective on potential antibody-dependent enhancement of sars-cov-2 intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes how innate immune mechanisms contribute to antibody-enhanced viral infections altered reactivity to measles virus. atypical measles in children previously immunized with inactivated measles virus vaccines field evaluation of a respiratory syncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins anti-sars-cov igg response in relation to disease severity of severe acute respiratory syndrome anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcgammar pathway risk factors in dengue shock syndrome: a prospective epidemiologic study in rayong, thailand. i. the 1980 outbreak maternal antibody and viral factors in the pathogenesis of dengue virus in infants effect of dengue serostatus on dengue vaccine safety and efficacy detection of post-vaccination enhanced dengue virus infection in macaques: an improved model for early assessment of dengue vaccines a shorter time interval between first and second dengue infections is associated with protection from clinical illness in a school-based cohort in thailand dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity neutralization and antibody dependent enhancement of dengue viruses shock associated with dengue infection. i. clinical and physiologic manifestations of dengue hemorrhagic fever in thailand immunologic enhancement of dengue virus replication in vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody pathologic highlights of dengue hemorrhagic fever in 13 autopsy cases from myanmar the good, the bad, and the shocking: the multiple roles of dengue virus nonstructural protein 1 in protection and pathogenesis association of endothelial glycocalyx and tight and adherens junctions with severity of plasma leakage in dengue infection dengue virus ns1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components dengue in vietnamese infants--results of infectionenhancement assays correlate with age-related disease epidemiology, and cellular immune responses correlate with disease severity a t164s mutation in the dengue virus ns1 protein is associated with greater disease severity in mice pathogenesis of feline infectious peritonitis: nature and development of viremia the severe acute respiratory syndrome time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars pathogenesis of severe acute respiratory syndrome t-cell immunity of sars-cov: implications for vaccine development against mers-cov pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology the macrophage in the pathogenesis of severe acute respiratory syndrome coronavirus infection dengue 1 virus and dengue hemorrhagic fever primary severe acute respiratory syndrome coronavirus infection limits replication but not lung inflammation upon homologous rechallenge rubella: reinfection of vaccinated and naturally immune persons exposed in an epidemic human challenge studies to accelerate coronavirus vaccine licensure an urgent need for "common cold units" to study novel coronavirus disease (covid-19) tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining observations related to pathogenesis of dengue hemorrhagic fever. vi. hypotheses and discussion reduced risk of disease during postsecondary dengue virus infections observations related to pathogenesis of dengue hemorrhagic fever. iv. relation of disease severity to antibody response and virus recovered observations related to pathogenesis of dengue hemorrhagic fever. iii. virologic studies of fatal disease virus role during intraepidemic increase in dengue disease severity anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection a c c e p t e d m a n u s c r i p t 12 key: cord-270709-jahnjvyk authors: hasford, joerg title: large simple double-blind randomized trials for the rapid assessment of the effectiveness of covid-19 vaccines date: 2020-08-26 journal: j infect dis doi: 10.1093/infdis/jiaa456 sha: doc_id: 270709 cord_uid: jahnjvyk nan large simple double-blind randomized trials for the rapid assessment of the effectiveness of covid-19 vaccines to the editor-the coronavirus disease 2019 (covid-19) pandemic has brought not only far too many losses of human lives but an economic crisis as well. thus, effective treatments and vaccines for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) are urgently needed. eyal et al have discussed challenge studies [1] to accelerate the assessment of vaccine effectiveness. in a challenge study, all participants are vaccinated with placebo or with the test vaccine and are then intentionally exposed to doses of sars-cov-2. there is already a worldwide initiative to register volunteers for such studies [2] . as all participants have been exposed, the effectiveness of a vaccine can be assessed with smaller sample sizes and possibly more quickly compared to the conventional trial with community participants; however, challenge studies are accompanied by serious ethical issues [3] . first, the characteristics of a distinct group of volunteers without risks for fatal progression or serious late complications of covid-19 need to be reliably known. second, a highly effective and safe treatment should be available for patients with covid-19, so that fatalities and persistent adverse consequences can be avoided. both these problems are not yet solved. third, the consent of the volunteer must be with their full understanding of comprehensive information, including appreciation of potential long-term consequences. it may be questioned, however, whether someone at age 20 years or so can imagine the consequences of a scarred lung occurring many years later. finally, it cannot be taken for granted that a vaccine that works in a challenge study with young, healthy volunteers will work in elderly patients with possible comorbid conditions [4] . thus, even the social value of challenge trials can be questioned. fortunately, there is an ethically more acceptable alternative for an accelerated evaluation of sars-cov-2 vaccines. the large, simple, randomized trial (lsrt), as proposed by yusuf et al in 1984, is a reliable, methodologically and ethically sound alternative [5] . characteristics of this design include: wide, simple eligibility criteria; central randomization; recording of only few baseline data; simple and short-term treatment; reduced or no follow-up visits; and outcome assessments of hard endpoints, preferably by registries. using this design, truly large randomized trials with sample sizes beyond 40 000 patients have been carried out and answered important questions [6] . probably due to the increasing bureaucratization of clinical research activities, including very costly monitoring, in the last 20 years there has been an almost complete disappearance of this trial design. at the current stage of evaluation of the effectiveness of sars-cov-2 vaccines, this design should be revived as it is ideally suited for this task. there would be wide eligibility criteria with very few exclusion criteria as the vaccine should become available for almost everybody. the investigational vaccine is a 1 or 2-time treatment only and no follow-up visits are needed. the outcomes, covid-19 or death, can be collected either by registries available in many countries (eg, in the united states, united kingdom, or scandinavia) or by patient reporting. vaccine safety information can be collected by established systems like the vaccine safety datalink in the united states [7] , prescription event monitoring programs (eg, the drug surveillance research unit in the united kingdom [8] ), or by direct patient safety reporting on websites, including those accessible with smartphones [9] , which can be specifically designed for vaccine trials. among the advantages of using the lsrt design are that it allows central randomization of large numbers of volunteers within a short time and rapid collection of the relevant outcomes at a low cost compared to the conventional phase 3 trials with many follow-up visits and extensive monitoring. adaptive design features (eg, modification of the eligibility criteria considering the accruing safety information) are feasible as well. given the wide entry criteria, the results provide external validity for large parts of the population compared to any challenge trial, which would need to focus on participants with extremely low risks for developing serious covid-19. as there will be very many people who would like to participate in such a vaccination trial, the sample sizes needed should be achieved within a very short time. when the lsrt double-blind design is used, the validity of the results is assured and it does not generate the serious ethical issues inherent in challenge trials. regarding the salk vaccine, large randomized trials with sample sizes of more than 70 000 were done in the early 1950s [10] and such lsrts should be feasible in 2020. thus, the sponsors of vaccine trials and the drug regulatory agencies should start the preparatory work now to be ready once an investigational vaccine is ready to be administered on a large scale. disclaimer. the opinions expressed do not necessarily represent those of the association of medical ethics committees in germany. potential conflicts of interest. author certifies no potential conflicts of interest. the author has submitted the icmje form for disclosure of potential human challenge studies to accelerate coronavirus vaccine licensure covid-19 human challenge trials experimental infections in humans-historical and ethical reflections vaccines to prevent infectious diseases in the older population: immunological challenges and future perspectives why do we need some large, simple randomized trials isis-3: a randomised comparison of streptokinase vs tissue plasminogen activator vs anistreplase and of aspirin plus heparin vs aspirin alone among 41,299 cases of suspected acute myocardial infarction vaccine safety datalink patent safety making history: thomas francis, jr, md, and the 1954 salk poliomyelitis vaccine field trial key: cord-288756-r96izsyq authors: wu, zhiqiang; yang, li; ren, xianwen; zhang, junpeng; yang, fan; zhang, shuyi; jin, qi title: orf8-related genetic evidence for chinese horseshoe bats as the source of human severe acute respiratory syndrome coronavirus date: 2016-02-15 journal: j infect dis doi: 10.1093/infdis/jiv476 sha: doc_id: 288756 cord_uid: r96izsyq several lineage b betacoronaviruses termed severe acute respiratory syndrome (sars)–like covs (sl-covs) were identified from rhinolophus bats in china. these viruses are characterized by a set of unique accessory open reading frames (orfs) that are located between the m and n genes. among unique accessory orfs, orf8 is most hypervariable. in this study, the orf8s of all sl-covs were classified into 3 types, and, for the first time, it was found that very few sl-covs from rhinolophus sinicus have orf8s that are identical to that of human sars-cov. this finding provides new genetic evidence for chinese horseshoe bats as the source of human sars-cov. the severe acute respiratory syndrome (sars) pandemic in 2002-2003 spread to 29 countries, caused 8098 cases, and led to 774 deaths. a novel coronavirus (cov), termed sars-cov, was identified as the etiological agent. sars-cov belongs to lineage b in the genus betacoronavirus (beta-cov) of the family coronaviridae [1] . although 12 years have passed without a recurrent sars outbreak, the search for the original animal reservoir for human sars-covs is ongoing. researchers have discovered lineage b beta-covs related to sars-covs in insectivorous rhinolophus and chaerephon bats in china. the nucleotide sequences in the orf1ab, e, m, and n genes in these bat-borne lineage b beta-covs are 89%-93% similar to those in the sars-covs from humans. the covs were thus named "sars-like covs" (sl-covs). the finding of diverse sl-covs in bats led to the hypothesis that rhinolophus bats were the natural reservoirs of sars-covs [2] [3] [4] . however, this hypothesis is challenged by the significant differences in nucleotide and amino acid sequences in certain hypervariable regions. these regions include the s1 domain of the viral spike glycoprotein (s) and the open reading frames (orfs) that encode a set of accessory proteins, particularly orf8. although a functionally similar bat-origin s gene was recently identified in the sl-covs of rhinolophus sinicus (chinese horseshoe bats) and rhinolophus affinis (wiv1, rs3367, and lyra11), it had less sequence similarity to the human-origin s gene [2, 5] . furthermore, genetic evidence for the identical orf8 is needed to trace the origin of sars-covs to bat sl-covs. all genome sequences were submitted to genbank. the accession numbers for all viruses are kj473811-kj473822, jx993987, jx993988, and kf636752. the ga ii sequence data were deposited into the national center for biotechnology information sequence read archive under accession number sra051252. the nucleotide sequences of the genomes and the amino acid sequences of the orfs were deduced by comparing them with the sequences in other covs. the conserved protein families and domains were predicted using pfam and interproscan 5 (available at: http://www.ebi.ac.uk/services/proteins). routine sequence alignments were performed using clustal omega, needle (available at: http://www.ebi.ac.uk/tools/), megalign (lasergene, dnastar, madison, wisconsin), and t-coffee with manual curation. mega5.0 (phoenix, arizona) was used to align the nucleotide sequences and the deduced amino acid sequences, using the muscle package and default parameters. the best substitution model was then evaluated using the model selection package. finally, we constructed a maximum-likelihood method, using an appropriate model to process the phylogenetic analyses with 1000 bootstrap replicates. in this study, a systematic survey of bat-borne covs was performed using bat virome data from throughout china, described in our previous report [6] , to obtain genetic evidence indicating the source of sars-covs. fifteen sl-covs were identified from 9 bat species in 11 provinces ( figure 1a ) [6] . throat and anal swab specimens from 22 wild and 124 farmed palm civets from guangxi, hunan, and fujian provinces were also used for virome analysis. however, we did not detect any cov-related sequences in the civet samples. compared with other bat-borne alpha-covs and beta-covs, the bat lineage b beta-covs (or sl-covs) are characterized by a set of unique accessory orfs (ua-orfs; figure 1b ). these ua-orfs encompassed approximately 1085-1095 base pairs and were located between the m and n genes. the ua-orfs encoded putative orf6, orf7a, orf7b, and orf8 proteins from the 5′ terminus to the 3′ terminus. these ua-orfs were absent or significantly different from those of all other alpha-covs, beta-covs, gamma-covs, and delta-covs [7] [8] . the sequence analysis of the identified lineage b beta-covs revealed that, in ua-orfs from all human sars-covs and sl-covs, orf6 and orf7s are highly conserved (90.8%-96.9% nucleotide sequence identities for orf6 and orf7a, and 85.2%-99.3% nucleotide sequence identity for orf7b). however, orf8 is hypervariable (approximately 48.6% nucleotide sequence identity; supplementary tables) [6] . the phylogenetic analysis of the sequences of orf8 from all available batborne lineage b beta-covs suggested that orf8s can be divided into 3 types (figure 2a ). type i orf8s shared high intra-type sequence similarity (97.6% nucleotide sequence identity) and low inter-type sequence similarities to the type ii orf8s (approximately 80.3% nucleotide sequence identity) and type iii orf8s (<52% nucleotide sequence identity). the type ii orf8s showed >97% intra-type identity, whereas the type iii orf8s showed 80%-95% intra-type identity and <50% nucleotide sequence identity with the type ii orf8s. type ii and type iii orf8s but not type i orf8s were previously detected in bat lineage b beta-covs. in this study, we observed that most of the orf8s in bat lineage b beta-covs are either type ii or type iii. all type ii orf8s were found in rhinolophus ferrumequinum. however, type iii orf8s were detected in multiple bat species, including r. sinicus. we are the first to have found a type i orf8 in bat lineage b beta-covs. type i orf8 is rare. in the country-wide screening, we found that only 2 covs collected from r. sinicus in kunming city in yunnan province (rs-betacoronavirus/yunnan2013) and hezhou city in guangxi province (rs-betacoronavirus/guang-xi2013) contained type i orf8s. thus, according to the currently available data, r. sinicus is the only bat species that harbors sl-covs with type i orf8. additionally, r. sinicus is also the only bat species with sl-covs containing 2 different types of orf8s. the type iii orf8 is the dominant type; type i is the minor type. the orf8s of sars-covs are also type i. the orf8s found in sars-covs from human patients in the early phase of the first epidemic of sars in 2003 (represented by the gz02 and gd01 isolates) and the 4 patients during the 2003-2004 outbreak (represented by the gz0401 isolate) are nearly identical to those of the 2 newly identified covs, rs-betacoronavirus/ yunnan2013 and rs-betacoronavirus/guangxi2013, with a few single-nucleotide mutations (98% and 99% nucleotide sequence identities, respectively; figure 2b ). this region of sars-cov experiences ongoing adaptive evolution in humans with gradual deletions (29-nucleotide, 82-nucleotide, or 415nucleotide deletions) after transmission to humans [9] [10] . the undeleted region of viruses with the 29-nucleotide deletion (the 29-nucleotide deletion splits orf8 into orf8a and orf8b, represented by the gz-a, urbani, and tor2 isolates) and viruses with the 82-nucleotide deletion (represented by the zs-a and hgz8l1-b isolates) also showed approximately 98% nucleotide sequence identities with the type i orf8, with a few single-nucleotide mutations. the nearly identical orf8s between sars-covs and sl-covs from chinese horseshoe bats identified in this study suggests a critical role for chinese horseshoe bats in the maintenance of sars-covs. the discovery of sl-covs in several bat species (including r. ferrumequinum, r. sinicus, rhinolophus pusillus, rhinolophus macrotis, r. affinis, and chaerephon plicata) and the characterization of ua-orfs shared only by sars-covs and sl-covs in the family coronaviridae established a genetic relationship between bats and human sars-covs. the orf8 nearly identical to that in sars-cov was found only in sl-covs from r. sinicus and traces the source of sars-covs to chinese horseshoe bats. functional studies for the proteins of orf8, orf8a, and orf8b have been reported. the 8a protein enhances sars-cov replication and induces caspase-dependent apoptosis [11] . the expression of the 8b protein is related to dna synthesis and the degradation of e protein [12] [13] . the orf8 protein may be functional in sl-covs from r. ferrumequinum [14] . however, the orf8 region may code for a functionally unimportant protein for human sars-covs, because gradual deletions in this region found in the early phase, the middle phase, and the late phase of the epidemic of sars did not apparently affect the survival of the virus [9] . thus, changes in this region can act as fingerprints to trace the genesis of sars-covs. the identical orf8s found in sl-covs from r. sinicus and sars-covs from patients in the early phase of the sars epidemic provides a link indicating that the first human infection with sars-cov may have originated from bats. although an s gene identical to that of sars-cov has not been found in any bat species, the distinctive diversity of the s region in sl-covs from r. sinicus may imply that sl-covs in r. sinicus are prone to recombine within r. sinicus or with covs from other hosts [6] . the identification of the s protein from sl-wiv1 from r. sinicus greatly increases the possibility of recombination of different sl-covs to generate sars-cov in r. sinicus. as the original site of the sars pandemic, guangdong province is the primary region in china in which wildlife (including bats) is consumed. the supply of wildlife in guangdong comes from surrounding provinces, such as guangxi, yunnan, hunan, and fujian. the sl-covs from r. sinicus have nearly identical orf8s and similar backbone genes to those in sars-covs in yunnan and guangxi provinces. furthermore, the observation that sl-covs from r. sinicus are prone to recombine with covs from other hosts may suggest that the wildlife markets in guangdong may provide an ideal incubator for the genesis of sars-covs. moreover, human consumption of wildlife increases the possibility of human exposure to viruses carried by wildlife. sl-covs closely related to human sars-covs are still present in nature, and the custom of wildlife consumption is ongoing. thus, there is an ongoing risk of sars reemergence or the emergence of a similar zoonotic infectious disease in humans. although palm civets were once suspected to be the natural reservoirs of human sars-cov, the isolation and genome sequencing of sars-covs in civets was limited to those present in the marketplace of the epidemic area of the sars outbreak and only during the outbreak period [10, 15] . in our study in 2012, we used the same metagenomic methods to detect sl-cov genomes in civets from guangxi, hunan, and fujian provinces that supply palm civets to the markets in guangdong. however, we did not detect any sars-cov or sl-cov sequences in any samples. this finding is consistent with a non-civetorigin cov reported prior to sars and after the pandemic. supplementary materials are available at http://jid.oxfordjournals.org. consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. financial support. this work was supported by the program for changjiang scholars and innovative research team in university of china (irt13007); the national s&t major project "china mega-project for infectious disease," people's republic of china (grants 2011zx10004-001 and 2014zx10004001); and the national natural science foundation of china (grants 81501773 and 31570382). potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. epidemiological and genetic analysis of severe acute respiratory syndrome isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor bats are natural reservoirs of sars-like coronaviruses characterization of a novel coronavirus associated with severe acute respiratory syndrome identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in china deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases the sars coronavirus: a postgenomic era coronavirus diversity, phylogeny and interspecies jumping molecular evolution of the sars coronavirus during the course of the sars epidemic in china cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome-associated coronavirus the human severe acute respiratory syndrome coronavirus (sars-cov) 8b protein is distinct from its counterpart in animal sars-cov and down-regulates the expression of the envelope protein in infected cells sars coronavirus orf8 protein is acquired from sars-related coronavirus from greater horseshoe bats through recombination isolation and characterization of viruses related to the sars coronavirus from animals in southern china key: cord-011718-hcyluzkx authors: gaglani, manjusha; spencer, sarah; ball, sarah; song, juhee; naleway, allison; henkle, emily; bozeman, sam; reynolds, sue; sessions, wendy; hancock, kathy; thompson, mark title: antibody response to influenza a(h1n1)pdm09 among healthcare personnel receiving trivalent inactivated vaccine: effect of prior monovalent inactivated vaccine date: 2014-06-01 journal: j infect dis doi: 10.1093/infdis/jit825 sha: doc_id: 11718 cord_uid: hcyluzkx background. few data are available on the immunogenicity of repeated annual doses of influenza a(h1n1)pdm09-containing vaccines. methods. we enrolled healthcare personnel (hcp) in direct patient care during the autumn of 2010 at 2 centers with voluntary immunization. we verified the receipt of a(h1n1)pdm09-containing monovalent inactivated influenza vaccine (miiv) and 2010–2011 trivalent inactivated vaccine (tiv). we performed hemagglutination inhibition antibody (hi) assays on preseason, post-tiv, and end-of-season serum samples. we compared the proportion of hcps with hi titer ≥40 against a(h1n1)pdm09 per receipt of prior-season miiv, current-season tiv, both, or neither. results. at preseason (n = 1417), hi ≥ 40 was significantly higher among those who received miiv (34%) vs those who did not (14%) (adjusted relative risk [arr], 3.26; 95% confidence interval [ci], 2.72–3.81). at post-tiv (n = 865), hi ≥ 40 was lower among hcp who received miiv and tiv (66%) than among those receiving only tiv (85%) (arr, 0.93 [95% ci, .84–.997]). at end-of-season (n = 1254), hi ≥ 40 was 40% among those who received both miiv and tiv and 67% among those receiving only tiv (arr, 0.76 [95% ci, .65–.88]), 52% among those who received miiv only, and 12% among those receiving neither. conclusions. hcp immunization programs should consider effects of host immune response and vaccine antigenic distance on immunogenicity of repeated annual doses of influenza vaccines. performed a prospective cohort study during 2010-2011 at 2 medical centers offering voluntary hcp immunization to examine factors that influence immunogenicity against laboratory-confirmed influenza. the administration of monovalent vaccines in 2009-2010 presented an opportunity to examine the hi response against the 2009 influenza a(h1n1) pandemic virus [a(h1n1)pdm09] among hcp who may have received vaccine containing the same antigen. our hypothesis was that receipt of 2010-2011 seasonal a(h1n1)pdm09-containing vaccines should produce an hi response similar to that reported in clinical trials [16] [17] [18] [19] [20] , regardless of receipt of prior-season vaccine. a prospective cohort was enrolled after the 2009-2010 influenza a(h1n1) pandemic season in autumn 2010 at scott & white healthcare (swh) in temple/round rock, texas, and kaiser permanente northwest (kpnw) in metropolitan portland, oregon. eligible hcp were aged 18-65 years, working ≥32 hours per week, receiving care from site for >12 months, and providing direct patient care. details on cohort recruitment are presented in thompson et al [21] . announcements regarding a "respiratory illness in healthcare workers" study were sent to all employees before and during voluntary immunizations. study protocol was approved by the sites' institutional review boards. participants completed an internet-based questionnaire at enrollment/preseason that included demographics, health, occupation, and work setting. we assessed self-rated health status with a 5-level rating of overall health [22, 23] , and calculated body mass index. we extracted data from electronic health records to characterize participants with high-risk conditions during the prior year [24] . we confirmed 2010-2011 and prior-season influenza immunization from medical/employee health records. blood was drawn at 3 time points: at enrollment from all participants ( preseason serum), approximately 30 days after receipt of 2010-2011 vaccine, ( post-trivalent inactivated vaccine [tiv] serum), and approximately 7 months after enrollment from all participants (end-of-season serum). from 18 december 2010 to 30 april 2011, participants completed weekly internet or computer-assisted telephone surveys assessing for acute respiratory illness (ari; cough and fever/ feverishness/chills, onset ≤7 days). internet reports were common (78%). noncompliant participants received email/telephone reminders to complete surveys. health records were monitored daily for ari diagnoses (international classification of diseases, ninth revision, clinical modification codes 460-466, 480-488). identified aris prompted a visit at home or the clinic where nasal, nasopharyngeal, and oropharyngeal swabs were collected, then tested for influenza a and b using real-time reverse transcription polymerase chain reaction (rrt-pcr) assay, with primers, probes, and reagents from the centers for disease control and prevention. a positive result from any swab was accepted. each site offered both tiv and live attenuated influenza vaccine (laiv) during both seasons. the monovalent a(h1n1)pdm09 vaccines contained a/california/7/2009 (h1n1)-like virus, which was the predominant virus in 2009-2010 at both sites [25] . the 2010-2011 seasonal trivalent vaccines also contained a/california/7/2009 (h1n1)-like virus. the predominant circulating viruses at the sites in 2010-2011 were similar to that in the united states: 74% of all positive results were influenza a, a(h3n2) 62% of all subtyped, and the rest a(h1n1)pdm09 [26] . at kpnw, a single lot (a) of tiv from manufacturer w was administered to 88% of vaccinated hcps for whom all 3 serum samples were taken; remaining lots were from w and x. at swh, 2 lots (b and c) from manufacturer y accounted for 91%; remaining lots were from manufacturer z. in 2009-2010, both sites had administered many lots of monovalent inactivated influenza vaccine (miiv) from w, x, z, or unknown manufacturers. of 489 hcp who received 2009-2010 miiv and 2010-2011 tiv, 146 of 242 (60%) from kpnw received a single lot of miiv and 193 of 247 (78%) from swh received 2 lots from the same manufacturer z. serum hi assays for each hcp were run in duplicate simultaneously using standard technique at the battelle laboratory (aberdeen, maryland), after completing a centers for disease control and prevention (cdc) proficiency panel [27, 28] . a standard turkey red blood cell (rbc) suspension was prepared, and serum samples were treated with receptor-destroying enzyme to remove nonspecific inhibitors. nonspecific agglutinins were removed by serum adsorption with packed rbcs. serum was diluted 2-fold starting from 1:10. the hi titer was the reciprocal of the serum dilution in the last well with complete hemagglutination inhibition. the geometric mean titer (gmt) from duplicate results was reported; hi < 10 was considered to be 5 for gmt calculation. we performed the primary analysis at 3 time points. for time point 1, we included 1417 hcp who had a serum specimen at preseason. because the predominant vaccine used at each site during both seasons was inactivated and we were examining the effect of second annual homologous revaccination on a(h1n1)pdm09 hi titers, we excluded monovalent laiv recipients. for time point 2, we included hcp who had a specimen at preseason, received 2010-2011 tiv, and had a specimen approximately 30 days (from 14 to 63 days only) post-tiv. some members of this group had received a(h1n1)pdm09 miiv and others had not. for time point 3, we included hcp who had serum samples taken at both preseason and end-ofseason. this included some who received only 2009-2010 miiv or 2010-2011 tiv, some who received both, and some who received neither. for the end-of-season analysis, we excluded h1n1pdm09-infected hcp during 2010-2011 evidenced by a positive rrt-pcr test or seroconversion (between preseason and end-of-season serum for unvaccinated, and between post-tiv and end-of-season serum for tiv recipients). seroconversion was defined as preseason hi < 10 and post-tiv/end-of-season hi ≥ 40; or preseason hi ≥ 10 and a minimum 4-fold rise for post-tiv/end-of-season serum (us food and drug administration definition). for the 3 primary analyses, we examined hcp characteristics: demographics, health status, vaccination history, timing of serum sampling, site, proxies for influenza exposure, and hi titer preseason. means were compared utilizing the wilcoxon rank-sum or 2-sample t test, and proportions by χ 2 or fisher exact test. a p value of <.05 indicated statistically significant difference. we constructed logistic regression models for each analysis with outcome variable a(h1n1)pdm09 hi ≥ 40, adjusting for hcp characteristics. as preseason hi titers correlate with post-tiv titers, controlling for baseline/preseason hi titer is recommended for single-season studies [29] . however, because we examined effects over 2 seasons, we did not adjust for preseason titers, because of a lack of true baseline before receipt of all vaccines (miiv receipt was prior to 2010-2011 preseason titer) and doing so would have removed prior h1n1 infection and/or miiv effect. an hi ≥ 40 is a generally accepted laboratory correlate for at least 50% protection against influenza infection [30] [31] [32] . we thus compared the proportion of hcp with hi ≥ 40 for preseason, post-tiv, and end-of-season serum samples by vaccination history. we also assessed hi response for post-tiv and end-ofseason serum samples by preseason hi < 40 or hi ≥ 40; by hi < 10, hi 10 to <40, and hi ≥ 40; and by vaccination history. because the outcome variable hi ≥ 40 was common, the odds ratio (or) is not a good approximation to the relative risk (rr), so we converted adjusted ors to adjusted relative risks (arrs) using the following equation, where p 0 = the incidence of disease in the nonexposed group: rr = or/([1 -p 0 ] + [or* p 0 ]) [33] . this conversion is recommended for cohort designs such as ours; however, we also validated the arr conversion using poisson regression with robust covariance, adjusting for covariates. we conducted 4 sets of secondary analyses to confirm our findings. first, we ran logistic regression models for each site for the 3 primary analyses. second, we compared hcp characteristics and hi titers for those who received the 3 most common lots of vaccine at our 2 sites. third, we compared descriptive characteristics for tiv-vaccinated hcp by receipt or not of prior miiv. fourth, as a sensitivity analysis for the subgroup of vaccinated hcp with all 3 serum samples, we applied a linear mixed effects model. for this analysis, hi titers were first rounded down to 2-fold reciprocal serum dilutions. only a small number of gmts that fell in between levels were rounded down (eg, gmt of 7 or 28 was rounded down to 5 or 20, respectively). for assessment as a continuous variable, hi titers were then converted to ordinal hi levels from 0 to 9 for each 2fold increase from 5 to 2560 by log transformation: log 2 (hi titer/5) [32, 34] . the linear mixed-effects model assessed ordinal hi level as primary outcome, controlling for time and other covariates. post-tiv days were represented with linear and quadratic time terms. we considered different errorcovariance structures to account for within-subject correlation over time and selected a first-order autoregressive with randomintercept model because it had the smallest bayesian information criterion. we evaluated the effect of prior receipt of miiv on ordinal hi levels for the 3 serum samples. sas version 9.2 (sas institute) was used for analyses. supplementary figure 1 shows that 1417 hcp in direct patient care were enrolled and gave preseason sera. of these, 936 (66%) received tiv and 865 (59%) had a blood specimen approximately 30 days post-tiv; the specimen was drawn between 14 and 42 days for 822 (95%) and between 43 and 63 days for 43 (5%). when compared with hcp included preseason (table 1) , those post-tiv were more likely to be aged 50-65 years, have high-risk conditions, and receive 2009-2010 seasonal vaccines, and less likely to work in the emergency department. of 1417 hcp from preseason, 1254 (89%) had end-of-season serum samples assessed for a(h1n1)pdm09 hi titers. the proportion of hcp who had prior-season miiv was similar among those providing preseason (42%) and end-of-season (43%) serum samples, but higher among those providing post-tiv sera (57%) ( table 1 ). there were modest but statistically significant differences between the 2 study sites in hcp characteristics at enrollment (supplementary table 1 ). in the time 1/preseason analysis, an hi ≥ 40 for a(h1n1) pdm09 was found in 34% of those receiving prior-season miiv vs 14% for those without (arr = 3. 26 . for post-tiv-serum samples, hi ≥ 40 was associated with female sex and swh site and inversely associated with age and direct patient care hours (data not shown). in the time 3/end-of-season analysis, we found a statistically significant interaction between receipt of miiv and tiv and therefore stratified the analysis into 4 groups ( table 2) . among all 1254 hcps without lab-confirmed a(h1n1)pdm09 infection in 2010-2011, hi ≥ 40 was maintained in 40% among those who received both miiv and tiv, 67% among those . among those with preseason hi < 40, end-of-season hi ≥ 40 was much less common in those who received both tiv and miiv (15%) vs those receiving only tiv (63%) ( table 2) . among the 834 vaccinated hcp with all 3 sera, the mean foldchange in hi titers was highest among those with preseason hi < 10 (table 3) . post-tiv serum hi ≥ 40 was acquired in more than half of hcp with preseason hi < 10 (61%) and hi 10 to <40 (79%). however, at end-of-season, these proportions dropped to 35% and 43%, respectively. in contrast, 96% of those with preseason hi ≥ 40 remained so through end-ofseason (table 3) . among these 834 vaccinated hcp, history of receipt of miiv was associated with lower hi gmts for post-tiv and end-of-season regardless of preseason hi gmts ( figure 1 ). this finding was seen at both sites but was more pronounced at swh (supplementary figure 2) . table 4 presents the descriptive characteristics of 865 hcp who received tiv by receipt or not of prior miiv, including high-risk conditions and pcr-confirmed h1n1 infections in 2010-2011. the preseason hi gmts of those with no prior miiv were significantly lower than those receiving 2009-2010 miiv. when we controlled for preseason hi titers in the logistic regression models, even though hi ≥ 40 post-tiv and endof-season was associated with preseason baseline titers, the effect of receipt of 2009-2010 miiv on hi titers noted in table 2 increased (data not shown). the linear mixed-effects model included 2502 hi titers, 3 for each of 834 vaccinated hcp (who provided all 3 serum samples). the model confirmed results from our primary analyses (data not shown). after controlling for covariates, those receiving miiv had higher preseason ordinal hi level (gmt ratio estimate: 2.31) than those who had not (table 5 ). however, they also had lower ordinal hi level (gmt ratio estimates: 0.57 and 0.74 for post-tiv and end-of-season, respectively), than those who had not. there was no significant difference in preseason hi titers for hcp receiving 3 major tiv lots (at kpnw: lot a, n = 238; at swh: lot b, n = 291 and lot c, n = 221 when hi titers were measured at end-of-season, compared to those who had never received any a(h1n1)pdm09-containing vaccine, hcp who were vaccinated both seasons had significantly higher probability for maintaining hi ≥ 40. however, those vaccinated with tiv in 2010-2011 but not miiv in 2009-2010 ended the season with significantly higher odds of hi ≥ 40 than hcp vaccinated both seasons. thus, hcp who were naive to the a(h1n1)pdm09 vaccine antigen in 2010-2011 had the best hi response post-tiv and at end-of-season. we noted similar trends for seroconversion post-tiv, and these effects were expected and noted at both study sites (data not shown). periodically, there have been reports of reduced immunogenicity and effectiveness associated with consecutive annual vaccination [9] [10] [11] [12] [13] [14] [15] . nabeshima et al [15] found lower immunogenicity of revaccination in 2003 among japanese hcp compared with those unvaccinated in 2002, unrelated to prevaccination hi titers [15] . other studies have reported better serologic response among previously unvaccinated adults [13, [35] [36] [37] although findings regarding revaccination and vaccine effectiveness have been mixed [38, 39] . antibody and effector and memory b-cell responses were greater in tiv recipients than in laiv recipients. prior-season tiv recipients had significantly higher preseason hi titers, but lower hi response after vaccination with either tiv or laiv in the study season compared with those who were not vaccinated during the previous season. these subjects also had a lower effector b-cell (antibody-secreting cell) response to new tiv but not laiv. a possible mechanism is that some of the injected hemagglutinin protein in tiv could form antigen-antibody complexes with preexisting hi antibodies, which could reduce the amount of hi antigen available for stimulating b cells [40] . in our study, we observed differences in hi antibody response based on receipt of miiv even in participants with preseason hi < 10, suggesting that hcp with hi < 10 despite prior miiv were primary vaccine nonresponders who were also less likely to respond to repeat vaccination. we speculate that this nonresponse may be related to exhaustion of memory b cells from prior influenza infections. another explanation is the "antigenic distance hypothesis" [41] . by comparing the predictions from a computer model to 7 influenza outbreaks from 2 studies [9, 42] , smith et al [41] accurately predicted year-to-year variations in vaccine efficacy, and specifically predicted that revaccination would negatively interfere with serologic response when the antigenic distance between strains in consecutive vaccines is small. because miiv and 2010-2011 tiv contained an identical a(h1n1)pdm09 antigen, this theory would predict lower response among those receiving a second annual vaccination with a(h1n1)pdm09, which fits what we observed. in a recent report of t-cell and antibody responses against influenza a(h1n1)pdm09, similar cd8 recall t-cell responses to h1n1 from 1934 and 2009 implied cross-reactive t-cell responses [43] . almost 60% of a toronto cohort had crossreactive memory t-cell responses to influenza virus at 1 year after the pandemic. the size of the long-lived pandemic h1n1reactive memory t-cell pool was not different between infected, vaccinated, and unvaccinated individuals, suggesting that the memory t-cell response increased only transiently postinfection and was not boosted by adjuvanted miiv. on the basis of the findings in a single donor, the authors postulate that these t cells could expand significantly postinfection. also, 46% of vaccinated and 15% of unvaccinated donors from their seroprevalence cohort had hi ≥ 40 during summer 2010, suggesting that antibody levels were not maintained at high levels postvaccination/infection. similar to our hcp cohort, those vaccinated with 2009-2010 miiv were more likely to have preseason hi ≥ 40 compared with those unvaccinated in their entire cohort [43] . hcp characteristics and tiv vaccines used differed at the 2 sites. the major tiv lot from kpnw was associated with lower mean fold-change postvaccination for those with low or intermediate hi titers at preseason compared with the response to the 2 lots from swh. this effect was at least partially mediated by kpnw hcp characteristics and does not impact the interpretation of our results overall. this is so because when we stratified analyses by site, the same inverse association between receipt of miiv and hi response to subsequent tiv was found, statistically significant for swh albeit not for kpnw. the proportion of hcp with preseason hi ≥ 40 against a(h1n1)pdm09 was 14% among 819 hcp not receiving miiv, suggesting that some had past infection (or cross-reactive antibody). our finding is similar to previous studies reporting 19% of emergency department providers who had hi ≥ 40 after the first wave [44] and to an estimated 20% of the us population infected with a(h1n1)pdm09 prior to the 2010-2011 season [45] . randomized clinical trials reported that 90%-100% of healthy adults had hi ≥ 40 post-miiv [16] [17] [18] [19] [20] . for our cohort, the proportion of hcp with hi ≥ 40 after a(h1n1)pdm09containing tiv was 74% (95% ci, 71%-77%) overall; 85% (95% ci, 81%-88%) among those who had only tiv and 66% (95% ci, 61%-70%) among those who had both miiv and tiv. however, the overall proportion we observed is higher than that reported for hcp in hong kong [46] of 54% (95% ci, 44%-63%) and in japan [47] of 38% (95% ci, 33.2%-42.9%) after receipt of 1 unadjuvanted miiv dose. what we observed is closest to hi ≥ 40 of 80% among hcp in the netherlands, after 1 mf-59 adjuvanted miiv dose [48] . strengths of our study include its large sample size, and the ability to compare findings across study sites and major vaccine lots. there are also several limitations to our study. first, although hi ≥ 40 is considered a surrogate marker of protection for licensure of influenza vaccines [29, 34] , its association with vaccine effectiveness is limited and the clinical meaningfulness of the differences we observed is unknown. second, the observational nature of this study introduced differences in timing of blood draws, site, and participant characteristics, which could only be adjusted for statistically in multivariate models and by subsetting our data by site. because hcp aged ≥50 years and those with high-risk conditions are more likely to be revaccinated, we cannot rule out residual confounding. although we included medically attended acute respiratory illness as a covariate in models, we are limited by variable prior exposure to influenza viruses and vaccines, and underlying health and immunologic status of our participants. other potentially serious limitations include selection bias, as miiv or tiv recipients were not randomized, and possible variable potency of many lots of 2009-2010 miiv. there are possibly other unknown biases inherent to any nonrandomized study. additional study is needed to ascertain if our finding of lower immunogenicity to a(h1n1)pdm09 antigen with second annual inactivated vaccination applies to other antigens or laiv. whether or not lower hi titers have clinical significance also requires further research, through vaccine-effectiveness studies, especially among hcp. finally, although annual vaccination for hcp remains a safe and effective prevention strategy [2, 24, 49] , the imperfect immune response and afforded protection [38, 39] imply that hcp should remain vigilant for wintertime respiratory illness irrespective of vaccination history to limit transmission of influenza to their patients. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org/). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. incidence of influenza in healthy adults and healthcare workers: a systematic review and metaanalysis influenza vaccination of health-care personnel: recommendations of the healthcare infection control practices advisory committee (hicpac) and the advisory committee on immunization practices (acip) interim results: influenza a (h1n1) 2009 monovalent and seasonal influenza vaccination coverage 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university hospital in japan immunogenicity, boostability, and sustainability of the immune response after vaccination against influenza a virus (h1n1) 2009 in a healthy population prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices (acip)-united states, 2012-13 influenza season acknowledgments. the authors thank the following persons at the influenza division, national center for immunization and respiratory diseases, cdc, for critical review of this manuscript: joe miller, phd; jackie katz, phd; david shay, md, mph; alicia fry, md, mph; joe breese, md; and jerry tokars, md. we appreciate the research teams at scott & white healthcare, kaiser permanente center for health research, and abt associates. we also thank the healthcare personnel in direct patient care at the study sites who volunteered to participate in this study.disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the cdc, abt associates, inc, kaiser permanente center for health research, or scott & white healthcare.financial support. this work was supported by the cdc (contract 200-2010-f-33396 to abt associates inc). this research was supported in part by an appointment to the research participation program at the cdc administered by the oak ridge institute for science and education through an interagency agreement between the us department of energy and the cdc.potential conflicts of interest. m. g. has received research funding from medimmune and novartis, and a. n. has received research funding from glaxosmithkline. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-277735-a9gkath5 authors: leung, danny tze ming; chi hang, tam frankie; chun hung, ma; sheung chan, paul kay; cheung, jo lai ken; niu, haitao; tam, john siu lun; lim, pak leong title: antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid date: 2004-07-15 journal: j infect dis doi: 10.1086/422040 sha: doc_id: 277735 cord_uid: a9gkath5 the recent outbreak of severe acute respiratory syndrome (sars) provided an opportunity to study the antibody response of infected individuals to the causative virus, sars coronavirus. we examined serum samples obtained from 46 patients with sars, 40 patients with non-sars pneumonia, and 38 healthy individuals, by use of western blotting (wb), enzyme-linked immunoassay (elisa), and immunofluorescence assay, using both native and bacterially produced antigens of the virus. we found a highly restricted, immunoglobulin g-dominated antibody response in patients with sars, directed most frequently (89% by elisa) and predominantly at the nucleocapsid. almost all of the subjects without sars had no antinucleocapsid antibodies. the spike protein was the next most frequently targeted, but only 63% of the patients (by elisa) responded. other targets of the response identified by use of wb included antigens of 80 and 60 kda. several nonstructural proteins cloned were not antigenic, and the culture-derived nucleocapsid appeared to be specifically degraded. peptidase n [8] . after this initial attachment, the viral e protein fuses with the plasma membrane of the host cell, and a cascade of intracellular events follows, including interaction between the m and n proteins. this interaction is important for packaging of the progeny virions [9] . after infection, patients with sars develop antibodies to the virus, and 190% of them experience seroconversion within 20 days [10] . however, little else is known about the antibody responses in these patients. the conventional antibody test, which detects antibodies to antigens present in virus-infected cells by use of immunofluorescence assay (ifa) [3] , does not reveal which viral antigens are targeted by the immune response. knowing the viral targets is important for several reasons. first, this allows the development of cell-free antibody tests that are less cumbersome and subjective than ifa and that can be used for mass screening in times of epidemics. second, this enhances our understanding of the immunopathology of the disease, and moreover, helps in design of a vaccine. both the humoral and cellular arms of the adaptive immune response are presumed to be important in controlling 2 or preparation u reacted with a serum sample from a patient with sars showing the highly reactive antigens-nucleocapsid (n) 1, n2, and n3-in the former. mw, molecular weight markers (in kda). c, wb results of 6 patients with sars (s) and 4 patients with non-sars pneumonia (p), showing strong reactivities of the n1-n3 antigens and lesser reactivities of the spike and the 80-and 60-kda proteins or the partial reactivity (n1) or total lack of reactivity. the infection. as with other covs, antibodies can function by blocking the adsorption or entry of the virus to the target cell [11] [12] [13] or by interfering with viral transcription [14] . in the present study, we identify the viral antigens to which our patients with sars had responded. patients and control subjects. we diagnosed sars according to the world health organization criteria [15] , on the basis of the following symptoms: acute onset of fever (138њc) accompanied by frequent chills or rigor, dyspnea, headache, myalgia, hypoxemia, and radiological evidence of pneumonia. in addition, the patients with sars had anti-sars cov igg antibodies detected by use of an ifa [3] , which showed seroconversion or a 4-fold increase in titer. in this assay, which uses virus-infected monkey kidney (vero) cells and which is performed routinely in our laboratory, a serum titer of у1:40 was considered to be positive. all 46 patients (15 males and 31 females; age range, 8-68 years; age, years) had contact with mean ‫ע‬ sd 35.9 ‫ע‬ 13.0 patients with confirmed sars within 10 days of the onset of their symptoms and were admitted to the prince of wales hospital, hong kong, during the sars outbreak that started in early march 2003. two of the patients died, 5 required intensive care but eventually recovered, and the rest had a mild clinical course. because methods of detection were not available early during the outbreak, the sars cov was cultured from only 1 patient, and viral rna was detected by use of reverse-transcription polymerase chain reaction (pcr) [16] from the respiratory or stool samples from 4 patients. the serum samples used in the present study were obtained 7-35 days ( , days) mean ‫ע‬ sd 17.1 ‫ע‬ 6.4 after the onset of fever. control subjects included 40 inpatients (21 males and 19 females; age range, 8-84 years; mean age, 43.3 years) who had presented at the prince of wales hospital with clinical features of atypical pneumonia and were treated accordingly at the hospital in 2000 before the emergence of sars and 38 healthy individuals (blood donors and students). institutional approval for both human and animal ethics was obtained for the study, and the institutional guidelines were followed. viral culture and crude native antigens. vero cells (atcc crl-1586) were grown in dulbecco's modified eagle medium containing 5% fetal calf serum at 37њc in a 5% co 2 humidified incubator. the cuhk-w1 sars cov strain (genbank accession no. ay278554) used was isolated from a patient in our hospital. ). after incubation for 1 h on ice, the supernatant obtained from the cell lysate by centrifugation was heated for 30 min at 55њc, to inactivate any live virus present, and was stored at ϫ70њc until use. control cell lysate was similarly prepared from uninfected vero cells. recombinant viral antigens. total rna extracted from sars cov-infected vero cells by use of a qiamp viral rna kit (qiagen) was reverse-transcribed with random hexamers (applied biosystems), and the cdna obtained was used to generate the various gene segments. the following forward (f) primers (containing a bamh1 site, underlined) and reverse (r) primers (containing an ecor1 site, underlined) were used in pcrs described elsewhere [17] for the corresponding gene segments (sizes the pcr conditions used were 3 min at 94њc, 34 cycles of 1 min at 94њc, 1 min at 55њc, 1 min at 72њc, and 15 min at 72њc. the gel-and affinity-purified fragments were cloned into the bacterial expression vector pgex-2t (amersham bioscience), which is fused to the bacterial glutathione s-transferase (gst) gene. the ligated vector was transfected to escherichia coli bl21. recombinant antigens were recovered from selected transformants induced with isopropyl-b-d-thiogalactopyranoside. in brief, the cell pellet was resuspended in ice-cold lysis buffer (25 mmol/l tris-hcl, 100 mmol/l nacl, 0.1% triton x-100, 1 mmol/l pmsf, and 1ϫ protease inhibitor cocktail [ph 8.0; sigma]) and was sonicated (30 mm). the recombinant antigen was recovered from the supernatant of the lysate by use of affinity chromatography using glutathione-coupled agarose (amersham) and, for elution, 25 mmol/l tris-hcl buffer (ph 8.0) containing 20 mmol/l reduced-form glutathione, 100 mmol/l nacl, 0.1% triton x-100, and 5 mmol/l ddt. the eluted antigen was examined on sds-page gels stained with coomassie blue (expected size and good purity were observed in all cases; data not shown). in sds-page, the antigen preparation was heated for 5 min at 100њc in loading buffer (0.25 mol/l tris-hcl [ph 6.8], 20% 2-mercaptoethanol, 40% glycerol, 8% sds, and 0.01% bromophenol blue) and electrophoresed (150 v for 80 min at room temperature [rt]) on 10% polyacrylamide. antigens of interest (pn2, pn3, and ps) were located in the unstained gel by use of a parallel gel stained with coomassie blue and were recovered with an eluter (harvard bioscience) at 40 v overnight in 25 mmol/l tris buffer (ph 8.3) containing 192 mmol/l glycine, 20% methanol, and 0.5% sds. in other experiments, the whole gel was electroblotted onto a 0.22-mm polyvinylidene fluoride membrane (bio-rad) and was used for protein sequencing (protein facility, iowa state university) or for wb. in the latter assay, the membrane was blocked with skim milk and incubated (for 1 h at rt) with the unknown human or mouse serum (diluted 1: 200 in 2 ml of pbs containing 5% skim milk) [8] . after washing, the blot was incubated (for 1 h at rt) with peroxidase-labeled goat anti-human igg or anti-mouse ig (all classes) (bd biosciences) and later with the chemiluminescence substrate ecl (amersham). the assay was developed by exposure to hyperfilmb max (amersham). in inhibition wb, the unknown serum (10 ml) was preincubated with the inhibiting antigen (600 mg/ml) in 50 ml of pbs containing 5% skim milk overnight at 4њc. elisa. the native viral antigens, either crude or purified (pn2, pn3, and ps; 1:200 stock dilution), or the recombinant antigens (rna, rsa, rsb, rsc, rnsp12a, rnsp12b, rnsp13, and rnsp9; 1 mg/ml) were coated onto 96-well immunon-2 plates (dynex) in bicarbonate buffer (ph 9.6) overnight at 4њc, and the assay was performed as described elsewhere [17] . in brief, 100 ml of the unknown human serum diluted 1:50 in pbs (containing 1.3% bovine serum albumin, 0.25% casein, and 0.05% tween-20) were added to the wells and incubated for 30 min at rt. the plates were washed and incubated (for 15 min at rt) with horseradish peroxidase-labeled goat anti-human ig (igg, igm, or iga specific) (bd biosciences). after washing, substrate (3,3 ,5,5 -tetramethylbenzidine) was added, and reaction was allowed for 15 min at rt. the results were read at 450 nm in a dynex mrx ii reader. mouse immunization. balb/c mice (3 mice/group) were injected intraperitoneally with the affinity-purified, alum-precipitated antigen (200 mg/mouse) in complete freund's adjuvant and received booster injections with 50 mg of the antigen in incomplete freund's adjuvant 2 weeks later. blood was obtained from the retro-orbital plexus 4 days after administration of the booster injection. statistics. the relationships of the (rna) elisa with other immunoassays or with sampling times were examined by use of regression analysis (graphpad prism 3; graphpad software). we first examined the antibody response of the patients with sars by use of wb. figure 1b shows the gel-separated crude extract of the culture-grown virus. several virus-specific antigens are discernible by comparing the control (uninfected) extract with the viral extract, particularly ones at 48, 46, and 44 kda molecular weight. the antigenicity of these proteins, labeled n1, n2, and n3, respectively, is shown by immunoblotting with serum samples obtained from patients with sars ( figure 1b) . the relative abundance of n1-n3 varied among batches of the extracts, but n3, which is actually a doublet, was consistently lowest in quantity. the wb results from representative patients with sars and patients with non-sars pneumonia are shown in figure 1c . these results were based on the igg response; the igm reactions were either weak or absent. the n1-n3 antigens were the most reactive antigens found in the patients with sars but were absent in patients with non-sars pneumonia. these 3 antigens always appeared as a triplet. one of the non-sars serum samples reacted weakly with an antigen at the position of n1. lessreactive antigens found at 150, 80, and 60 kda were not seen in the non-sars serum samples. the molecular size of n1 suggests that it is the nucleocapsid, but n2 and n3 could not be identified in this way. protein sequencing of these antigens was not successful, but it confirmed the 150-kda "s" antigen (sdldr) to be the s protein. on the basis of wb results for a total of 43 patients with sars, 23 patients with non-sars pneumonia, and 16 healthy subjects, most (79%) of the patients with sars produced antibodies to the n1-n3 antigens, but only 40% produced antibodies to the s protein ( figure 2 ). in addition, 35%, 26%, 19%, and 5% of the patients with sars produced antibodies to the 80-, 60-, 32-, and 24-kda antigens, respectively (data not shown). none of the control subjects produced antibodies to any of these antigens, although 13% of the serum samples from patients with non-sars pneumonia showed weak reactivity with the (presumably) n1 antigen (see below). the sars serum samples were positive for viral antibodies when examined by use of an ifa using infected cells, although some (17%) had low titers (!1:80) only (figure 2). none of the serum samples from the 2 control cohorts examined were positive by either ifa or wb. using a bigger study group (46 patients with sars, 40 patients with non-sars pneumonia, and 38 healthy individuals), we examined the antibody responses further by use of elisa. first, when the crude viral extract was used as antigen, 91% of the patients with sars were positive for antibodies, compared with !6% of the combined control subjects (figure 2). all 4 patients with sars who were negative for antibodies had low ifa titers (!1:80). all serum samples were not reactive with the control antigen, gst (data not shown). the results are based on the igg response. the igm response, in contrast, was less robust and less frequent (43%) among the patients with sars (figure 2) and less discriminatory between the sars and non-sars cohorts. second, we made a recombinant antigen of the n-terminal half of the n protein (rna), and, when it was used in an igg elisa, we found results almost identical to those found with the crude viral extract (89% sensitivity and 94%-95% specificity), including 4 negative cases in common ( figure 2) . the igm responses were similarly low and infrequent among the patients with sars (data not shown). we investigated the possibility that the 20% of serum samples that were negative or weakly positive in the elisa but were ifa positive might have antibodies directed to antigens other than the nucleocapsid or those present in the crude extract. we thus examined the recombinant antigens made from several nsps of the virus. however, although nsp12 (both subunits) and nsp9 were found to be nonantigenic, nsp13 showed only weak reactivities with some (11%) of the serum samples from patients with sars (data not shown). native s antigen that was purified and used in an elisa detected antibody responses in 63% of the patients with sars (figure 2), slightly more than those detected by use of wb. however, most of the responses were weak. using recombinant s antigens, we found that only the c-terminal end (rsc) of the protein was antigenic and that only a small number (13%) of serum samples from patients with sars were reactive, similar to results for serum samples from patients with non-sars pneumonia (figure 2). we purified the native n2 and n3 antigens together from the gel-separated crude extract and used these in an elisa. the results obtained were very similar to those of the rna elisa, particularly with respect to the negative or positive cases ( figure 2 ). this suggests that n2 or n3 or both might be antigenically related to rna. to investigate this possibility, we performed wb experiments using rna as inhibitor. rna was indeed found to reduce the reactivity of not only n1, but also of n2 and n3 (figure 3a). in fact, inhibition was greatest with n3 and least with n1. of interest, there appeared to be a fourth, minor n fragment (n4) with a molecular weight of 32 kda (figure 3a) that was seen with some serum samples (e.g., s13; figure 1 ). similar results were obtained when the purified n2 and n3 antigens were used as the inhibitor in the wb analysis ( figure 3a) . in contrast, when the s antigen, rsc, was used as inhibitor, reactivity at the 150-kda region ("s"), but not that of n1-n4 , was affected (i.e., abolished) ( figure 3a) . we proved further that n1, n2, n3, and n4 are all n antigens. mouse serum made against the recombinant antigens rna, rsa, and rsc (rsb was not available at the time) were used in wb against the crude viral extract. all 3 mice immunized with rna produced antibodies that reacted specifically with n1, n2, n3, and n4, but not with other antigens (figure 3b). in figure 2 . comparison of the efficiency of various detection assays for severe acute respiratory syndrome (sars). individual serum samples from each group (46 patients with sars, 40 patients with non-sars pneumonia, and 38 healthy individuals) were examined by use of immunofluorescence assay (ifa), western blotting (wb), or elisa. antigens used included native spike (s), native nucleocapsid (n), crude antigen, crude viral extract, recombinant nucleocapsid (rna), gel-purified native n2 and n3 antigens (pn2,3), gel-purified native spike (ps), and recombinant spike (rsc). except where marked "igm", all tests are based on detection of igg. in each test, the cutoff for positivity is shown by the shaded bar; for elisa, this is based on the sd value of the combined cohorts of patients with non-sars pneumonia and healthy individuals. in wb, the intensity of the mean + 1 reaction was arbitrarily scored by eye (3, strongest) . serum samples that were not examined because of a lack of antigen or serum or because the results were not readable are shown by a dot. contrast, neither of the s antigens produced any antibodies against the crude extract (figure 3b). the n antigen of sars cov stands out as the most important diagnostic antigen of the virus. the majority of our patients who developed sars (89% by elisa) produced antibodies to this antigen. when present, these antibodies are also the most abundant of the antibodies made to the virus. it is not clear why the n antigen is so immunogenic, but we have found that even a bacterially produced fragment of it induced good production of antibodies in mice. the n antigen also has been found to be highly immunogenic in the elk cov [18] and in the infectious bronchitis virus [19] . we have found the n antigen to be the predominant antigen in the crude viral extract. this is shown by the good correlation between the rna and crude antigen elisas (figures 2 and 4a). in contrast, there was only weak correlation between the rna elisa and the ifa performed on these serum samples ( figure 4b ). this result was not due to the fact that we used a singledilution measurement in the elisa, rather than titrating the serum ( figure 4c ). it is possible that some antigens cannot be extracted from the cell or become degraded during the extraction, but this was not the case with nsp12 or nsp9. it is noteworthy that the serum samples from the 3 patients with non-sars pneumonia, which reacted with the (presumably) n1 component by wb, were negative by the rna elisa. this result suggests that the reactive epitope is located elsewhere in the n antigen or in a nonviral (vero) antigen. as expected, detection of the anti-n antigen antibodies in patients with sars by use of the rna elisa improved with increasing intervals between the time samples were obtained and the onset of fever ( figure 4d ). thus, in 3 of the 5 negative cases, the serum was obtained within 10 days after the onset of fever. on the other hand, 4 other serum samples obtained during the same period were found to be positive. although this means that more than half of the early sars cases (and all of the late [121 days] cases) could be detected, it is important to note that we used serum samples that were ifa selected. of note with regard to the anti-n antibody response in the patients with sars is the predominance of igg antibodies over igm antibodies, even early (second week) during the course of the disease. this result implies that there was strong t helper cell involvement. an exaggerated t cell response probably also accounts for the pneumonia-associated immunopathology seen in patients with sars [10] . the n antigen may be important here. it may indeed be both a potent b cell and a potent t cell immunogen; that is, an important candidate for a vaccine. although antibodies to the nucleocapsid are nonneutralizing, whether such antibodies can be protective in other ways, as observed for mouse hepatitis virus [20] and rotavirus [21] , needs to be addressed formally. the latter phenomenon may be due to the ability of the iga antibodies to enter the infected cell by the iga-transcytosis pathway, thereby interfering with viral transcription [14] . we found evidence that the n antigen is degraded in cell cultures of the virus. the degradation is specific, since the 3 major n antigens, n1-n3, were found in every preparation of the crude extract that we used. a minor antigen, n4, was also sometimes seen. n1 is presumably the full-length protein, and the others are fragments of n1 that lack varying lengths of the c-terminal end. because of the high specificity seen, we do not consider the degradation to be an artifact of the antigen-extraction procedure. rather, we suspect that n1 is cleaved by caspases in the vero cells as the cells undergo apoptosis, similar to what eleouet et al. [22] observed for the n antigen from the transmissible gastroenteritis cov. after the n antigen, the s antigen is the one most often targeted by the immune response of the patients with sars. however, !63% (by elisa) of the patients responded, and their responses were generally weak. it is possible, on the other hand, that the responses were underestimated because the s antigen contained in the crude extract used for wb or elisa was degraded or underrepresented and the recombinant antigen used for elisa that was produced in bacteria lacked the necessary glycosylation normally associated with the native antigen (see: http://www.cbs.dtu.dk). other antigens of the virus targeted by the immune response are presumably minor. examples are the 80-and 60-kda antigens. the latter could be nsp13, recently postulated to be an mrna cap-1 methyltransferase [23] . the 24-kda antigen to which some patients responded could be the m protein. both this and the e protein of the virus (the latter was too small [8.5 kda] to assess in our wb gels) are presumed to be important in protection. identification of severe acute respiratory syndrome in canada coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov-229e characterization of the coronavirus m protein and nucleocapsid interaction in infected cells clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein protection from lethal coronavirus infection by immunoglobulin fragments interference of coronavirus infection by expression of immunoglobulin g (igg) or iga virus-neutralizing antibodies inhibition of rotavirus replication by a non-neutralizing, rotavirus vp6-specific iga mab world health organization. case definitions for surveillance of severe acute respiratory syndrome (sars) human metapneumovirus detection in patients with severe acute respiratory syndrome a human and a mouse anti-idiotypic antibody specific for human t14 + anti-dna antibodies reconstructed by phage display production, characterization, and uses of monoclonal antibodies against recombinant nucleoprotein of elk coronavirus recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice protective effect of rotavirus vp6-specific iga monoclonal antibodies that lack neutralizing activity the viral nucleocapsid protein of transmissible gastroenteritis coronavirus (tgev) is cleaved by caspase-6 and -7 during tgev-induced apoptosis mrna cap-1 methyltransferase in the sars genome we thank peggy fung for excellent secretarial help. key: cord-270205-fw555w1u authors: cillóniz, catia; dominedò, cristina; magdaleno, daniel; ferrer, miquel; gabarrús, albert; torres, antoni title: pure viral sepsis secondary to community-acquired pneumonia in adults: risk and prognostic factors date: 2019-10-01 journal: j infect dis doi: 10.1093/infdis/jiz257 sha: doc_id: 270205 cord_uid: fw555w1u we investigated the risk and prognostic factors of pure viral sepsis in adult patients with community-acquired pneumonia (cap), using the sepsis-3 definition. pure viral sepsis was found in 3% of all patients (138 of 4028) admitted to the emergency department with a diagnosis of cap, 19% of those with cap (138 of 722) admitted to the intensive care unit, and 61% of those (138 of 225) with a diagnosis of viral cap. our data indicate that males and patients aged ≥65 years are at increased risk of viral sepsis. we investigated the risk and prognostic factors of pure viral sepsis in adult patients with community-acquired pneumonia (cap), using the sepsis-3 definition. pure viral sepsis was found in 3% of all patients (138 of 4028) admitted to the emergency department with a diagnosis of cap, 19% of those with cap (138 of 722) admitted to the intensive care unit, and 61% of those (138 of 225) with a diagnosis of viral cap. our data indicate that males and patients aged ≥65 years are at increased risk of viral sepsis. keywords. sepsis; viral sepsis; virus; community-acquired pneumonia. improved molecular diagnostic techniques have increasingly revealed a high prevalence of viral pneumonia over recent years. globally, it is now estimated that 100 million cases of viral pneumonia occur annually, with the incidence varying by seasonality, geographic location, and age group [1] . respiratory viruses are detected as etiological agents in almost one third of cases of community-acquired pneumonia (cap) [2] [3] [4] [5] and in 7%-36% of patients with severe cap with a defined microbial etiology [2, 3] . recently, jain et al [2] analyzed 2320 cases of pneumonia detected by intensive microbiological diagnosis, including viral molecular techniques. a microbial etiology was identified in 853 cases (38%). the 3 main causes were respiratory viruses (23%), bacteria (11%), and coinfections (3%), indicating the clear prominence of a viral etiology. cap is often complicated by sepsis, which is a multifactorial process for which staging is necessary to provide personalized treatments that target individual needs [6] . viral sepsis has been defined as a severe inflammatory response to viral infection [7] , and unlike bacterial sepsis, its prevalence in adults with cap is unknown. we aimed to investigate the prevalence, risks, and prognostic factors associated with pure viral sepsis in adult patients with cap, using the third international consensus definitions for sepsis and septic shock (sepsis-3) criteria [6] . we performed a retrospective observational study of consecutive adult patients with a diagnosis of cap admitted to the hospital clinic of barcelona from the emergency department between 2005 and 2017. we excluded nonhospitalized patients and those with severe immunosuppression, active tuberculosis, viral bacterial coinfection, and unavailable data. we selected patients with pure viral cap and compared those with and without sepsis. severe cap was defined according to the american thoracic society/infectious diseases society of america guidelines [8] . sepsis was defined as the presence of pneumonia and an increase of ≥2 points in the sequential organ failure assessment score [6] . diagnosis of respiratory virus infection was made on the basis of results of serologic analysis, immunofluorescence assay, and cell cultures from 2005 to 2007. however, diagnosis was based on results of polymerase chain reaction (pcr) and/or culture of nasopharyngeal swab samples from 2008 to 2017. two independent nested multiplex real-time pcr tests were used to detect human influenza viruses (a, b, and c), respiratory syncytial virus, adenoviruses, parainfluenza viruses (1-4), coronaviruses (229e and oc43), enteroviruses, and rhinoviruses (a, b, and c). the criteria for etiological diagnosis are available in a previous report [3] . the main clinical outcome was in-hospital mortality. secondary outcomes included length of hospital stay, intensive care unit (icu) admission, mortality among patients admitted to the icu, length of icu stay, need for mechanical ventilation, 30-day mortality, and 1-year mortality. patients were followed for one year. for publication purposes, the study was approved by the ethics committee of our institution (comité ètic d'investigació clínica; registration no. 2009/5451). the need for written informed consent was waived because of the noninterventional study design. logistic regression analyses were used to examine the association between sepsis and risk factors. first, each risk factor was tested individually. then, all risk factors that showed an association in the univariate model (p < .10) were added to the multivariable model. finally, backward stepwise selection (p in < .05 and p out > .10) was used to determine factors associated with sepsis. generalized linear model analyses were performed to determine the influence of the risk factors on in-hospital mortality. models were defined using a binomial probability distribution and a logit link function, using inverse probability of treatment weights (iptws) to account for biases due to observed confounders. first, each risk factor was tested individually. second, a propensity score for patients with sepsis was developed. iptw used the propensity score to form a weight. finally, the weight and the year of admission were incorporated in the multivariable weighted logistic regression model for in-hospital mortality, which included all risk factors and showed an association in the univariate analyses (p < .10), and backward stepwise elimination was performed to detect the factors associated with in-hospital mortality. we used the multiple imputation method for missing data in the multivariable analyses. the level of significance was set at 0.05 (2-tailed). all analyses were performed using ibm spss statistics, version 25.0 (armonk, ny). we identified 4028 consecutive patients admitted to the emergency department with a diagnosis of cap during the study period. a total of 2760 patients (68%) were hospitalized, of whom 225 (8%) were found to have a pure viral cap. thirty-six patients (23%) had severe cap. among the 225 cases of pure viral cap, the most common respiratory viruses were influenza a virus ( [15] ), and coronavirus (2% [4] ). we did not observe any change in the prevalence of viral cap over the study period (p = .65). the mean age (±sd) was 66 ± 19 years, and the sex of 126 (56%) was male. most patients (66% [146]) had ≥1 comorbidity, with chronic respiratory disease (in 37%) and diabetes mellitus (in 22%) being the most frequent. despite bacterial pathogens were not isolated, patients received empirical antibiotic therapy. monotherapy was reported for 84 patients (40%); fluoroquinolones and β-lactams were the most common agents administered. a total of 127 patients (60%) received combination therapy, with the most frequent combinations comprising a β-lactam plus a macrolide (27% [56 patients]) and a β-lactam plus a fluoroquinolone (26% [54]). the median length of hospital stay was 7 days (interquartile range, 5-12 days); in-hospital mortality was 7% (16 patients). a total of 43 patients (19%) were admitted to the icu, of whom 23 (53%) required mechanical ventilation; the median length of icu stay was 7 days (interquartile range, 4-12 days), and icu mortality was 7% (3 patients). thirty-day mortality was 4% (10 patients), and 1-year mortality was 8% (17). among all patients with a diagnosis of pure viral cap, 138 (61%) presented with sepsis, and 9 (7%) presented with septic shock at admission. table 1 summarizes the main clinical characteristics. the sepsis group had a greater mean age, a greater proportion of males, and a greater prevalence of comorbidities (especially chronic respiratory diseases), compared with the nonsepsis group. there was no statistically significant difference in symptoms (fever, cough, pleuritic pain, purulent expectoration, or dyspnea) between the 2 groups. at admission, a greater proportion of patients in the sepsis group presented with an elevated respiratory rate and lower lymphocyte levels, compared with patients in the nonsepsis group. there was no statistically significant difference in the distribution of respiratory viruses between the 2 groups. thus, we did not find any association between the type of virus and the presence or absence of sepsis (in the nonsepsis group, influenza virus was found in 59% [51 patients] and non-influenza virus in 41% [36], compared with 59% [82] and 41% [56], respectively, in the sepsis group; p > .99). more patients in the sepsis group were classified as having a pneumonia severity index of iv-v, indicating severe cap. overall, 92 patients (41%) received antiviral therapy with oseltamivir. the percentage of patients who received antiviral therapy was similar between the 2 groups (47% vs 42%; p = .43). forty-four patients (33%) with sepsis were treated empirically with antibiotic monotherapy. the sepsis group received fluoroquinolone-based monotherapy less frequently than the nonsepsis group (27% vs 44%; p = .008). antimicrobial therapy was inappropriate (ie, nonconcordant with published guidelines) in 4 cases (3%) in the sepsis group, but there was no significant difference from the nonsepsis group (4%). among the variables associated with viral sepsis in the univariate logistic regression analysis, age ≥65 years and male sex remained independent risks factors for viral sepsis in the multivariable analysis (table 2 ). internal validation of the logistic regression model by using bootstrapping with 1000 samples demonstrated robust results for all variables included in the model, with small 95% confidence intervals (cis) around the original coefficients. no statistically significant difference was observed between the two groups in terms of in-hospital mortality, icu mortality, length of icu stay, 30-day mortality, and 1-year mortality (table 1) . however, patients with sepsis showed longer length of hospital stay, were more frequently admitted to icu and needed (10) .032 more frequently invasive mechanical ventilation than patients without sepsis. in the propensity-adjusted logistic regression multivariable analysis of in-hospital mortality using the weighted data, after exclusion of patients with septic shock at admission and those with do-not-resuscitate orders, pure viral sepsis was not associated with in-hospital mortality (odds ratio, 0.77; 95% ci, .18-3.17). all variables remained significant after the bootstrapping procedure, with a small 95% cis around the original coefficients. this study has 3 main findings. first, pure viral sepsis defined according to the sepsis-3 criteria was found in 3% of all patients admitted with a diagnosis of cap, 19% of those admitted to abbreviations: ci, confidence interval; copd, chronic obstructive pulmonary disease; or, odds ratio. a the variables analyzed in the univariate analysis were as follows: age, sex, smoking status, alcohol consumption, influenza vaccination, pneumococcal vaccination, previous inhaled corticosteroid therapy, previous systemic corticosteroid therapy, previous antibiotic therapy in last week, chronic pulmonary disease, chronic cardiovascular disease, chronic renal disease, chronic liver disease, diabetes mellitus, chronic neurologic disease, and nursing home admission (p = .51). defined as the probability of being in the sepsis group divided by the probability of being in the nonsepsis group. c based on the null hypothesis that all ors relating to an explanatory variable equal unity (ie, that there was no effect). patients who initially received noninvasive ventilation but subsequently needed intubation were included in the invasive mechanical ventilation group. the icu, and 61% of those with a diagnosis of pure viral cap. second, male sex and age ≥65 years were shown to be risk factors for pure viral sepsis. third, pure viral sepsis was not found to be a risk factor for in-hospital mortality. sepsis is a life-threatening organ dysfunction due to the host's overwhelming response to infection. although respiratory viruses are reported to be important causative agents of severe cap [9] , the prevalence of pure viral sepsis is not fully known. a recently published study investigated the role of virus detection by multiplex pcr of nasopharyngeal samples from clinically septic patients during a winter season [10] . the authors reported that respiratory viruses, including influenza a virus, human metapneumovirus, coronavirus, and respiratory syncytial virus were detected in 70% of adult patients with sepsis. in another study, montull et al [11] investigated the predictors of severe sepsis in patients with cap and found that 38% of patients presented with severe sepsis and that 0.5% were identified to have respiratory viruses as casual agents. the proportion of patients with pure viral sepsis was slightly higher in our study population, but we think that this was due to our use of the new sepsis-3 definition. montull et al also highlighted the association between older age and development of viral sepsis, which was in line with our finding that viral sepsis affected 64% of patients (88) aged ≥65 years. these results are consistent with data showing that, because of the increased prevalence of chronic conditions and age-related changes in the immune system, elderly patients are more susceptible to infectious diseases and sepsis. it is also possible that the endothelium is fragile in this population [12] . male sex was another risk factor for pure viral sepsis, consistent with data that men typically have more chronic comorbidities and a higher incidence of cap than women [13] . we observed that viral sepsis was not a risk factor for in-hospital mortality in patients without septic shock. our data support those of previous studies in which respiratory viruses were frequently found in critically ill patients with pneumonia but mortality rates did not significantly differ between patients with bacterial infection and those with viral infection [9, 10, 14] . this highlights the need to identify patients at higher risk of viral sepsis and the importance of a complete microbiological diagnosis in cases of cap. we could not find other studies addressing the issue of pure viral sepsis (defined according to the sepsis-3 criteria) in case of cap in a large inpatient adult cohort. finally, we observed that 41% of patients with viral cap received oseltamivir therapy, without differences between patients with and those without sepsis. compared with other previous studies [5, 15] , our population received a higher proportion of antiviral therapy. however, future studies are needed to investigate why the frequency of antiviral therapy use among patients hospitalized with cap is not high, since current guidelines strongly recommend early treatment with oseltamivir in patients with influenza [8] . some limitations must be addressed. first, although the protocol used for cap diagnosis in our hospital did not change substantially during the 12-year study, we cannot discount the effect of changes in microbiological diagnosis over this period. second, regarding microbiological diagnosis, morerapid pcr diagnostic tests for influenza virus and respiratory syncytial virus were used during the influenza season. third, the indications for oseltamivir therapy were only extended in 2009, before which it was only used to treat severe cases of viral infection. in conclusion, in our cohort, pure viral sepsis affected 61% of patients with a diagnosis of viral cap, supporting the importance of stratifying patient risk for viral sepsis and making a complete microbiological diagnosis in cases of cap. notes viral pneumonia cdc epic study team. community-acquired pneumonia requiring hospitalization among u.s. adults microbial aetiology of community-acquired pneumonia and its relation to severity viral infection in community-acquired pneumonia: a systematic review and meta-analysis an international perspective on hospitalized patients with viral community-acquired pneumonia the third international consensus definitions for sepsis and septic shock (sepsis-3) viral sepsis in children infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults viral infection in patients with severe pneumonia requiring intensive care unit admission respiratory viral infections are underdiagnosed in patients with suspected sepsis nac calidad group. predictors of severe sepsis among patients hospitalized for community-acquired pneumonia shared features of endothelial dysfunction between sepsis and its preceding risk factors (aging and chronic disease) risk factors for community-acquired pneumonia in adults in europe: a literature review lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia oseltamivir use among children and adults hospitalized with community-acquired pneumonia we thank all medical and nursing colleagues for their assistance and cooperation in this study.financial support. this work was supported by ciber de enfermedades respiratorias (ciberes cb06/06/0028), 2009 support to research groups of catalonia 911; idibaps. dr cilloniz is the recipient of a postdoctoral grant (strategic plan for research and innovation in health-peris 2016-2020 to c. c.) and separ fellowship 2018 (to c. c.).potential conflicts of interest. all authors: no reported conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-269383-1tyorrb0 authors: lai, christopher k c; chen, zigui; lui, grace; ling, lowell; li, timothy; wong, martin c s; ng, rita w y; tso, eugene y k; ho, tracy; fung, kitty s c; ng, siu t; wong, barry k c; boon, siaw s; hui, david s c; chan, paul k s title: prospective study comparing deep-throat saliva with other respiratory tract specimens in the diagnosis of novel coronavirus disease (covid-19) date: 2020-08-01 journal: j infect dis doi: 10.1093/infdis/jiaa487 sha: doc_id: 269383 cord_uid: 1tyorrb0 background: self-collected specimens has been advocated to avoid infectious exposure to healthcare workers. self-induced sputum in those with a productive cough, and saliva in those without a productive cough have been proposed, but sensitivity remains uncertain. methods: we performed a prospective study in two regional hospitals in hong kong results: we prospectively examined 563 serial samples collected during the virus shedding periods of 50 patients: 150 deep-throat saliva (dts), 309 pooled-nasopharyngeal (np) and throat swabs, and 104 sputum. dts had the lowest overall rt-pcr positive rate (68.7% vs. 89.4% [sputum] and 80.9% [pooled np and throat swabs]), and the lowest viral rna concentration (mean log copy/ml 3.54 vs. 5.03 [sputum] and 4.63 [pooled np and throat swabs]). analyses with respect to time from symptom onset and severity also revealed similar results. virus yield of dts correlated with that of sputum (pearson correlation index [95% ci]: 0.76 [0.62 – 0.86]). we estimated the overall false-negative rate of dts could be 31.3%, and increased 2.7 times among patients without sputum. conclusion: dts produced the lowest viral rna concentration and rt-pcr positive rate compared to conventional respiratory specimens in all phases of illness. self-collect sputum should be the choice for patients with sputum. since december 2019, the novel coronavirus disease (covid-19) put healthcare systems all over the world under unprecedented stress. while an all-round control strategy consists of border control, quarantine measures, isolation, social distancing and contact tracing; an early case detection by sensitive laboratory diagnosis test is indispensable to achieve an effective control of pandemic [1] . international authorities recommend laboratory diagnosis of sars-cov-2 infection should base on real-time pcr (rt-pcr) detection of viral rna in respiratory specimens [2, 3] . conventional respiratory tract specimens consist of nasopharyngeal swab or aspirate, throat swab, sputum, and tracheal aspirate. however, collection of respiratory tract specimens carries a risk of inducing cough, and thus poses additional risk to healthcare personnel. since covid-19 patients do not always have productive cough [4] , other types of self-collect specimens have been explored [5] [6] [7] [8] . saliva is a self-collect specimen. the collection process is non-invasive. it has long been explored for diagnosis of viral infections including hiv, hepatitis c, and dengue fever; but with variable degrees of success [9] [10] [11] [12] . the diagnostic value of saliva for sars-cov-2 has recently been examined. posterior oropharyngeal saliva was shown to be a good alternative to nasopharyngeal specimens for diagnosis [5] [6] [7] [8] , and for monitoring of viral load [13, 14] . while positive results aid prompt treatment and isolation, false-negative results carry a risk of inadvertent spread of infection in the community [15] . here, we evaluated the diagnostic performance we performed a prospective study in two regional hospitals in hong kong. all recruited patients had sars-cov-2 infection confirmed by two rt-pcr targeting different regions of the rdrp gene performed respectively by the local hospital and public health laboratory service. asymptomatic patients included in our study were admitted for isolation after testing positive for sars-cov-2 through active case finding as part of border control measures or contact investigations. all patients provided informed consent. the joint chinese university of hong kong -new territories east cluster clinical research ethics committee approved the study. the study was registered with nct (nct04325919) database. we collected serial conventional respiratory tract specimens including sputum and pooled nasopharyngeal (np) and throat swabs. in addition, patients were asked to self-produce -saliva‖ according to a specific instruction to collect -deep-throat‖ secretions. early morning samples before tooth brushing and breakfast were preferred. patients were instructed to refer to a video tutorial produced by the centre for health protection, hksar [16] . in brief, patients first cleared their throat by gargling with their own saliva, and then spitted out the dts into a sterile bottle. sputum samples were self-collected. patients were asked to cough out sputum, and spitted into a sterile plastic bottle. pooled np and throat swabs were collected by healthcare workers under strict infection control precautions, using flocked swabs (floqswabs, copan, italy) contained in a sterile bottle with viral transport medium. rna was extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany). sars-cov-2 rna was detected and quantified by real-time reverse-transcriptase polymerase chain reaction (rt-pcr), with primers and probes targeting the n gene of sars-cov-2 as described previously [17, 18] . according to the policy at that time, all patients were required to remain in isolation care until two consecutive rt-pcr-negative respiratory specimens were obtained at least 24 hours apart. we defined the respiratory viral shedding period as the period inclusive and between the first and last respiratory specimens tested positive by rt-pcr. clinical severity was classified as previously described to mild, moderate, severe, and critically ill [4, 18] . sars-cov-2 viral rna concentration data and positive rates were analyzed in relation to symptom onset date. for the purpose of analysis in this study, the day of diagnosis was taken as symptom onset for asymptomatic cases. data were presented as percentage, mean or median. comparisons of positive rates and viral loads between groups were performed using mann-whitney u test and wilcoxon signed-rank test as appropriate, with p ≤ 0.05 considered as statistically significant. false-negative rate was calculated by a c c e p t e d m a n u s c r i p t 7 number of negative samples for each specimen type over the respiratory viral shedding period divided by the number of each type of specimen collected over the same period. between february 6 and april 10, 2020, a total of 50 patients, including 27 females and 23 males aged between 16 and 72 years old were recruited. one patient had critical illness, two had severe disease, 14 had moderate disease, 31 had mild disease, and two were asymptomatic. a total of 563 specimens collected within the respiratory shedding period of the corresponding patients were included for analysis. specimens collected ≥ 23 days after symptom onset were excluded from this study, as those specimens were dominated by a few prolonged shedders. each patient provided 2 to 32 specimens. all patients provided deep-throat saliva specimens (dts) (range 1-10 per patient) and pooled np and throat swabs (1-16 per patient). in addition, 26 patients provided sputum specimens (1-9 per patient). samples were collected between one to 22 days (mean, standard given the observation that dts rt-pcr positive rate correlated with viral rna concentration in sputum, we further analyzed dts rt-pcr false-negative rates among sputum producers and nonsputum producers. in this analysis, we focused on events occurred during the first week from illness onset, as this is the most critical period for diagnosis. we divided patients into sputum producers (n = 15) and non-producers (n = 35) according to their symptoms reported during the first week of illness. of note, there were an additional 11 patients who developed productive cough after the first week, making a total of 26 patients had experienced productive cough when the whole study period was considered. among the rt-pcr results of 48 dts collected during day 1-7 after symptom onset, we found that the dts rt-pcr false-negative rate of non-sputum producers were 2.6 times higher than countries have strived to improve both accessibility and capacity of laboratory testing for sars-cov-2. drive-thru testing is implemented in the us and south korea [19, 20] , where individuals drive their vehicle to the testing sites, which are usually open, naturally ventilated areas for healthcare workers to collect nasopharynx plus throat swabs in full gear personal protective equipment (ppe) of surgical mask, face shield, gloves, and gown. hong kong has adopted an alternate approach of homebased screening utilizing self-collect dts specimens. there are several advantages of self-collected dts specimens. it alleviates the demand of ppe during specimen collection and negative pressured rooms or open space required during nasopharyngeal specimen collection by healthcare workers. this is especially valid during the hike of pandemic period where supply of ppe is scarce in many parts of the world. in hong kong, the centre for health protection implemented a policy using early morning dts (also referred to as posterior oropharyngeal saliva) collected at home to test for sars-cov-2 among persons who do not require hospitalization, and inbound travelers at the hong kong international airport [16, 21] . the appropriateness of the type of samples to be collected for diagnosis of covid-19 should be further investigated. in two previous studies [13, 22] , the authors analyzed 173 respiratory specimens collected from 23 patients throughout the disease course. ten patients had severe symptoms and 13 had mild symptoms. they found that rt-pcr was negative in the saliva specimens of 3/23 (13%) patients. in another study by the same research group [23] , they tested archived nasopharyngeal swabs and posterior oropharyngeal saliva specimens from 58 confirmed covid-19 patients using xpert® xpress sars-cov-2 assay. they found that 6/58 (10.3%) of saliva specimens were falsely negative. however, in these two studies, the authors did not scrutinize clinical severity and time of specimen collection. another group studied two hundred sample pairs of nasopharyngeal and throat swabs and saliva samples from 200 individuals attended an acute respiratory infection clinic. nineteen patients a c c e p t e d m a n u s c r i p t 12 were diagnosed with covid-19. the sensitivity and specificity of rt-pcr using saliva specimen were 84.2% and 98.9% respectively, when using nasopharyngeal and throat swabs as reference standard [24] . in another retrospective analysis, 95 patient-matched dts and nasopharyngeal swab specimens from 62 patients tested for sars-cov-2 by rt-pcr were analyzed. the authors concluded that the performance of dts was equivalent to nasopharyngeal swab specimens [25] . to date, our current study provides the largest number of patients with prospectively collected saliva specimens throughout the clinical course and with head-to-head comparison of dts to both upper and lower tract respiratory samples. we involved two major acute hospitals and included patients from a wide spectrum of illness ranging from asymptomatic, mild, moderate, to severe and critical; and specimens were collected at different time from symptom onset.. our findings are in concordance with others, where we observed that viral rna concentration was highest during early phase of illness, and gradually decreases over time. this observation is also consistent with our previous findings [18] . more importantly, dts produced the lowest viral rna concentration and lowest rt-pcr positive rate compared to conventional respiratory specimens throughout the early, mid and late phases following symptom onset. we therefore do not recommend the use of dts when other specimen types are feasible, namely hospitalized patients and designated screening centers where ppe can be provided and infection risk can be controlled. we found that sputum, an option of self-collect specimen, provided the highest diagnostic yield. however, not all sars-cov-2-infected patients are sputum producers. as in our study cohort, there were patients with very mild symptoms without cough at all; or those with cough but could not produce sputum. under these circumstances, other choice of specimen is needed. a c c e p t e d m a n u s c r i p t 13 it would be advisable to be specific on the terminology used for -saliva‖ specimens. we suggest dts to be differentiated from saliva specimens collected from anterior buccal cavity, or those collected by the -drooling technique‖ where saliva was collected intraorally with the use of a pipette [26] . we found that saliva specimens were more closely correlated to sputum than to pooled np and throat swabs. in addition, the consistency of dts was often observed to be thick and mucoid and sputumlike. these findings suggest dts represents lower respiratory tract secretions that are propelled up from lower airway by the ciliated cells of the respiratory epithelium. although we found that the diagnostic performance of dts was improved in sputum producers, it is not clinically relevant as for these patients a self-collect sputum would be a better choice. our findings caution the use of dts. one must be fully aware of its limitations before advocating its widespread use. dts contains lower viral rna concentration and is less sensitive in detecting sars-cov-2 infection than sputum and pooled np and throat swabs. collection of good dts specimens demands certain technique which could vary between individuals, especially for home-collected specimens. sensitivity of dts is likely to be influenced by factors including the volume of saliva collected, the time of day of specimen collection, collection technique, and being a sputum producer or not. clear instructions must be given to ensure acceptable specimen quality. our study had several limitations. firstly, out of the 563 samples analyzed, only 170 were from synchronized samples. the comparison could be more meaningful if more synchronized samples were available. secondly, we did not have data on the time of collection for the dts samples. we postulate the timing could affect the sensitivity of dts, and with a higher viral rna concentration from early morning specimens than at later times of the day. also, we did not directly supervise the collection of dts to ensure adherence to instructions, and this might influence the diagnostic accuracy of dts. nevertheless, this means our results reflect the expected performance of self-collect dts. lastly, only a c c e p t e d m a n u s c r i p t 14 two patients in our study were asymptomatic. asymptomatic persons are more likely to be the target of screening, and their dts specimens could give an even lower sensitivity. in conclusion, with the highest overall detection rate and viral rna concentration, we believe selfcollected sputum is the specimen of choice for sputum-producing patients. dts has an obvious advantage of being self-collected and with a low infectious risk during specimen collection. however, since dts has both the lowest viral rna concentration and detection rate amongst the three specimen types analyzed in the current study, one must bear in mind its limitations before advocating its widespread use. covid-19 epidemic in switzerland: on the importance of testing, contact tracing and isolation laboratory testing for 2019 novel coronavirus (2019-ncov) in suspected human cases center of disease control and prevention. interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (covid-19). available at clinical characteristics of coronavirus disease 2019 in china gargle lavage as a safe and sensitive alternative to swab samples to diagnose covid-19: a case report in japan comparison of gargle samples and throat swab samples for the detection of respiratory pathogens saliva as a diagnostic specimen for testing respiratory virus by a point-of-care molecular assay: a diagnostic validity study posterior oropharyngeal saliva for the detection of sars-cov-2 value of routine dengue diagnostic tests in urine and saliva specimens demonstration of hepatitis c virus genome in saliva and urine of patients with type c hepatitis: usefulness of the single round polymerase chain reaction method for detection of the hcv genome comparison of hiv oral fluid and plasma antibody results during early infection in a longitudinal nigerian cohort detection of sars associated coronavirus in throat wash and saliva in early diagnosis temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study saliva is more sensitive for sarscov-2 detection in covid-19 patients than nasopharyngeal swabs false negative tests for sars infection -challenges and implications update on the list of areas with active community transmission of covid-19 and further enhancement on the surveillance at clinics of private medical practitioners. available at: p t (2019-ncov) real-time rrt-pcr panel primers and probes viral dynamics of sars-cov-2 across a spectrum of disease severity in covid-19 covid-19 drive through testing: an effective strategy for conserving personal protective equipment testing on the move: south korea's rapid response to the covid-19 pandemic covid-19 testing extended accessed 2 consistent detection of 2019 novel coronavirus in saliva evaluating the use of posterior oropharyngeal saliva in a point-of-care assay for the detection of sars-cov-2 saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease 2019: a cross sectional study deep throat saliva as an alternative diagnostic specimen type for the detection of sars-cov-2 saliva is a reliable tool to detect sars-cov-2 key: cord-286596-p10t0dta authors: erard, veronique; chien, jason w.; kim, hyung w.; nichols, w. garrett; flowers, mary e.; martin, paul j.; corey, lawrence; boeckh, michael title: airflow decline after myeloablative allogeneic hematopoietic cell transplantation: the role of community respiratory viruses date: 2006-06-15 journal: j infect dis doi: 10.1086/504268 sha: doc_id: 286596 cord_uid: p10t0dta we conducted a 12-year retrospective study to determine the effects that the community respiratory-virus species and the localization of respiratory-tract virus infection have on severe airflow decline, a serious and fatal complication occurring after hematopoietic cell transplantation (hct). of 132 hct recipients with respiratory-tract virus infection during the initial 100 days after hct, 50 (38%) developed airflow decline ⩽1 year after hct. lower-respiratory-tract infection with parainfluenza (odds ratio [or], 17.9 [95% confidence interval {ci}, 2.0–160]; p=.01) and respiratory syncytial virus (or, 3.6 [95% ci, 1.0–13]; p=.05) independently increased the risk of development of airflow decline ⩽1 year after hct. the airflow decline was immediately detectable after infection and was strongest for lower-respiratory-tract infection with parainfluenza virus; it stabilized during the months after the respiratory-tract virus infection, but, at ⩽1 year after hct, the initial lung function was not restored. thus, community respiratory virus–associated airflow decline seems to be specific to viral species and infection localization perienced significant airflow decline and a significantly increased mortality risk that were attributable to this syndrome [1] . this analysis also identified community respiratory-virus infections during the initial 100 days after hct as being a significant risk factor for development of severe airflow decline but did not differentiate between specific viral infections or between infection localization in the upper respiratory tract (urt) or lower respiratory tract (lrt) [1] . given the known association between infections with specific community respiratory viruses and other forms of airways disease [2, 3] , we investigated the contributions that the virus species and infection localization made to the development of this syndrome. a well-characterized cohort of patients who received their first allogeneic transplantation at the fred hutchinson cancer research center (fhcrc) between 1 january 1990 and 31 december 2000 were evaluated by pulmonary function tests (pfts) 16 (1) community respiratory-virus infection 132 (12) note. data indicate no. (%) of 1131 subjects, unless otherwise indicated [1] . fev 1 , first-1-s forced expiratory volume; fvc, forced volume capacity; gvhd, graft-versus-host disease; hct, hematopoietic stem cell transplantation. a disease at hct: low-risk diseases included chronic myeloid leukemia in chronic phase, refractory anemia, and aplastic anemia; intermediate-risk diseases included chronic myeloid leukemia in accelerated phase or in chronic phase after blast phase, acute leukemia or lymphoma in remission, refractory anemia with excess blasts, chronic lymphocytic leukemia, and paroxysmal nocturnal hemoglobinuria; and highrisk diseases included chronic myeloid leukemia in blast phase, juvenile chronic myeloid leukemia, acute leukemia or lymphoma in relapse, refractory anemia with excess blasts in transformation, and myeloma. b de novo chronic gvhd indicates the absence of prior acute gvhd, and chronic gvhd indicates a history of acute gvhd that either did or did not resolve before the onset of chronic gvhd. prior to hct and р1 year after hct. the analysis was restricted to patients who survived to 6 months after hct and for whom pft results were available both before (baseline pft) and after that period. clinical variables were collected prospectively as part of the usual clinical care and were stored in a centralized database. disease risk for all malignancies at the time of hct was classified as low, intermediate, or high, on the basis of previously published criteria [4] . donor type was determined according to donor-recipients abo compatibility and hla-a, hla-b, and hla-dr status. acute graft-versushost disease (gvhd) was graded using standard criteria [5] and was categorized as yes-acute gvhd (grades [3] [4] or noacute gvhd (grades 0-2). the diagnosis and staging of chronic gvhd were established using previously published criteria [6] . acute and chronic classes of gvhd were then integrated and categorized as no-acute gvhd or chronic gvhd, acute gvhd alone, de novo chronic gvhd (not preceded by acute gvhd), and chronic gvhd (preceded by acute gvhd that was or was not followed by a period of quiescence). the impact that community respiratory-virus urt infection and lrt infection had on severe airflow decline was examined. in addition, cytomegalovirus (cmv) pneumonia and pulmonary aspergillosis (definite and probable) also were evaluated as infections not typically associated with airflow decline. the fhcrc institutional review board approved the study. respiratory specimen and virology procedures. throughout the study period, all hct recipients with urt infection symptoms, such as rhinorrhea, pharyngitis, or coryza, during the first 100 days after hct underwent nasopharyngeal-throat wash and swab testing. all patients with radiographic signs of lrt infection underwent a bronchoalveolar lavage (bal). nasopharyngeal-throat wash and swab samples, bal specimens, and/or tissue-biopsy or autopsy material were tested for community respiratory viruses (respiratory syncytial virus [rsv], parainfluenza virus types 1-3, influenzavirus a and b, adenovirus, and rhinovirus), by direct immunofluorescence (dfa) testing, shell vial centrifugation culture, and conventional culture. cultures were inoculated into tissue cultures containing rhesus monkey kidney, human foreskin fibroblasts, and a549 cells. type-specific respiratory dfa smears were performed using commercially available, type-specific antiserum (bartels vrk; intracel) [7] . bal samples were also tested for cmv, by viral culture, shell vial centrifugation culture, and dfa. probable or proven aspergillus pulmonary infection was confirmed by a positive culture or by histopathological results of bal or lung biopsies [8] . community respiratory-virus urt infection was defined as the detection of any community respiratory virus from a nasopharyngeal-throat specimen, by culture, shell vial culture, or dfa, in conjunction with consistent symptoms in the absence of a new infiltrate detected by chest radiography. community respiratory-virus lrt infection was defined as the isolation of any respiratory virus, by culture or dfa from bal or lung biopsy or autopsy specimens, in conjunction with symptoms and a radiographically new or changing infiltrate [7, 9] . on the basis of the viruses detected in the clinical specimen, 3 categories of community respiratory-virus infection were defined: parainfluenza infection, rsv infection, and "other" infection, which included rhinovirus, adenovirus, and influenzavirus a or b. any patient with a parainfluenza infection, regardless of coinfections, was considered as having parainfluenza infection and was analyzed in the parainfluenza category only. the same criterion was applied to patients with rsv infections, unless parainfluenza virus was also detected, in which case patients were analyzed in the parainfluenza category. pft, airflow decline definition, and data analysis. pfts were performed as described elsewhere [1] . the first-1-s forced expiratory volume (fev 1 ) and forced vital capacity (fvc) were expressed as a percentage of predicted normal values, calculated using published equations for children and adults [10, 11] . baseline (before hct), day 100 (100 days after hct), and year 1 (1 year after hct) pft results were obtained routinely, regardless of the presence or absence of symptoms. significant airflow decline was defined as a 15%/year decline in percentage of predicted fev 1 during year 1, with the year 1 fev 1 :fvc ratio !0.8 [1] . the annualized rate of airflow decline (decline in percentage of predicted fev 1 ) by year 1 was calculated using a least squares regression method [1] . the annualized rate of decline in percentage of predicted fev 1 in patients who had developed respiratory infection with community respiratory viruses was also calculated from baseline to day 100 ([(day 100 fev 1 ϫ baseline fev 1 ) / (day 100 pft date ϫ baseline pft date)] ϫ 365) and from day 100 to year 1 ([(year 1 fev 1 ϫ day 100 fev 1 ) / (year 1 pft date ϫ day 100 pft date)] ϫ 365). statistical analysis. statistical analyses were performed using stata (version 8.0; statacorp) and the r statistical program [12] (available at: http://www.r-project.org). two-sided p values !.05 were considered to be statistically significant. univariate analyses were conducted using the x 2 test. the presence of significant airflow decline by year 1 was analyzed in a mul-tivariable logistic regression model. the outcome, significant airflow decline, was defined as a binary variable. the independent variable included the localization of the infection and the species of community respiratory virus. an additional analysis was performed to determine the risk of severe airflow decline associated with lrt cmv infection and pulmonary aspergillosis. the model was adjusted for age at hct, baseline fev 1 : fvc ratio, and gvhd category. these were covariates that had been found to be significant risk factors for airflow decline in the previous analysis [1] . the kruskall-wallis test was used to compare the median rate of decline in percentage of predicted fev 1 , of each viral-infection category, from baseline to day 100 and from day 100 to year 1. the mortality cohort consisted of patients for whom year 1 follow-up pft results were available. the probability of overall survival (kaplan-meier method) and the multivariable cox proportional-hazard analysis, comparing patients with severe airflow decline to patients without severe airflow decline, had been described in the initial study [1] . of the 3002 first-myeloablative allogeneic-hct recipients between 1 january 1990 and 31 december 2000, 1131 (38%) were eligible for the study, and 299 (26%) cases of severe airflow decline were identified when the recently validated definition of severe airflow decline was applied [1] . the clinical characteristics of the study population are shown in table 1. during the initial 100 days after hct, 132 patients (12%) had a documented viral infection with community respiratory viruses. of these 132 patients, 114 (86%) had a urt viral infection, and 38 (33%) of these 114 developed significant airflow decline by year 1. two additional common, lrt infections were evaluated regarding their ability to cause severe airflow decline. of long-term survivors, 9 patients (1%) were identified as having cmv pneumonia during the first 100 days (and 1 of these 9 was coinfected with rhinovirus), and 16 (1%) were identified as having pulmonary aspergillosis. severe airflow decline by year 1. of the 132 patients who had a documented viral infection, 18 (14%) had an lrt viral infection, and, of these 18 patients, 12 (67%) developed significant airflow decline by year 1. table 2 displays the distribution of the different categories of respiratory-tract virus infection, according to the presence or absence of significant airflow decline by year 1. the majority of patients with lrt parainfluenza-virus infection (86%) and lrt rsv infection (55%) during the first 100 days developed significant airflow decline by year 1. none of these patients were coinfected or developed a superinfection with a bacterial pathogen. multivariable analysis, including covariates previously determined to be significant risk factors for significant airflow decline [1] , demonstrated that the presence of community respiratory-virus lrt infection was associated with an increased risk of having a significant airflow decline by year 1 (odds ratio [ ). neither lrt p p .05 cmv infection nor lrt aspergillus infection was found to predict severe airflow decline (data not shown). fev 1 changes during study periods. in addition to the annualized rate of airflow decline, we estimated the impact that the infection status had on lung function, by using the change in percentage of predicted fev 1 from baseline to day 100 and from day 100 (when the viral infection was diagnosed) to year 1. by day 100, the median rates of decline in percentage of predicted fev 1 for patients who had lrt infection with either parainfluenza virus or rsv were 13% and 11%, respectively. all patients with urt viral infection only experienced a rate of decline in percentage of predicted fev 1 that was !5% ). the rate of decline in p p .09 percentage of predicted fev 1 for patients with lrt rsv infection did not reach statistical significance (when compared with that in the other groups). during the months after infection (day 100 to year 1), there was a slight improvement in the function, with the median rate of percentage of predicted fev 1 increasing from 1.6% to 5%, depending on the type of infection with which patients presented during the initial 100 days (figure 1). the median rate of improvement in percentage of predicted fev 1 was not statistically different between the different categories of infection ( ). p p .74 because of their ability to cause fatal pneumonia, community respiratory viruses are widely recognized as a significant cause of morbidity and mortality after hct [13] [14] [15] . the present study suggests that community respiratory-virus infections might contribute to increased overall mortality [7, 16] , not only by causing fatal pneumonia but also via a long-term decline in lung function. indeed, the initial study has revealed that severe airflow decline has a significant adverse effect on mortality after adjustment for other common causes of death among long-term survivors of hct [1] . we found that the association between community respiratory viruses and hct-related airflow decline may be influenced by the specific type and location of respiratory-virus infection. in multivariable analysis, lrt viral infection was found to significantly increase the risk of development of air-flow decline р1 year after hct, and this risk was particularly high for patients who developed an lrt parainfluenza-virus infection (table 3) . lrt rsv infection probably also increases this risk; however, the number of cases was too small to reach statistical significance. interestingly, even urt parainfluenzavirus infection was notably associated with airflow decline, suggesting a potential mechanism for the previously reported poor outcomes associated with urt parainfluenza-virus infection [7] . the presence of copathogens has been reported as being a significant risk factor for death in patients with parainfluenza pneumonia [17] . in the present study, none of the patients with lrt viral infection were infected with a copathogen, precluding us from assessing the impact that copathogens might have on airflow decline associated with respiratory-tract virus infection. the absence of copathogens in this study is probably due to the fact that only long-term survivors of respiratory-tract virus infections were included. we did, however, examine the impact that 2 common pulmonary pathogens had on airflow decline. as expected, neither lrt cmv infection nor lrt aspergillus infection increased the risk of severe airflow decline in patients who survived the infection. we also examined the timing of the development of airflow decline relative to the community respiratory-virus infection. the most significant decline of the percentage of predicted fev 1 was observed between the day of hct and day 100, during which time the respiratory-tract virus infection occurred. the significant decline observed for lrt infections compared with urt infections has to be considered with caution, because of the limitation inherent in performing multiple testing. although the percentage of predicted fev 1 of patients who experienced a significant fev 1 decline during the first 100 days after hct improved between day 100 and year 1, none of those patients' levels of fev 1 returned to their baseline, suggesting that, despite some recovery, lrt infection with community respiratory viruses can result in permanent loss of lung function. there are several possible explanations for this phenomenon. prolonged shedding of parainfluenza is known to occur in immunosuppressed patients [18] , and subclinical shedding of respiratory viruses has been documented in hct recipients [19] . thus, subclinical persistence of the virus in the respiratory tract after the initial infection might maintain sustained airway inflammation, which may ultimately lead to permanent loss of lung function, even after apparent clinical clearance of the infection. community respiratory viruses might also activate an inflammatory process in the lrt that, in the patients who survive the acute phase of an lrt virus infection, might lead to irreversible damage to the tissue of the small airways that is independent of viral replication [20] . the present study has strengths and limitations. its strengths include the large sample size, a standardized diagnostic approach for urt and lrt infections, and standardized pfts throughout the study period. one limitation is that the predictor of interest (community respiratory-virus infections) was assessed only during the first 100 days after hct. it is likely that respiratory-tract virus infections can also occur later, after patients have been discharged from the transplantation center and return to a less protected environment. in addition, other potential contributors, such as bacterial coinfection, were not examined. another possible limitation is that the conventional detection methods used in this study, because of their lack of sensitivity, may have underestimated the true rate of respiratory infections [21] . these assays were also unable to detect uncultivated pathogens, such as human metapneumovirus and coronaviruses [22] [23] [24] . however, both undetected pathogens and asymptomatic shedders [25, 26] would be included in the noninfected group and would therefore make the significance of our findings even stronger. a further possible limitation is that the number of cases was not large to provide adequate power to assess the individual role that viruses such as adenovirus, rhinovirus, and influenza virus play in the occurrence of significant airflow decline. finally, only patients who survived the infection were included in the estimation of the rate of airflow decline, resulting in a potential "healthy survivor effect." however, this limitation might actually have resulted in an underestimation of the impact that respiratory infection had on the rate of lung decline. in conclusion, the present study revealed that parainfluenza virus and rsv are associated with late severe airflow decline, which may suggest an additional mechanism by which some respiratory viruses may cause morbidity and perhaps even mortality after hct. these data deserve further investigation, which is focused on understanding the mechanisms by which viral infections may lead to the development of severe airflow decline after hct. such studies may ultimately lead to insight into the role that viral infections play in other, more common airway diseases, such as asthma. airflow obstruction after myeloablative allogeneic hematopoietic stem cell transplantation respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children progress in defining the role of rsv in allergy and asthma: from clinical observations to animal models therapy for chronic graftversus-host disease: a randomized trial comparing cyclosporine plus prednisone versus prednisone alone hematopoietic stem cell transplantation chronic graft-versus-host disease in 52 patients: adverse natural course and successful treatment with combination immunosuppression parainfluenza virus infections after hematopoietic stem cell transplantation: risk factors, response to antiviral therapy, and effect on transplant outcome defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus phase 1 evaluation of the respiratory syncytial virus-specific monoclonal antibody palivizumab in recipients of hematopoietic stem cell transplants reference spirometric values using techniques and equipment that meet ats recommendations ventilatory functions of normal children and young adults-mexican-american, white, and black. i. spirometry r: a language and environment for statistical computing community respiratory virus infections in bone marrow transplant recipients: the m.d. anderson cancer center experience diagnosis and epidemiology of community-acquired respiratory virus infections in the immunocompromised host respiratory virus infections in stem cell transplant patients: the european experience respiratory syncitial virus (rsv) infection in hematopoietic stem cell transplant (hct) recipients: risks factor for acquisition and lower respiratory tract disease, and impact on mortality of hematology annual meeting and exposition community-acquired respiratory syncytial virus and parainfluenza virus infections after hematopoietic stem cell transplantation: the fred hutchinson cancer research center experience molecular epidemiology of two consecutive outbreaks of parainfluenza 3 in a bone marrow transplant unit asymptomatic respiratory virus infection among hematopoietic cell recipients viral induction of a chronic asthma phenotype and genetic segregation from the acute response frequent detection of respiratory viruses in adult recipients of stem cell transplants with the use of real-time polymerase chain reaction, compared with viral culture detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness emerging viral infections after hematopoietic cell transplantation coronavirus infection in acute lower respiratory tract disease of infants preemptive treatment of pediatric bone marrow transplant patients with asymptomatic respiratory syncytial virus infection with aerosolized ribavirin prolonged outbreak of human parainfluenza virus 3 infection in a stem cell transplant outpatient department: insights from molecular epidemiologic analysis we thank barry storer for additional statistical analysis and arlo upton and kieren marr for providing information on aspergillus disease. key: cord-267015-mprsdi2e authors: zhu, zhongyu; bossart, katharine n; bishop, kimberly a; crameri, gary; dimitrov, antony s; mceachern, jennifer a; feng, yang; middleton, deborah; wang, lin-fa; broder, christopher c; dimitrov, dimiter s title: exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody date: 2008-03-15 journal: j infect dis doi: 10.1086/528801 sha: doc_id: 267015 cord_uid: mprsdi2e we have previously identified neutralizing human monoclonal antibodies against nipah virus (niv) and hendra virus (hev) by panning a large nonimmune antibody library against a soluble form of the hev attachment-envelope glycoprotein g (sg(hev)). one of these antibodies, m102, which exhibited the highest level of cross-reactive neutralization of both niv and hev g, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavy-chain variable domain and panning against sg(hev). one of the selected antibody fab clones, m102.4, had affinity of binding to sg(hev) that was equal to or higher than that of the other fabs; it was converted to igg1 and tested against infectious niv and hev. it exhibited exceptionally potent and cross-reactive inhibitory activity with 50% inhibitory concentrations below 0.04 and 0.6 μg/ml, respectively. the virus-neutralizing activity correlated with the binding affinity of the antibody to sg(hev) and sg(niv). m102.4 bound a soluble form of niv g (sg(niv)) better than it bound sg(hev), and it neutralized niv better than hev, despite being originally selected against sg(hev). these results suggest that m102.4 has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. it could be also used for prophylaxis and diagnosis, and as a research reagent we have previously identified neutralizing human monoclonal antibodies against nipah virus (niv) and hendra virus (hev) by panning a large nonimmune antibody library against a soluble form of the hev attachment-envelope glycoprotein g (sg hev ). one of these antibodies, m102, which exhibited the highest level of cross-reactive neutralization of both niv and hev g, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavychain variable domain and panning against sg hev . one of the selected antibody fab clones, m102.4, had affinity of binding to sg hev that was equal to or higher than that of the other fabs; it was converted to igg1 and tested against infectious niv and hev. it exhibited exceptionally potent and cross-reactive inhibitory activity with 50% inhibitory concentrations below 0.04 and 0.6 g/ml, respectively. the virus-neutralizing activity correlated with the binding affinity of the antibody to sg hev and sg niv . m102.4 bound a soluble form of niv g (sg niv ) better than it bound sg hev , and it neutralized niv better than hev, despite being originally selected against sg hev . these results suggest that m102.4 has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. it could be also used for prophylaxis and diagnosis, and as a research reagent. hendra virus (hev) and nipah virus (niv) are highly pathogenic paramyxoviruses that have recently emerged from flying fox populations to cause serious disease outbreaks in humans and livestock in australia, malaysia, singapore, bangladesh, and india [1] . hev emerged in queensland, australia, in 1994, killing 1 human and 14 horses [2] and the virus was responsible for at least 4 other sporadic outbreaks involving horses and humans between 1994 and 2006 [1] . the closely related niv emerged in 1998 -1999 in peninsular malaysia, resulting in the death of more than 100 people and the culling of more than one million pigs [3] . since then, several outbreaks of niv infection have been recorded in bangladesh and india [1, 4, 5] . several important observations have been made during these most recent outbreaks, such as a higher incidence of acute respiratory distress syndrome, higher rates of person-to-person transmission, and higher case fatality rates (60%-75%), compared with the malaysian outbreak (with case fatality rates of ϳ40%) in which the virus was initially discovered [6 -10] . there are currently no therapeutic modalities for treating niv or hev infections, and a vaccine for prevention of disease in human or livestock populations does not exist. the first antiviral antibody-based drug approved by the u.s. food and drug administration is a humanized antibody against respiratory syncytial virus (manufactured by medimmune), which has proven to be highly successful in reducing respiratory syncytial virus infection in infants and immunocompromised patients; this antibody has been recently improved [11] . in this context, the development of neutralizing human mabs (hmabs) against henipaviruses could have important implications for prophylaxis and passive immunotherapy. one of the challenges in the development of human antibodies for antiviral applications is the heterogeneity and mutability of rna viruses. it is, therefore, important to select antibodies that recognize epitopes that are highly conserved among different virus variants. previously, we reported the isolation and characterization of potent neutralizing recombinant hmabs that targeted the viral envelope glycoprotein g by use of a highly purified, oligomeric, soluble hev g glycoprotein (sg hev ) [12] as the antigen for the screening of a large, naive human phagedisplayed antibody library [13] . one of these antibodies, m102, exhibited cross-neutralizing activity against both hev and niv. in this article, we report the identification and characterization of a novel antibody, m102.4, derived from m102 by light-chain shuffling and heavy-chain variable domain random mutagenesis. this antibody exhibits exceptional potency against both, niv and hev, and being fully human antibody, it could be directly used for prophylaxis or treatment of humans exposed to or infected by hev or niv. such an antibody could also be used for improved diagnosis and as a research tool for better understanding of virus-host interaction. fine mapping of the hmabdefined epitope may also provide information useful for the rational development of candidate vaccines and small molecule drugs. hela-usu cells have been described elsewhere [12, 14] . vero cells were provided by the australian animal health laboratory. the human glioblastoma cell line u373-mg was provided by adam p. geballe, fred hutchinson cancer research center, seattle. hela-usu and u373 cells were maintained in dulbecco's modified eagle medium (dmem [quality biologicals]) supplemented with 10% cosmic calf serum (hyclone), and 2 mmol/l l-glutamine (dmem-10). vero cells were maintained in eagle minimal essential medium (emem) (invitrogen) supplemented with 10% fetal calf serum (invitrogen australia pty. ltd), 1 mmol/l hepes (invitrogen), and 2 mmol/l l-glutamine (invitrogen) (emem-10). all cell cultures were maintained at 37°c in a humidified 5% co 2 atmosphere. generation of phage-displayed fab libraries and selection of affinity-matured fabs. the original human fab phage display library, from which the antibodies m101-m107 were identified [13] , was used as the source for the light-chain variable domain (vl) repertoire in the shuffled library. the phagemid preparation from the original library was first digested with ncoi and spei, followed by electrophoresis on an agarose gel to delete the entire heavy-chain variable domain (vh) repertoire. the gene encoding the vh of m102 was amplified by error-prone polymerase chain reaction (pcr) (stratagene) to introduce random mutations and then fused with the ch1 gene fragment by use of splicing by overlapping extension pcr. the fused fragment was digested with ncoi and spei and purified from gel; it was then ligated into the purified backbone vector to create the vls-shuffled fab repertoire. escherichia coli tg1 cells were transformed with the ligation mixtures via electroporation. the transformed tg1 cells were plated on 2ϫ yeast extract-tryptone (2yt) agar plates containing 100 g/ml ampicillin and 2% glucose (2ytag). after incubation overnight at 37°c, all of the colonies grown on the plates were scraped into 5 ml of 2ytag medium, mixed with 1.2 ml of 50% glycerol (final concentration, 10%), aliquoted, and stored at ϫ70°c as the library stock. two rounds of biopanning were performed on sg hev conjugated magnetic beads as described for the original library panning [13] . eight clones were identified as affinity-maturated antibodies, and m102.4 was selected for further characterization. selected fabs and m102.4 igg1 expression and purification was performed as described elsewhere [14] . elisa binding assay. the sg hev glycoprotein (50 ng per well) was coated in a 96-well plate in 50 l pbs. serially diluted antibodies were added into the well after washing with 1ϫpbs with 0.05% tween 20. after incubation for 1 hour at room temperature and washing, horseradish-peroxidase conjugated secondary antibody was added and incubated for another hour. after washing, plates were developed and read at 450 nm in an elisa reader. stable cell line construction and fermentation. linearlized m102.4 pdr12 construct was transfected into cho k1 cells with polyfectin in accordance with the protocol from qiagen. a stable antibody-producing cell line was selected by screening in glutamine acid-free culture medium with 50 mol/l methionine sulphoximine. it was adapted to grow in suspension in serum-free medium. the antibody was produced by fermentation in a 15-l fermentor and purification was performed with protein a. expression and immunoprecipitation of alanine hev g mutants. alanine mutations were introduced at specific residues in myc-tagged hev g using site-directed mutagenesis (stratagene). expression and immunoprecipitation of all hev g mutants were as performed as described elsewhere [15] . binding and competition analysis using multiplex microsphere assays. multiplexed microsphere assays were performed as described elsewhere [16] . cell fusion assays. the inhibition assay of cell fusion by fabs and iggs was performed as described elsewhere [12] . virus neutralization assays. all live virus experiments were conducted under strict biocontainment procedures in a biosafety level 4 laboratory. a total of 2 ϫ 10 4 vero cells were added per well with 150 l emem-10 in 96-well plates and incubated overnight at 37°c in a humidified 5% co 2 atmosphere. the foci assay was performed as described elsewhere [12] . antibody pharmacokinetics in plasma and biological activity. four juvenile (ͻ12 months), male ferrets were anesthetized by mask induction with isofluorane and maintained on 2% isofluorane and 100% oxygen. a baseline blood sample was collected from an axillary vein, and a 20-g intravenous catheter was placed in the left jugular vein. the antibody m102.4 was administered via the catheter by slow infusion over 2 minutes; 2 ferrets received 25 mg of m102.4 and 2 ferrets received 5 mg of m102.4. the catheter was withdrawn, and animals were allowed to recover from the anesthetic. subsequently, the ferrets were anes-thetized on days 1, 4, 8, 12,18, 21, 28, 35, and 42 by intramuscular injection with ketamine (ketamil; ilium) and medetomidine (domitor; novartis) (reversed with atipamazole [antisedan; novartis]). blood was collected from an axillary or jugular vein while animals were under anesthesia. after collection of the final blood sample, the animals were euthanized by means of an intravenous barbiturate overdose. for all samples, serum was aliquoted and stored at ϫ80°c. ferret serum was diluted 1:1000 and assayed by use of the binding multiplex microsphere assay described above. an m102.4 standard curve ranging from 500 ng/ml to 0.5 ng/ml and all ferret serum samples were assayed simultaneously. ferret serum m102.4 concentrations were extrapolated from the standard curve using nonlinear regression analysis (graphpad software; graphpad). half-lives were estimated from the slopes of the natural logarithms of the antibody concentration as function of time by using the formulas ta ϭ 0.69/a[ln (2) respectively, a and b are measured as the slopes of ln (m102.4 concentration, g/ml) dependent on time. serum collected on days 1, 4, and 8 was evaluated in virus neutralization assays. all sera were diluted 1:5, 1:10, 1:20 and 1:40 and assayed in duplicate using the foci assay. complete neutralization was defined as no viral foci in either well at a particular dilution. previously, we reported the isolation of henipavirus-neutralizing recombinant hmabs by screening of a large nonimmune phage-displayed fab library against a soluble form of the hev g glycoprotein (sg hev ). one of these antibodies, m102, exhibited the highest level of crossreactivity and relatively better binding to niv g than to hev g. we reasoned that improving m102's binding to hev g could further increase its cross-reactive neutralizing activity by increasing its affinity to hev g. to mature in vitro m102, we constructed an antibody library-which contained approximately 2 ϫ 10 8 clones-by light-chain shuffling combined with heavychain vh random mutations introduced by error-prone pcr. two rounds of panning against sg hev conjugated to magnetic beads demonstrated sufficient enrichment (data not shown), and 190 random colonies were screened by phage elisa. the 24 best binders were selected for sequencing analysis. they represented 8 different clones, which were designated m102.1-m102.8. although there were no amino acid sequence changes, silent mutations occurred in the vh regions in 3 of the 8 clones (data not shown), indicating that the error-prone pcr had worked. seven of the 8 different light chains were from v subfamily iii, which is the same as that of m102; 1 clone (m102.8) was from the v subfamily i. a sequence analysis of these light chains showed that all clones contained mutations in all 3 complementarity-determining regions; of the 7 clones from subfamily iii, m102.4 had the largest number of mutations (data not shown). all 8 clones were expressed as fabs, purified, and analyzed by elisa for binding to the selecting antigen sg hev . the elisa data confirmed that all 8 fab clones displayed a higher level of binding to sg hev than did the parental m102 fab (figure 1). clone m102.4, which had binding affinity equal to or higher than that of the other clones, was selected for further characterization and converted to an igg1 format. binding of igg1 m102.4 to cognate antigens. to investigate the binding of igg1 m102.4 to hev g and niv g and its ability to block receptor-g interactions, we used 2 multiplex microsphere assays that we recently developed [16] . as shown in figure 2a , m102.4 binds to both sg hev and sg niv in a dosedependent fashion. at relatively low concentrations (in the range of 50 ng/ml or less), the binding reached 50% of its maximum, indicating strong binding to both soluble g proteins. furthermore, the multiplex assays also demonstrated that m102.4 is highly efficacious in blocking the binding of ephrin-b2 ligand (efnb2) to both sg hev and sg niv (figure 2b). it is important to note that the increase in affinity did not alter the specificity of the fab; it could still bind to both g proteins very well. it is also interesting to note that m102.4 binds sg niv better than sg hev , a result that was also reflected in its slightly better efficiency at blocking efnb2-sg niv binding. these results suggest that m102.4 is a cross-reactive, high-affinity binder to the soluble forms of both hev g and niv g glycoproteins. hev env-mediated cell fusion in both formats, as fab and as igg1. as expected, fab m102.4 was significantly more potent than fab m102, and igg1 m102.4 was more potent than fab m102.4 ( figure 3) . a comparison with a previously identified antibody, m101, which is specific for hev and the most potent inhibitor of infectious hev [13] , suggested that m102.4 and m101 had comparable activity in both fab and igg1 formats ( figure 3b) . interestingly, and similar to the multiplexed results, the inhibitory activity against niv g-mediated cell fusion was higher than that against hev g-mediated fusion although m102.4 was selected by using sg hev . these results suggest m102.4 possesses exceptional cross-reactivity and potency against hev and niv env-mediated cell fusion and syncytia formation. potent cross-reactive neutralization of live viruses. igg1 m102.4 exhibited exceptionally potent and cross-reactive inhibitory activity against infectious niv and hev with ic 50 values fig. 4 . immunofluorescence-based syncytia assay of hendra virus (hev) and nipah virus (niv) infection. vero cells were plated into 96-well plates and grown to 90% confluence. virus and antibodies were premixed for 30 min at 37°c prior to addition to the cell monolayers. cells were incubated in the presence of antibody-virus mixtures for 24 h, fixed in methanol, and immunofluorescently stained for p protein prior to digital microscopy. all images were obtained at an original magnification of 20ϫ. a, hev without antibody; b, hev with m101 at 50 g/ml; c, hev with m101 at 10 g/m; d, hev with 102.4 at 50 g/ml; e, hev with 102.4 at 10 g/ml; f, niv without antibody; g, niv with m101 at 50 g/ml; h, niv with m101 at 10 g/ml; i, niv with 102.4 at 50 g/ml; j, niv with 102.4 at 10 g/ml. below 0.04 and 0.6 g/ml, respectively (table 1) . these data suggest a strong correlation between binding to the antigens, inhibition of fusion, and neutralization of infectious virus and confirm the exceptional potency and cross-reactivity of m102.4. igg1 m102.4 was also evaluated in the sensitive vero cell-fociimmunostaining assay overnight side by side with igg1 m101 ( figure 4) . in this case, not only was the amount of virus per well high (4000 tcid 50 for hev and 2000 tcid 50 for niv), but the antibody-virus mixtures were incubated on vero cell monolayers overnight. if no antibody was present (panels a and f), there was massive coalesced syncytia for both viruses. in the presence of m101, hev was neutralized to localized foci at 10 g/ml, which was further reduced to individual infected cells at 50 g/ ml. importantly, if this assay was carried out as a standard cytopathic effect-based neutralization assay, these wells would essentially look uninfected. by comparison, m102.4 neutralized 100% of hev and niv at either 10 g/ml or 50 g/ml. this extended neutralization window demonstrates the exceptional potency of m102.4 and may have important implications for postexposure therapeutic efficacy. to characterize the epitope of the affinity-maturated m102.4 -compared with m101-on the hev g glycoprotein, a panel of 17 hev g alanine-scanning mutants constructed in a previous study [15] were expressed and tested for binding to m101 and m102.4 by immunoprecipitation. the binding of hev g mutants d260a, g439a, k443a, and k465a to both m101 and m102.4 was almost absent, whereas the mutations g449a and d468a significantly decreased the binding of both antibodies, although they did so to varying degrees ( figure 5 ). interestingly, one mutation, k560a, almost completely eliminated the binding of the hevspecific antibody m101 but did not have any effect on the binding of the cross-reactive antibody m102.4. bishop et al. [15] showed that all 4 mutations-d260a, g439a, k443a and k465a-that eliminated the binding of both mabs in this study are detrimental for the binding of hev g to the receptor ephrin-b2. these results suggest that the m102.4 epitope overlaps the receptor-binding domain of hev g and the epitope of m101. in vivo plasma half-life and biological activity. as hmab m102.4 has the potential to be a potent henipavirus therapeutic agent, we next assessed its in vivo half-life and toxicity. we chose to use ferrets for these studies because they have been shown to be susceptible to niv-mediated disease (k. bossart, j. bingham, and d. middleton; unpublished data) and have become important animal models for several other human respiratory viruses, including sars coronavirus and highly pathogenic avian influenza virus. ferrets received 1 of 2 different doses of m102.4 (25 or 5 mg), as detailed above. animals were closely observed for at least 2 hours after recovery, and no adverse effects were noted. blood samples were collected over a 42-day period, and antibody concentrations were measured. a typical antibody concentration over time is shown in figure 6 , which shows 2 slopes in a logarithmic scale. half-lives calculated from these slopes did not vary with antibody dose. average distribution and elimination half-lives of 1.48 days and 3.53 days, respectively, were calculated, with relatively small individual differences. to determine whether the relatively short elimination half-life was the result of immune responses, we tested the ferret serum for anti-m102.4 ferret antibodies. we were not able to detect such antibodies in ferret serum samples after administration of m102.4 (data not shown). to demonstrate that m102.4 measured in plasma was biologically active, serum collected on days 1, 4, and 8 was evaluated using virus neutralization assays, as described above (data not shown). importantly, for all ferrets, niv was completely neutralized by 1:5 diluted serum collected on day 1 after antibody administration. when serum samples collected on day 8 were assayed, 2 samples demonstrated complete virus neutralization, and a third serum sample demonstrated 50% neutralization. although negative on day 8, the fourth serum sample showed 50% neutralization on day 4. taken together, these data demonstrate that m102.4 can remain biologically active in vivo for at least 8 days. the major finding of this study is the identification of a novel, exceptionally potent, cross-reactive neutralizing hmab, m102.4. this antibody has significantly improved potency, compared with the parent antibody m102 and with other hmabs identified and characterized in our previous study [13] . importantly, the substantial gain in potency was achieved without decreasing cross-reactivity. to our knowledge, this is the first fully human antibody that is capable of potently neutralizing both infectious hev and niv. an interesting observation made during the present study was that m102.4 had better binding to sg niv than to sg hev , despite the fact that sg hev was used as the selector antigen during the original library screening [13] and in the maturation panning procedures. the better binding to sg niv correlated with better neutralizing activity against niv, compared with hev. further studies are required to understand the mechanism underlying this unexpected observation. although the epitope mapping by alanine-scanning mutagenesis indicated that m102.4 and m101 share most of the residues on the hev g glycoprotein that affect their binding, a dramatic difference was observed for the k560a mutation, which completely abolished the binding of m101 to hev g, but did not affect m102.4's binding. it was previously shown by bishop et al. [15] that k560a had no effect on the binding of hev g to the henipavirus receptors ephrin-b2 and ephrin-b3. thus, the m101 epitope may contain contact site(s) located outside the receptor binding site. because the m102.4 epitope does not include this site, one could hypothesize that it overlaps the receptor binding site to larger extent than the m101 epitope does. such a hypothesis is in agreement with m102.4's much higher observed degree of cross-reactivity, compared with that of m101. thus, one could further hypothesize that the m102.4 epitope closely mimics the receptor binding site and, therefore, that the generation of escape mutants in the presence of this antibody would be less likely, compared with m101 and indeed any other known hmab. we have previously made similar observation for our potent cross-reactive neutralizing hmab m396, which is predicted to be effective against all sars coronavirus isolates with known sequences [17] . the m396 binding site overlaps extensively with the receptor binding site on the sars coronavirus spike (s) glycoprotein, as shown by the crystal structure of the m396-s complex [18] . thus, targeting the conserved and functionally important receptor binding site that is critical for virus entry into cells is a promising strategy for the development of cross-neutralizing antibodies. the igg1 m102.4 was well tolerated in ferrets, and no any adverse effects were noted for the relatively short time (42 days) of the experiment. the antibody pharmacokinetics consisted of 2 phases. the estimated distribution half-life of ϳ1.5 days is typical for iggs. the elimination half-life was significantly shorter than that for human iggs in humans (typically 2-3 weeks). we hypothesized that this could be the results of immune responses, specifically the elicitation of anti-human igg antibodies, which typically develop after 1-2 weeks. however, our attempts to detect such ferret antibodies against m102.4 did not result in any measurable quantities above the background (data not shown). this could be because of the low levels of such antibodies during the relatively short period of observation. further studies are required to clarify the answer to this question. we also found that m102.4 demonstrates reasonable stability and retains its biological activity in vivo. it has been previously shown that serum from hamsters immunized with vaccinia viruses that expressed niv envelope glycoproteins can protect the animals from challenge with niv [19] . this important study by guillaume et al. [19] further supports the notion that biologically active m102.4 would be able to protect animals and humans from henipavirus infections. in summary, m102.4 appears to be close to an ideal candidate for further development into an immunotherapeutic agent for henipavirus infection because it possesses many of the properties desired in such a therapeutic modality. it is a fully human mab; it retains its biological activity in vivo; it does not cause toxicity in ferrets; it has a distribution half-life typical for iggs, and its elimination half-life is likely to be significantly longer in humans; it cross-neutralizes both hev and niv; it has a muchimproved potency, compared with m102, its parental antibody; and it targets the g glycoprotein region, which largely overlaps with the receptor binding site. this mab may also prove useful in the development of diagnostics, small molecule drugs, and vaccines, and as a research reagent. hendra and nipah viruses: different and dangerous a morbillivirus that caused fatal disease in horses and humans nipah virus: a recently emergent deadly paramyxovirus nipah virus encephalitis reemergence emerging viruses: coming in on a wrinkled wing and a prayer emerging infectious diseases. nipah virus (or a cousin) strikes again fatal fruit bat virus sparks epidemics in southern asia nipah virus encephalitis reemergence nipah virus outbreak(s) in bangladesh person-to-person transmission of nipah virus in a bangladeshi community ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble g glycoprotein of hendra virus potent neutralization of hendra and nipah viruses by human monoclonal antibodies ephrin-b2 ligand is a functional receptor for hendra virus and nipah virus identification of hendra virus g glycoprotein residues that are critical for receptor binding neutralization assays for differential henipavirus serology using bio-plex protein array systems potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody nipah virus: vaccination and passive protection studies in a hamster model we wish to thank tim hancock from the australian animal health laboratory for his help with the in vivo ferret work. key: cord-270335-8vqi9c68 authors: seifert, stephanie n; letko, michael c; bushmaker, trenton; laing, eric d; saturday, greg; meade-white, kimberly; van doremalen, neeltje; broder, christopher c; munster, vincent j title: rousettus aegyptiacus bats do not support productive nipah virus replication date: 2019-11-04 journal: j infect dis doi: 10.1093/infdis/jiz429 sha: doc_id: 270335 cord_uid: 8vqi9c68 nipah virus (niv) is a bat-borne zoonotic pathogen that can cause severe respiratory distress and encephalitis upon spillover into humans. nipah virus is capable of infecting a broad range of hosts including humans, pigs, ferrets, dogs, cats, hamsters, and at least 2 genera of bats. little is known about the biology of niv in the bat reservoir. in this study, we evaluate the potential for the egyptian fruit bat (efb), rousettus aegyptiacus, to serve as a model organism for studying niv in bats. our data suggest that niv does not efficiently replicate in efbs in vivo. furthermore, we show a lack of seroconversion against niv glycoprotein and a lack of viral replication in primary and immortalized efb-derived cell lines. our data show that despite using a conserved target for viral entry, niv replication is limited in some bat species. we conclude that efbs are not an appropriate organism to model niv infection or transmission in bats. nipah virus (niv) is a zoonotic pathogen that can cause acute respiratory illness and fatal encephalitis upon spillover into human populations. since its discovery in malaysia in 1998, niv has emerged as a persistent public health problem in southeast asia warranting inclusion on the world health organization blueprint list of priority diseases for which research toward effective countermeasures is urgently needed [1] . unlike other paramyxoviruses, niv has a broad host tropism with multiple taxa supporting viral replication including ferrets, hamsters, cats, dogs, african green monkeys, squirrel monkeys, and pigs [2] [3] [4] [5] . nipah virus uses 2 major envelope glycoproteins to enter the host cell: the receptorbinding protein (g) and the fusion protein (f) [6] . after attachment of the niv g to the host cell receptor, ephrin-b2 or ephrin-b3, niv f fusogenic activity is triggered leading to the merger of the virion and host cell membranes and subsequent virus infection [7] [8] [9] [10] [11] . ephrins are highly conserved across mammalian taxa given their key role in development of the central nervous system [12] , likely contributing to the unusually broad host range of niv. fruit bats in the genus pteropus have been identified as the primary reservoir hosts for niv [13] . spillover of niv from bats to humans is thought to occur through ingestion of food or liquids contaminated with infected bat urine [14] . pteropus spp bats are known to feed from the spigots of date palm sap collection jars, often urinating into and contaminating the collection jars before human consumption [14, 15] . although pteropus spp bats are implicated as the primary reservoir for niv, they comprise only a fraction of the fruit bat visitation to date palm sap collection jars in bangladesh [15] . several other fruit bat species overlap with the distribution of pteropus spp bats and visit date palm sap collection jars, suggesting that if other bats are potential secondary reservoirs for the virus, then they may contribute to nipah virus spillover. less is known about the infection and enzootic transmission dynamics of niv in reservoir populations or between coroosting bat species. nipah virus has been isolated from the urine of both wild caught [16] [17] [18] and experimentally challenged [19] pteropus spp bats. nipah virus ribonucleic acid (rna) has also been detected in an insectivorous bat, hipposideros larvatus [20] , and serological evidence of niv infection has been detected in several other species of frugivorous and insectivorous bats, including rousettus leschenaultia [21, 22] . despite efforts to understand enzootic transmission dynamics, many questions remain regarding the biology of niv infection in bats. studies of wild caught pteropus spp suggest potential for viral recrudescence [16, 23] ; however, the hypothesis that niv may persist in an individual bat and re-emerge under times of stress has yet to be confirmed experimentally. pteropus spp bats are suboptimal model organisms for studying niv due to size and availability. in contrast, the egyptian fruit bat (efb), rousettus aegyptiacus, belongs to the same taxonomic family as pteropus spp, pteropodidae, and has been successfully used to model marburg virus transmission [24, 25] and serological cross-reactivity after filovirus challenge [26] . egyptian fruit bats are common in zoological settings because they are small, amenable to handling, and reproduce readily in captivity. the efb transcriptome is well annotated [27] , and there have been recent efforts to analyze the genome in context of antiviral immunity [28] . in this study, we evaluate efbs as a model system for niv infection in bats. all work with niv was conducted in the biosafety level (bsl) 4 facility at the rocky mountain laboratories, division of intramural research, national institute of allergy and infectious diseases, national institutes of health following standard operating procedures as approved by the institutional biosafety committee. egyptian fruit bats were sourced from a us-based zoological facility. all animal experiments were approved by the rocky mountain laboratories institutional animal care and use committee (asp no. 2018-042e) and performed following the guidelines of the association for assessment and accreditation of laboratory animal care, international (aaalac) by certified staff in an aaalac-approved facility. nipah virus was obtained through the special pathogens branch of the centers for disease control and prevention (atlanta, ga). nipah virus bangladesh was isolated from a throat swab from a human patient in 2004 (genbank accession number ay988601); the virus was propagated on vero-e6 cells and passaged a total of 2 times. the virus stock was deep sequenced at the rocky mountain laboratories genomics core unit before the start of this study to confirm that no fungal or bacterial contaminants were present. primary efb cell lines were generated as previously described [29] , with modifications, from kidney (raksm) and lung (ralu) tissue samples obtained from an efb euthanized under bsl2 conditions. in brief, tissue homogenates were washed in phosphate-buffered saline and resuspended in primary cell culture (d12) media containing dulbecco's modified eagle's medium (dmem)/f-12 supplemented with nonessential amino acids, 12% fetal bovine serum (fbs), 1 mm l-glutamine, 50 u/ml penicillin, 50 μg/ml streptomycin, 1 μg/ml amphotericin, and 1 mm sodium pyruvate. after 1 passage, amphotericin was excluded from the d12 media. cells were not maintained after 3 passages. jordan at probiogen ag, berlin, germany) were obtained and grown in d12 media without amphotericin. vero-e6 cells were used as a positive control. each cell line was seeded in triplicate in 12-well plates and inoculated with niv at a multiplicity of infection of 0.1 in dmem supplemented with 2% fbs, 1 mmol/l l-glutamine, 50 u/ml penicillin, and 50 μg/ml streptomycin. supernatants were collected at 0, 24, 48, and 72 hours postinoculation and stored in avl buffer (qiagen) at −80°c until inactivation and rna extraction. after inactivation of the virus with avl and ethanol as described in [30] , rna extraction was performed on the qiacube (qiagen) with the machery-nagel nucleospin virus core kit (machery-nagel). we then performed quantitative real-time reverse-transcription polymerase chain reaction (qrtpcr) as described in [31] on the quantstudio 5 real-time pcr system (thermo fisher scientific) with the inclusion of a serially diluted known concentration of niv on each plate to calculate tissue culture infectious dose (tcid 50 /ml) equivalent for each sample. sequences for the niv receptors, ephrin-b2 and ephrin-b3, that have been experimentally demonstrated to support niv entry [32] were downloaded from national center for biotechnology information (ncbi) in addition to the efb ephrin-b2 and ephrin-b3 sequences. the sequences for each ephrin were translated before alignment using the mafft v7.388 [33] plugin implemented in geneious prime 2019.0.4 (biomatters ltd). nipah virus was diluted in sterile dmem, and 10 5 tcid 50 /ml was administered to each of 12 adult r aegyptiacus bats via the intraperitoneal route of inoculation in a final volume of 200 μl. oronasal, urogenital, and rectal swabs were collected daily in addition to swabbing the excreta pan of each cage for the first 14 days followed by twice-weekly sampling through 28 days postinoculation (dpi). temperature and weight for each bat was taken at the time of sampling. blood was drawn before inoculation, then at 7 dpi, 14 dpi, and 21 dpi for survivors in addition to terminal blood draws at 3 dpi, 7 dpi, and 28 dpi for serological analyses. tissue samples were taken at necropsy and either stored at −80°c until rna extraction or placed in 10% formalin for histopathology and immunohistochemistry analysis. the rna extraction and qrtpcr were conducted as described in [31] and performed on a quantstudio 5 real-time pcr system (thermo fisher scientific). sera were analyzed for presence of immunoglobulins (ig) specific to the niv-g using a luminex xmap-based multiplex bead assay adapted from [34] . in brief, blood was collected into serum-separating tubes before centrifugation at 1000 ×g for 10 minutes; serum was then collected and frozen at −80°c. each sample received a dose of 8 mrads irradiation while on dry ice before heat-inactivation at 56°c for 30 minutes. soluble niv-g (niv-sg) was produced in a freestyle 293-f stable cell-line expression system before purification as described [35] and coupling to bio-plex pro magnetic cooh beads (bio-rad). we diluted each serum sample 1:250, and each serum sample was run in duplicate with the bio-plex 200 system (bio-rad) with purified rabbit igg against niv-sg diluted to 1:1000 as the positive control. in vitro replication kinetics showed no appreciable increase in niv titer over a 72-hour period on the 4 efb cell lines tested including the 2 primary efb cell lines and the 2 immortal efb cell lines (figure 1 ). the hammer-headed fruit bat cell line supported moderate niv replication relative to the vero-e6 cell line (figure 1 ). an alignment of the niv host receptors, ephrin-b2 and ephrin-b3, show no unique amino acid changes in the critical g protein-binding (g-h) loop between the efb sequences and the sequences of species that have been confirmed to facilitate niv entry (figure 2) . the efbs showed no significant change in temperature or weight throughout the study period, although variability was high for both metrics ( figure 3a and b) . we did not detect niv rna in any of the tissue samples or swab samples tested by qrtpcr, with no samples amplifying within 40 thermal cycles ( the naive mean fluorescence intensity; however, 1 bat showed a slight increase in mean fluorescence intensity relative to the naive bat serum at 21 and 28 dpi (figure 4) . we confirmed that the inoculum contained 10 5 tcid 50 /ml through back-titrations of the diluted viral stock (data not shown). none of the sectioned tissue samples showed pathology associated with niv infection, and no niv antigen was detected via immunohistochemistry staining ( figure 5 ). our data show a lack of productive niv replication in efbs. the lack of detectable viral rna in both the swabs and the tissues across all time points suggests that the bats did not shed virus nor was viral replication detected in any of the tested tissue types (table 1) ; viral replication and shedding are qualities associated with natural hosts [36, 37] . back-titrations of the viral inoculum confirmed that the efbs received a 10 5 tcid 50 dose niv, which is higher than the 5 × 10 4 tcid 50 niv challenge that resulted in productive viral replication in guinea pigs and pteropus bats [19] . a lack of detectable virus in any efb tissue samples 3 days postinoculation suggests that the virus was rapidly cleared; halpin et al [13] report that henipavirus inoculum is cleared within 48 hours. the bats did not seroconvert against niv g in the given timeframe of 28 days, although 1 individual had a slight increase in mean fluorescence intensity at 21 and 28 dpi, which may have increased above our cutoff if given more time (figure 4 ). previous studies have demonstrated that efb cells are permissive to ebola virus, but experimentally challenged bats did not shed virus or support productive replication [38, 39] despite compatibility between the ebola virus glycoprotein and the host receptor, npc1 [40] . these data suggest that productive viral replication is blocked by a mechanism other than compatibility with the host receptor. likewise, van doremalen et al [41] reported a lack of efficient viral replication in efbs when challenged with bat severe acute respiratory syndrome-like coronavirus wiv1 despite in vitro receptor compatibility. given the lack of unique variation in either efb ephrin-b2 or ephrin-b3 relative to compatible host ephrin sequences (figure 2) , it is likely that niv virus replication is not inhibited by a lack of binding between niv g and the efb ephrin-b2 or ephrin-b3, nor the subsequent f-mediated activation and membrane fusion process. indeed, niv f and g can mediate productive cell-cell fusion in a variety of different mammalian species including several that are negative or refractory to productive infection, such as rabbit and mouse [32, 42] . because we did not detect viral replication or shedding, we conclude that efbs are not a suitable model system for modeling niv transmission dynamics in bats. however, follow-up studies to determine the mechanism of inhibition of viral replication in efbs would be valuable in elucidating the evolution of niv in its natural reservoirs. few controlled studies have been conducted using bats as a model organism, and, as such, there are few reagents commercially available for studying the immunobiology of bats in response to viral infection. further research is urgently needed to expand upon the current capacity to conduct research in bats, particularly when considering that 5 of 9 viruses associated with the world health organization's blueprint list of priority diseases [1] likely originated as batborne zoonoses. recent studies have applied machine learning algorithms to prioritize surveillance for high-impact pathogens such as niv and ebola virus using data on ecological traits, life history, demographic traits, and species distributions [43, 44] . although it is important to consider broader ecological characteristics in determining potential for a host species to contribute to virus spillover and maintenance, understanding the limitations to reservoir potential on a mechanistic level for high-impact pathogens such as niv would further improve predictive modeling work. testing viral entry and replication through in vitro assays are important first steps in determining host potential, but this should be followed by in vivo experiments when possible. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. the who r&d blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a golden hamster model for human acute nipah virus infection development of an acute and highly pathogenic nonhuman primate model of nipah virus infection experimental nipah virus infection in pigs and cats functional properties of the fusion and attachment glycoproteins of nipah virus role of endocytosis and cathepsin-mediated activation in nipah virus entry ephrin-b2 ligand is a functional receptor for hendra virus and nipah virus ephrinb2 is the entry receptor for nipah virus, an emergent deadly paramyxovirus two key residues in ephrinb3 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infection in bats (order chiroptera) in peninsular malaysia antibodies to nipah or nipahlike viruses in bats henipavirus ecology research group. evidence for nipah virus recrudescence and serological patterns of captive pteropus vampyrus oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) modelling filovirus maintenance in nature by experimental transmission of marburg virus between egyptian rousette bats comparative analysis of serologic cross-reactivity using convalescent sera from filovirusexperimentally infected fruit bats de novo transcriptome reconstruction and annotation of the egyptian rousette bat the egyptian rousette genome reveals unexpected features of bat antiviral immunity bat airway epithelial cells: a novel tool for the study of zoonotic viruses effective chemical inactivation of ebola virus comparison of the pathogenicity of nipah virus isolates from bangladesh and malaysia in the syrian hamster functional studies of host-specific ephrin-b ligands as henipavirus receptors mafft multiple sequence alignment software version 7: improvements in performance and usability serologic evidence of fruit bat exposure to filoviruses expression system for recombinant henipavirus glycoproteins replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) bats: important reservoir hosts of emerging viruses experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera experimental inoculation of egyptian fruit bats (rousettus aegyptiacus) with ebola virus establishment of fruit bat cells (rousettus aegyptiacus) as a model system for the investigation of filoviral infection sarslike coronavirus wiv1-cov does not replicate in egyptian fruit bats (rousettus aegyptiacus) membrane fusion tropism and heterotypic functional activities of the nipah virus and hendra virus envelope glycoproteins undiscovered bat hosts of filoviruses prioritizing surveillance of nipah virus in india we thank marcel a. müller (institute of virology, charite -universitätsmedizin berlin, germany) and ingo jordan (probiogen ag, berlin, germany) for providing the immortalized bat cell lines used in this study, the centers for disease control and prevention for providing the initial nipah virus (niv) stock, and friederike feldmann (laboratory of virology, national institute of allergy and infectious diseases [niaid]) for preparing and maintaining the niv stocks. we thank spencer l. sterling and lianying yan (uniformed services university) for invaluable technical assistance with soluble niv-sg expression and purification. we thank emmie de wit (laboratory of virology, niaid) for advice and laboratory support. finally, we thank drs. patrick hanley, jamie lovaglio, and dana scott and the animal caretakers of the rocky mountain veterinary branch, niaid for support.disclaimer. the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. the views expressed in the manuscript are solely those of the authors, and they do not represent official views or opinions of the department of defense or the uniformed services university of the health sciences.financial support. this research was funded by the intramural research program of niaid, national institutes of health.potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. key: cord-011712-fyrbe8tw authors: venkatesan, sudhir; myles, puja r; bolton, kirsty j; muthuri, stella g; al khuwaitir, tarig; anovadiya, ashish p; azziz-baumgartner, eduardo; bajjou, tahar; bassetti, matteo; beovic, bojana; bertisch, barbara; bonmarin, isabelle; booy, robert; borja-aburto, victor h; burgmann, heinz; cao, bin; carratala, jordi; chinbayar, tserendorj; cilloniz, catia; denholm, justin t; dominguez, samuel r; duarte, pericles a d; dubnov-raz, gal; fanella, sergio; gao, zhancheng; gérardin, patrick; giannella, maddalena; gubbels, sophie; herberg, jethro; higuera iglesias, anjarath lorena; hoeger, peter h; hu, xiao yun; islam, quazi t; jiménez, mirela f; keijzers, gerben; khalili, hossein; kusznierz, gabriela; kuzman, ilija; langenegger, eduard; lankarani, kamran b; leo, yee-sin; libster, romina p; linko, rita; madanat, faris; maltezos, efstratios; mamun, abdullah; manabe, toshie; metan, gokhan; mickiene, auksė; mikić, dragan; mohn, kristin g i; oliva, maria e; ozkan, mehpare; parekh, dhruv; paul, mical; rath, barbara a; refaey, samir; rodríguez, alejandro h; sertogullarindan, bunyamin; skręt-magierło, joanna; somer, ayper; talarek, ewa; tang, julian w; to, kelvin; tran, dat; uyeki, timothy m; vaudry, wendy; vidmar, tjasa; zarogoulidis, paul; nguyen-van-tam, jonathan s title: neuraminidase inhibitors and hospital length of stay: a meta-analysis of individual participant data to determine treatment effectiveness among patients hospitalized with nonfatal 2009 pandemic influenza a(h1n1) virus infection date: 2020-02-01 journal: j infect dis doi: 10.1093/infdis/jiz152 sha: doc_id: 11712 cord_uid: fyrbe8tw background: the effect of neuraminidase inhibitor (nai) treatment on length of stay (los) in patients hospitalized with influenza is unclear. methods: we conducted a one-stage individual participant data (ipd) meta-analysis exploring the association between nai treatment and los in patients hospitalized with 2009 influenza a(h1n1) virus (a[h1n1]pdm09) infection. using mixed-effects negative binomial regression and adjusting for the propensity to receive nai, antibiotic, and corticosteroid treatment, we calculated incidence rate ratios (irrs) and 95% confidence intervals (cis). patients with a los of <1 day and those who died while hospitalized were excluded. results: we analyzed data on 18 309 patients from 70 clinical centers. after adjustment, nai treatment initiated at hospitalization was associated with a 19% reduction in the los among patients with clinically suspected or laboratory-confirmed influenza a(h1n1)pdm09 infection (irr, 0.81; 95% ci, .78–.85), compared with later or no initiation of nai treatment. similar statistically significant associations were seen in all clinical subgroups. nai treatment (at any time), compared with no nai treatment, and nai treatment initiated <2 days after symptom onset, compared with later or no initiation of nai treatment, showed mixed patterns of association with the los. conclusions: when patients hospitalized with influenza are treated with nais, treatment initiated on admission, regardless of time since symptom onset, is associated with a reduced los, compared with later or no initiation of treatment. seasonal influenza epidemics and pandemics increase pressure on hospital bed capacity. early initiation of monotherapy with neuraminidase inhibitors (nais) reduces illness duration in patients with uncomplicated influenza [1] [2] [3] ; associated reductions in complications, hospitalizations, and mortality are supported by systematic reviews of observational data [4] [5] [6] [7] [8] . the evidence is less clear that nai treatment reduces length of stay (los) in hospitalized patients with influenza, compared with supportive care without antiviral treatment [9] [10] [11] [12] [13] [14] [15] . minimizing the los is important in managing hospital surge and limiting healthcare costs due to seasonal influenza epidemics and pandemics. we undertook a one-stage individual participant data (ipd) [16] meta-analysis to explore the association between nai treatment of patients hospitalized with 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09) infection and the los during the 2009-2010 influenza pandemic. details regarding identification of study centers and inclusion of patients have been published previously [6] . briefly, we requested data from multiple clinical centers worldwide on patients admitted to hospital with laboratory-confirmed or clinically diagnosed a(h1n1)pdm09 infection for whom a minimum data set was available. of the ipd that we received, we excluded patients who had laboratory-confirmed absence of a(h1n1) pdm09 infection, retaining only patients who had laboratoryconfirmed a(h1n1)pdm09 infection and patients with clinically diagnosed pandemic influenza (ie, those for whom the clinical suspicion and working diagnosis was one of pandemic influenza but laboratory confirmation was not performed) [6] . the pride study protocol was registered with the prospero register of systematic reviews (crd42011001273) prior to data collection [17] . this states that the study will investigate the impact of nai treatment on multiple outcomes of public health interest in a(h1n1)pdm09-infected patients, using mixedeffects models. after collection and standardization of the data, sufficient data existed to assess 2 indicators of "severe hospital outcomes"-requirement for ventilatory support (ie, intensive care unit [icu] admission) and los. in this article, we present the findings relating to the los. we standardized data from individual data sets before we pooled the data (supplementary table 1 ). the primary outcome was the los (in whole days). we excluded patients with known receipt of nai treatment before admission, to ensure uniform potential for treatment to influence the los. we excluded patients with continuing postdischarge nai treatment; patients with a los <1 day, on the grounds that they would have received a maximum of 2 doses of nai inpatient treatment and that their admission may have been precautionary; and patients with nosocomial influenza (defined as influenza with symptom onset after the hospital admission date; figure 1 ). finally, since rapid deterioration and early death during hospitalization would be an adverse outcome associated with a paradoxically short los, those who died in the hospital were excluded from analysis. the primary exposure variable was in-hospital nai treatment received on the day of hospital admission, compared with later or no nai treatment. additionally, where data were available, we defined 3 further exposure variables: nai treatment (at any time) versus no nai treatment, early nai treatment (initiated within ≤2 days after symptom onset) versus no nai treatment, and early nai treatment versus later treatment (initiated >2 days after symptom onset). we derived propensity scores via multivariable logistic regression for each exposure variable, as described by hirano and imbens [18] , separately for individual study centers, based on patient characteristics recorded on admission. propensity score derivation models included, a priori, the following variables: age, sex, comorbidity (yes/no), and an indicator of disease severity, plus any additional covariates (ie, obesity, smoking, pregnancy, asthma, chronic obstructive pulmonary disease, lung disease, heart disease, immunosuppression, neurological disease, renal disease, and/or diabetes) that remained statistically significant in a regression model. variables for which data from >25% of participants were missing were excluded from propensity score derivation. to investigate the impact of nai treatment on the los, we performed a one-stage ipd meta-analysis using a mixed-effects negative binomial regression model, including study center as a random intercept to account for clustering. a negative binomial model was chosen to account for overdispersion in the los data (as represented in supplementary figure 1 ). we tested a zeroinflated negative binomial regression model on a subgroup of the data and found that the model fit was inferior to that of a negative binomial regression model. in our primary analysis, we aimed to quantify the potential benefits of a pragmatic treat-on-admission policy (irrespective of the time elapsed since symptom onset), compared with patients who received no nai treatment and those whose treatment was delayed until after the day of admission. by way of sensitivity analysis, we restricted the comparator group to patients who did not receive nai treatment at any point. for both analyses, we adjusted for propensity score quintile, in-hospital antibiotic treatment, in-hospital corticosteroid treatment, and the delay between symptom onset and hospital admission. in addition, we performed secondary analyses for the following exposures: nai treatment (at any time) versus no nai treatment, early nai treatment (≤48 hours after symptom onset) versus later nai treatment (>48 hours after symptom onset), and early nai treatment versus no nai treatment, adjusting for propensity score, in-hospital antibiotic treatment, and corticosteroid treatment. we performed a priori-specified analyses for the following subgroups: patients with laboratory-confirmed a(h1n1) pdm09 infection, children (age, <16 years), elderly patients (age, ≥65 years), patients with chest radiography-confirmed influenza-related pneumonia (irp), and patients with confirmed absence of irp. we looked at pregnant women and obese patients as post hoc subgroups. furthermore, we investigated, by stratification, the impact of nai treatment on the total los in patients admitted to critical care facilities (ie, icus) at any point and patients treated exclusively by using standard ward-based care. both unadjusted and adjusted models were run, and results are presented as unadjusted incidence rate ratios (irrs) or adjusted irrs (airrs) with 95% confidence intervals (cis). missing data in the covariates were included in the analysis as dummy variable categories. using airr point estimates, we determined the difference in the los (in days) between a treated patient and an untreated patient with similar characteristics by scaling the model prediction for los without treatment by (airr-1). repeating this for all patients in our data set gave us a distribution of expected changes in the los due to treatment (with timing as defined for each regression analysis). this does not account for error in the estimates of model covariates, which would require a bayesian approach; however, it offers a clinically relevant interpretation of airrs. the statistical analyses were performed using stata (version 14.2; statacorp, college station, tx). we identified 29 234 patients admitted to the hospital between 2 january 2009 and 14 march 2011 with laboratory-confirmed or clinically diagnosed a(h1n1)pdm09 infection [4] . the analysis population included 18 309 patients (62.6%; figure 1 ). the included patients came from 70 clinical centers in 36 countries across all 6 world health organization regions. the americas contributed the most data (46.2% of patients), followed by europe (for 33.3%). the country that contributed the most to the pooled data set was mexico (28.8% of patients), followed by spain (8.6%), the united states (7.6%), and the united kingdom (7.5%). among patients in the final study population, 67.4% were adults, and 81.1% had laboratoryconfirmed a(h1n1)pdm09 infection; general characteristics of the included population are further described in table 1 . among the 8621 patients (47.1%) for whom data on the timing of nai treatment were available, 3678 (42.7%) received early nai treatment, and 4816 (55.9%) initiated treatment on the day of admission. the median delay from illness onset to hospital admission was 2 days (interquartile range [iqr], 1-5 days), and among patients with data on the timing of treatment, 42.7% presented ≤48 hours after symptom onset; the median los was 5 days (iqr, 3-9 days; supplementary figure 1 ). in patients whose nai treatment was initiated on the day of hospital admission, the median interval between symptom onset and admission was 2 days (iqr, 1-4 days). in our primary analysis, we observed that nai treatment started on the day of admission was associated with a 19% overall reduction in the los (airr, 0.81 [95% ci, .78-.85]; median decrease, 1.19 days [iqr, 0.85-1.55 days]), compared with no or later initiation of nai treatment. this association was of similar magnitude and remained significant in all subgroups (table 2 and supplementary table 3 ). in the sensitivity analysis, we observed that nai treatment on the day of hospital admission was associated with an 8% reduction in the los among patients not admitted to the icu ( after adjustment, nai treatment at any time was associated with an 11% overall increase in the los (airr, 1.11 [95% ci, 1.07-1.16]; median increase, 0.74 days [iqr, 0.60-1.05 days]), compared with no nai treatment. by exploring subgroups, we identified corresponding statistically significant findings in patients with laboratory-confirmed a(h1n1)pdm09 infection, children, patients admitted to the icu, and patients with confirmed irp but not in the elderly, patients requiring non-icu care, or patients with confirmed absence of irp (table 2) . we did not find any evidence of effect modification by pandemic influenza vaccination (p = .68) or by in-hospital antibiotic treatment (p = .20); however, a borderline significant effect modification was observed for in-hospital corticosteroid treatment (p = .05), the irr was further adjusted for time from onset to admission. c statistically significant (p < .05). d data are for patients admitted to the icu at any point. the irr was calculated for the total length of hospital stay, not time in the icu. our sensitivity analyses and secondary analyses must be interpreted with caution because they may be affected by various time-dependent biases and patients with confirmed irp ( table 3 ). our study extends the existing literature by offering data on the association between nai treatment and the los in >18 000 adult and pediatric patients, of whom >80% had a laboratoryconfirmed diagnosis of a(h1n1)pdm09 infection. we found a mixed pattern of association between nai treatment and los, depending on the delay to initiation of treatment, age, and case severity. the most pragmatic and important question is whether nai treatment, started on admission, irrespective of delay since symptom onset, reduces the los in hospitalized patients with influenza. clinically, this is important because there can be significant uncertainty in ascertaining symptom onset, even by the attending physician. the uncertainty in ascertaining symptom onset could mean prescribing nai treatment outside the recommended (licensed) window of ≤48 hours after symptom onset. however, there is evidence pointing to the effectiveness of nai therapy, albeit reduced, even when given >48 hours after symptom onset [6] . statistically, by defining our exposure variable on the basis of treatment decisions made on admission, we avoided introducing correlations between exposure and los that can lead to survivorship bias in linear regression models of time-to-event data [19, 20] . additionally, this approach ensures that the propensity scores, modeled on symptom severity at admission, should appropriately correct for treatment bias [21] . however, this choice of exposure variable also reflects the clinical reality that patients present to the hospital at varying intervals after symptom onset (ranging from 0-20 days in our study) and that clinicians and policy makers want to know whether a so-called treat-at-the-door policy applied to patients admitted to the hospital with clinically recognized influenza will be beneficial, compared with no nai treatment or a watch-and-see approach. this was addressed by our primary analysis, which revealed that initiation of nai treatment on the day of admission was associated with a 19% reduction in the los (median decrease, 1.19 days), compared with later or no treatment, with similar statistically significant findings across all patient subgroups including children, pregnant women, and obese patients. these findings emphasize the importance of presumptive nai treatment in patients admitted to the hospital with suspected influenza, coupled with early diagnosis using standard laboratory or rapid diagnostic tests. in our sensitivity analysis, we found a significant reduction of 19% in the los (median decrease, 1.24 days) among patients with confirmed absence of irp and a reduction of 8% (median decrease, 0.5 days) among patients who required supportive ward-based care. in contrast, nai treatment (compared with no treatment) was associated with a 28% increase in the los (median increase, 1.73 days) among patients with irp. these data suggest that nais may be more effective in reducing the los when patients do not have irp and are consistent with the fact that nais have no known antibacterial properties. in secondary analyses, we observed an 11% overall increase in the los associated with nai treatment, equivalent to a median increase of about 0.74 days and irrespective of the time between symptom onset and initiation of therapy. compared with no treatment, nai treatment initiated within 48 hours after symptom onset was associated with a 7% overall reduction in the los, equivalent to a median decrease of 0.40 days; this effect was not observed in children and patients requiring icu care. this finding is clinically important because it suggests that rapid access to antiviral treatment after symptom onset may influence the los in adults and elderly individuals; nevertheless, we did not observe the same result among patients requiring icu care. our results in children may be influenced by a higher a(h1n1) pdm09 viral load in children [22] than in adults, leading to prolonged hospital stay, suboptimal dosing in very young children [23] , increased likelihood of antiviral resistance emergence in children [24] , secondary bacterial infections, confounding by indication related to baseline illness severity [25] , or a combination of these factors. although we attempted to adjust for influenza severity by using propensity scores, we found icu care to be very strongly associated with a prolonged los (irr, 2.96; 95% ci, 2.84-3.09) and nai treatment to be associated with a higher likelihood of requiring icu care (adjusted odds ratio, 3.11; 95% ci, 2.42-3.98). furthermore, we found that patients who presented to the hospital >2 days after symptom onset were 73% more likely to eventually require icu care than patients who presented earlier (odds ratio, 1.73; 95% ci, 1.53-1.95). in addition, patients requiring icu care have frequently developed extrapulmonary manifestations of influenza and multiorgan decompensation; therefore, inhibition of virus replication may not correspond with rapid clinical recovery. we noted no association between nai treatment and los among hospitalized children with influenza when considering early treatment versus no treatment. the study may have been underpowered in children, but other factors might have contributed to our findings. the los is typically shorter among children, compared with adults; mortality and serious outcomes are less common among hospitalized children with influenza, compared with adults; and different discharge policies and thresholds for children could also influence the findings. in addition, vomiting is a recognized side effect of oseltamivir in children [3] , and this may have prevented discharge in some cases. previous studies examining whether use of nais in patients hospitalized with influenza affects the los have generally been of smaller size (<1300 individuals) as compared to our study and reached variable conclusions. of note, 8 studies [11] [12] [13] [14] [15] [26] [27] [28] (of which one [12] was a randomized trial) assessed nai treatment of hospitalized children, but only 2 (both with an observational design) concluded that the total number of hospital days in the nai-treated hospital cohort was reduced (by 18% [8.3 days]) [11, 28] , with the other 6 reporting no differences [12-15, 26, 27] . only 4 studies have addressed the same question in adults. in hong kong, a study of 356 adult patients hospitalized with laboratory-confirmed seasonal influenza showed that early oseltamivir treatment was associated with a reduced los in both unadjusted and multivariable analyses [9] , compared with no or later treatment, with the median los decreasing from 6 to 4 days; this accords with our primary analysis. a canadian study of adult patients with seasonal influenza found that oseltamivir treatment was not associated with the los among surviving patients [29] . a further study in 13 spanish hospitals involving 538 patients with laboratory-confirmed a(h1n1)pdm09 infection noted that the los increased by 7% (odds ratio, 1.07), after adjustment for confounders, if nai treatment was instigated <48 hours after symptom onset; however, this was of borderline statistical significance [10] . a recent american study analyzed data on 201 adult patients with laboratory-confirmed seasonal influenza, reporting that nai treatment was not associated with the los overall but was associated with a reduced los among vaccinated individuals (hazard ratio of discharge, 1.6; 95% ci, 1.0-2.4; p = .04) [30] . finally, 2 studies included patients of all age groups. one of them, performed in 813 hospitalized patients with a(h1n1)pdm09 infection in spain, found that early nai treatment reduced the los by 1.9 days (p = <.001) [31] . the other, an american study using insurance claims data from patients with seasonal influenza, reported that patients treated with nais spent fewer days in the hospital (p = <.0001) [32] . this study has a number of strengths and weaknesses. we combined data from geographically diverse centers, offering broad generalizability of our findings. we used propensity scores to adjust for major confounders. by excluding patients who died (10%), we removed the paradoxical possibility that a short los (a positive outcome in our analyses) was associated with an extremely unfavorable clinical outcome. however, a limitation of this approach is that it does not explain the impact of nai treatment on the relationship between los and in-hospital mortality. in our primary analysis, we adjusted for the delay between illness onset and admission, to address length bias [20] , and chose our exposure variable to avoid time-dependent/survivorship bias [19, 21] . however, our secondary analyses, which used time since onset to define the exposure variable, are subject to time-dependent biases and must therefore be interpreted with caution. indeed, the benefit of early versus late treatment ( table 2 ) will be partially driven by this bias [19] . all of our analyses may be subject to residual competing risk bias, which was not removed through adjustment; for example, we found a significant difference between propensity scores to receive nais in the hospital for surviving and nonsurviving patients in the data set (p < .05, by the kruskal-wallis test), signaling that our removal of nonsurviving patients altered the aggregate presenting patient characteristics for which our results hold. our data, generated during the 2009-2010 influenza pandemic, contained relatively few elderly patients and children, consistent with patterns of a(h1n1)pdm09 infection [33] , and differs in profile from seasonal influenza a(h3n2) virus infection, for which patients admitted to the hospital tend to be much older and to have a median los higher than the los of 5 days we observed [34, 35] . in addition, the prevalence proportions of clinically recorded obesity (12%) and pregnancy (23%) were both comparatively high. optimally, clinicians wish to treat patients with influenza within 48 hours after symptom onset, yet in many cases patients with influenza do not seek medical care during this therapeutic window. our data show that 57.3% of included patients were hospitalized >48 hours after symptom onset. what then matters is whether initiation of treatment upon hospitalization (on the day of admission), irrespective of the time elapsed since symptom onset, is effective and whether this is preferable to nontreatment or further delays in treatment. we revealed a 19% reduction in the los (median decrease, 1.19 days) among patients who were treated with an nai upon admission, compared with those who received no or later treatment; the trend was observed across all subgroups, including children. this treatment approach would avoid the uncertainties associated with ascertaining the symptom onset date. our data support current recommendations to treat adults hospitalized with clinically suspected influenza with nais as soon as possible upon admission; furthermore, this approach appears to be superior to no treatment or delayed treatment in terms of a reduced los. if used consistently, this strategy would contribute to the management of surge pressures and healthcare costs during seasonal influenza epidemics and pandemics. the pride consortium investigators are as follows (affiliations are listed in supplementary neuraminidase inhibitors for preventing and treating influenza in healthy adults and children. cochrane database syst rev oseltamivir treatment for influenza in adults: a meta-analysis of randomised controlled trials efficacy and safety of oseltamivir in children: systematic review and individual patient data meta-analysis of randomized controlled trials antivirals for treatment of influenza impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-2010 influenza a(h1n1) pandemic: a systematic review and meta-analysis in hospitalized patients effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h1n1pdm09 virus infection: a metaanalysis of individual participant data impact of outpatient neuraminidase inhibitor treatment in patients infected with influenza a(h1n1)pdm09 at high risk of hospitalization: an individual participant data metaanalysis oseltamivir effect on antibiotictreated lower respiratory tract complications in virologically positive randomized trial participants factors associated with early hospital discharge of adult influenza patients timing of oseltamivir administration and outcomes in hospitalized adults with pandemic 2009 influenza a (h1n1) virus infection oseltamivir shortens hospital stays of critically ill children hospitalized with seasonal influenza: a retrospective cohort study a randomized, double-blind, placebo-controlled trial evaluating the safety of early oseltamivir treatment among children 0-9 years of age hospitalized with influenza in el salvador and panama clinical characteristics of influenza b virus in children and the efficacy of oseltamivir: data from two university hospitals oseltamivir treatment for influenza in hospitalized children without underlying diseases clinical features, oseltamivir treatment and outcome in infants aged< 12 months with laboratory-confirmed influenza a in 2009 meta-analysis of individual participant data: rationale, conduct, and reporting a systematic review of the impact of neuraminidase inhibitor antiviral use on outcomes of public health importance during the 2009/10 (swine) influenza a/ h1n1v pandemic the propensity score with continuous treatments. applied bayesian modeling and causal inference from incomplete-data perspectives an easy mathematical proof showed that time-dependent bias inevitably leads to biased effect estimation time-dependent study entries and exposures in cohort studies can easily be sources of different and avoidable types of bias survivor treatment bias, treatment selection bias, and propensity scores in observational research correlation of pandemic (h1n1) 2009 viral load with disease severity and prolonged viral shedding in children national institute of allergy and infectious diseases collaborative antiviral study group. oseltamivir pharmacokinetics, dosing, and resistance among children aged <2 years with influenza five years of monitoring for the emergence of oseltamivir resistance in patients with influenza a infections in the influenza resistance information study confounding by indication in epidemiologic studies of commonly used analgesics the association between influenza treatment and hospitalization-associated outcomes among korean children with laboratoryconfirmed influenza ciberesp cases and controls in pandemic influenza working group. clinical features of influenza disease in admitted children during the first postpandemic season and risk factors for hospitalization: a multicentre spanish experience pandemic (h1n1) 2009 influenza in hospitalized children in manitoba: nosocomial transmission and lessons learned from the first wave toronto invasive bacterial diseases network. antiviral therapy and outcomes of influenza requiring hospitalization in ontario severe morbidity among hospitalised adults with acute influenza and other respiratory infections prognosis of hospitalized patients with 2009 h1n1 influenza in spain: influence of neuraminidase inhibitors oseltamivir and influenza-related complications, hospitalization and healthcare expenditure in healthy adults and children risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis complications and outcomes of pandemic 2009 influenza a (h1n1) virus infection in hospitalized adults: how do they differ from those in seasonal influenza? modelling estimates of age-specific influenzarelated hospitalisation and mortality in the united kingdom disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the uk government or the united states centers for disease control and prevention. the funder has had no role in protocol design, no opportunity to comment on it, and no opportunity to see it other than via the prospero website; no access to any data (and no rights to future access); no role in analysis or interpretation; no opportunity to preview results/findings before entry into the public domain; and no opportunity to contribute to, preview, or comment on manuscripts and presentations arising from this work. the research contract between the university of nottingham and the funder is freely available for inspection (with commercial details redacted) at: http://www.nottingham.ac.uk/research/groups/healthprotection/projects/pride.aspx. no data were provided or funded for collection by pharmaceutical companies.financial support. this work was supported by f. hoffmann-la roche (unrestricted educational grant to the pride study).potential supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord-289406-54vyzxjf authors: edwards, suzanne; small, j. david; geratz, joachim dieter; alexander, lorraine k.; baric, ralph s. title: an experimental model for myocarditis and congestive heart failure after rabbit coronavirus infection date: 1992-01-17 journal: j infect dis doi: 10.1093/infdis/165.1.134 sha: doc_id: 289406 cord_uid: 54vyzxjf in a model for virus-induced myocarditis and congestive heart failure, rabbit coronavirus infection was divided into acute (days 2–5) and subacute (days 6–12) phases on the basis of day of death and pathologic findings. during the acute phase, the principal histologic lesions were degeneration and necrosis of myocytes, myocytolysis, interstitial edema, and hemorrhage. the severity of these changes increased in the subacute phase. pleural effusion and congestion of the lungs and liver were also present at this time. myocarditis was detected by day 9 and peaked by day 12. heart weights and heart weight-to-body weight ratios were increased, and dilation of the right ventricular cavity became prominent early in infection and persisted. in contrast, dilation of the left ventricle occurred late in the subacute stage. virus was isolated from infected hearts between days 2 and 12. these data suggest that rabbit coronavirus infection progresses to myocarditis and congestive heart failure. viruses have long been recognized as important etiologic agents of heart disease in humans and experimental animals [1-3). epidemiologic evidence suggests that after viral infection, 2%-5% of a human population experience some degree of cardiac involvement [2, 3) . in humans and experimental animals, viruses commonly linked to heart disease include the picornaviruses, paramyxoviruses, myxoviruses, alphaviruses, and coronaviruses [2] [3] [4] [5] . viral infection of the heart may result in degeneration and necrosis of myocytes and lead to inflammation of the heart muscle. myocarditis may result in arrhythmias, conduction disturbances, circulatory collapse, and acute congestive heart failure [3] [4] [5] [6] [7] . acute viral infection of the heart muscle may also be an important factor in the development ofidiopathic dilated cardiomyopathy [8] [9] [10] [11] . coxsackie b virus and encephalomyocarditis virus (both enteroviruses) infections in mice are the best-characterized model systems for virus-induced heart disease [i, 5) . the exact mechanism for their pathogenesis is still controversial; however, considerable evidence suggests that the disease is primarily immune-mediated rather than the result of direct viral cytotoxicity to myocytes [12, 13] . both coxsackie band encephalomyocarditis virus infections in mice may progress to myocarditis and congestive heart failure, and some survi-vors may progress to a dilated cardiomyopathy later in life [5, [14] [15] [16] . the mechanisms by which viruses outside the enterovirus family cause heart disease are unclear. a model for virus-induced cardiomyopathy has also been described in rabbits [17] . rabbit cardiomyopathy is characterized by pulmonary edema, degeneration and necrosis of myocytes, and right ventricular dilation. similar findings have also been reported in rabbits infected with pleural effusion disease virus [18, 19) . the etiologic agent for rabbit cardiomyopathy is probably a rabbit coronavirus (rbcv) antigenically related to the human coronavirus strain 229e [ i 7] . we determined whether infection with rbcv would result in myocarditis and the development of congestive heart failure. animals and virus. rabbit coronavirus (rbcv) was originally obtained from a stock maintained by one of the authors (1.d.s.). viral stocks were diluted to 10 3_104 ridso/ml and stored at -140°c. male new zealand white rabbits (franklin rabbitry, wake forest, nc), weighing 2.5-3.0 kg, were housed at room temperature (21-24°c) and given water and rabbit diet (agway; grandville milling, creedmoor, nc) ad libitum. the animals were inoculated intravenously via the marginal ear vein with 0.2 ml of the 10 3-104 rid so viral stock. body weight and rectal temperature were recorded daily. animals were observed for signs of infection: dullness of the sclerae, severe congestion of the conjunctivae and irides, rectal temperatures >39°c, and weight loss. to assay viral titers in the heart muscle, moribund animals were intravenously injected with 50 mg/kg sodium pentobarbitol, and the hearts were perfused and washed extensively with pbs. two hundred micrograms of left ventricle were ground in 0.8 ml of pbs and centrifuged at 12,000 g for 10 min in an eppendorf centrifuge (fisher scientific, norcross, ga). the suexhibited moist rales and wheezing during the subacute stage. rabbits that died on days 10-12 had pleural effusion, pulmonary edema, ascites, enlarged hearts, dilated right and left ventricular cavities, and congestion in the lungs and liver. from these observations, we divided rbcv infection into an acute phase that was characterized by dilation of the right ventricle and pulmonary edema (days 2-5) and a subacute phase that was characterized by dilation of both ventricles and heart failure (days 6-12). heart weight and heart weight-to-body weight ratios during rbcv infection. body weight, heart weight, and heart weight-to-body weight ratios were measured on days 2-5, when myocardial degeneration and necrosis and pulmonary edema became apparent, and days 6-12, when heart failure was evident. body weights slowly decreased during the course of infection and were notably decreased (......,0.4 kg) during the subacute phase of the disease (data not shown). control rabbits sacrificed on days 2-5 (acute, n = 10) or days 6-12 (subacute, n = 10) had heart weights of 6.1 ± 0.3 g and 6.1 ± 0.5 g, respectively. after rbcv infection, heart weights were significantly increased, to 8.4 ± 1.4 g during the acute stage (n = 12) (p < .00 i) and 8.7 ± 1.6 g during the subacute phase of infection (n = 14) (p < .001). heart weight-to-body weight ratios were 0.0022 ± 0.0002 in the uninfected controls and were increased significantly, to 0.0031 ± 0.0003 (p < .00 i) and 0.0035 ± 0.0006 (p < .00 i) during the acute and subacute phases of infection, respectively. dimensions of the cardiac walls during rbcv infection. changes in the size of the heart and, in particular, dimensions of the ventricles were evident after rbcv infection (figure 2). to conclusively document the anatomic changes in the heart during infection, the thickness of the ventricular wall was measured through the coronal axis at the midpoint of the ventricles. the thickness of the right wall in uninfected controls was pernatant was serially diluted and inoculated into the marginal ear vein of rabbits. histologic studies. animals dying from rbcv infection were necropsied within 12 h of death. alternatively, moribund animals were sacrificed as described above. body weights were obtained to the nearest o. 10 kg. the heart was separated from the pericardial sac, trimmed of fat and extraneous tissue, and flushed with pbs. the heart was weighed to the nearest 0.1 g; the chambers were filled with 10% phosphate-buffered formalin (bf) and immersed in 10% bf for 24-48 h. the heart was removed and sectioned transversely at the widest dimensions of the ventricles. after additional fixation in bf, four paraffin-embedded 6-~m sections were cut at 150-~m intervals and stained with hematoxylin-eosin (h&e) stain. selected sections were also stained by masson's trichrome and the von kossa stains. sections ofthe lung, liver, thymus, kidney, and spleen were also stained with h&e. morphometric studies. a computerized zeiss videoplan-l digital morphometric system (carl zeiss, thornwood, ny) was used to measure the dimensions of the cardiac walls and cavities and to examine the area within each ventricular section. to measure the thickness of the right and left ventricular walls and interventricular septum, 15-20 measurements were taken at regular intervals across the ventricles. two or three different cardiac sections were measured from each animal, and the standard deviations were calculated and recorded. the dimensions of the ventricular cavities were measured at 15-20 points across the midpoint in each ventricle and then averaged between three or four consecutive cross-sections in the same animal. large differences between consecutive cross-sections were not detected. papillary muscle projections complicated measurements of the left ventricular cavity and wall. consequently, the area in each left and right ventricle cross-section was measured two or three times and averaged between different cross-sections in the same animal. animals dying during the acute or subacute phase ofinfection were grouped accordingly, and mean values were averaged and reported as mean ± so. student's t test for unpaired observations was used to evaluate the statistical significance of differences in cardiac dimensions. zealand white rabbits were inoculated with 0.2 ml of a 10 3 -10 4 rioso/ml stock and examined daily for clinical signs of infection. consistent with earlier studies [ i 7], mortality rates peaked at 4 days after infection, decreased, and then increased again between day 6 and 8. no animals died after 12 days after infection, and the overall mortality rate approached 64% (27% acute; 37% subacute) (figure i). animals dying early from infection had enlarged hearts characterized by striking dilation of the right ventricular cavity and accompanied by pulmonary edema. pleural effusion and congestion of the lungs and liver were occasionally present during the acute phase but were more commonly observed between days 6-9 after infection. clinically, rabbits between the uninfected controls and animals dying during the acute phase of the disease (p = .445). however, the thickness of the left ventricular wall was significantly reduced, by --15%, during the subacute phase (p < .05) (figure 3b) . dimensions of the ventricular cavities during rbcv infection. in uninfected controls, the width of the right ventricular cavity was --3100 jlm ( figure 4a ). in infected animals, the width of the right ventricular cavity was significantly increased, by --105% during the acute stage of to obtain an additional estimate of the dilation of the right and left ventricular cavities during infection, we averaged the area in several consecutive right and left ventricular cavity sections from animals dying during the acute or subacute phase. areas within the right and left ventricular cavity were similar between control animals sacrificed on days 3-5 or 6-12 ( figure 5a, b) . after rbcv infection, the area in the right ventricular cavity increased by --150% during the acute stage (p < .001) and 254% during the subacute phase (p < .001) ( figure 5a ). areas within the left ventricular cavity were not altered significantly during the early stages of infection (p = .278), but significant increases, of --30%, were noted during the subacute phase (p = .025) (figure 5b). pathologic findings during rbcv infection. rabbits were divided into two groups according to the day of death and pathologic findings in the host. with minor variations, lesions were similar to those previously reported [17] . the heart was the principal target organ, often with red streaks present on the epicardial and endocardial surfaces. pulmonary edema was always present, and accumulations of 10often present in the thorax. the frequency and volume of pleural effusion increased during the subacute period. late in the subacute period, a small amount of ascites fluid was seen in some animals. accentuation of the hepatic lobules was present in some rabbits dying during the subacute period; however, the liver margins remained sharp. microscopically, myocardial lesions in rabbits dying during the acute phase consisted of widening of intercellular spaces, scattered infiltrates of small numbers of heterophiles (rabbit neutrophils), increased granularity of myocyte cytoplasm, hemorrhage, and occasional degeneration and necrosis of myocytes ( figure 6a) . only rarely were foci of frank hemorrhage and calcification seen during this period. lesions were seen equally in the right and left ventricles and in the interventricular septum. as rabbits survived beyond 5 days, lesions progressed in size, number, and maturity. in several rabbits, necrotic foci had varying degrees of calcification ( figure 6b ). lesions were equally present in the left and right ventricles, interventricular septum, and papillary muscle. no lesions were seen in the heart valves or the vessels. interstitial edema increased. in addition to increased numbers of heterophils, macrophages and lymphocytes were seen. myocarditis was usually diffuse to focal and peaked by day 10-12 ( figure 6c ). alveolar epithelial cells were swollen. in a few rabbits of the subacute group, much of the intraalveolar fluid had been readsorbed, leaving fibrinous strands, macrophages, and heterophils. interalveolar blood vessels were distended ( figure 6d ). lymphoid tissue adjacent to bronchi was within normal limits, and bronchial epithelium was unremarkable. liver lesions were subtle during the acute phase. sinusoids were slightly distended, especially in areas surrounding central veins. in several rabbits the first row or two of hepatocytes surrounding some of the central veins were rounded up, hyalinized with indistinct nuclei, and deeply eosinophilic. with increased time to death in the subacute stage, b sinusoids became more distended, individual hepatocytes were compressed, the number of hepatocytes undergoing coagulative necrosis around the central veins increased, and the necrotic areas became increasingly hypercellular. portal triads were normal ( figure 6e) . isolation of infectious virus. infectious virus was isolated from the hearts of animals dying between days 2 and 12. virus was first detected in the heart muscle by day 2 (10 2-103 rid 50/g). peak' titers in the heart muscle occurred on days 3-5 (10 5-106 rid 50/g), and significant titers were detected viral infection of the heart muscle may result in degeneration and necrosis of myocytes, myocarditis, and congestive heart failure [15, 7] . viral infection may also be an important factor in the development ofidiopathic dilated cardiomyopathy [5, [20] [21] [22] . the incidence and mechanisms by which viruses cause heart disease in humans and animals are unclear, because these agents are rarely recovered from the patients and heart tissue at biopsy. since many different viral infections may result in cardiac damage, model systems are needed to examine the basis for virus-induced heart disease. rbcv infection results in degeneration and necrosis of myocytes, myocarditis, interstitial edema, hemorrhage, increased heart weight and heart weight-to-body weight ratios, and dilated ventricles. although dry weights of the hearts were not determined, pathologic findings suggest that the increase in heart weight is probably caused by interstitial edema. animals dying in the subacute stage of the disease develop congestion in the lungs and liver, suggesting that a significant percentage of these animals probably die from heart failure. manifestations of both left-and right-sided heart failure are clearly evident in the subacute phase of infection [4, 6, 7, 21] . previous studies in our laboratory clearly demonstrated the presence ofviral antigen in regions ofmyocardial degeneration and infectious virus in the hearts of infected animals, supporting the idea that changes in the myocardium are most likely caused by viral replication in the heart muscle [17] . the pathogen for this disease is morphologically and antigenically related to the group i human and porcine coronaviruses [17] . rbcv is also related to the etiologic agent responsible for pleural effusion disease in rabbits [17] [18] [19] 23] . both viruses have similar isolation histories and antigenic properties and produce similar diseases in vivo. here we demonstrated that infection with rbcv results in myocarditis. rabbits normally require 8-10 days to develop a strong lymphocytic infiltration after infection [24] . by the dallas classification system [25] , focal to diffuse myocarditis with fibrosis is clearly present by day 9 and reaches peak levels on days 10-12. in previous studies, little lymphocytic infiltration was noted because most of the animals dying from rbcv or pleural effusion disease virus infection were examined before day 9 [17, 18, 26] . it seems likely that pleural effusion disease virus infection also results in a significant percentage of animals dying from heart failure, since degeneration and necrosis of myocytes, pulmonary edema, pleural effusion, dilated ventricles, and congestion of the lungs, liver, and spleen are common [18, 26] . in humans, virus-induced myocarditis and congestive heart failure have been reported after infection with herpesvirus, enterovirus, paramyxovirus, myxovirus, alphavirus, flavivirus, and others [1-3, 27, 28]. complications associated with coronavirus infections in humans include myocarditis and perimyocarditis [4] . during the subacute phase of infection, myocardial lesions observed in rabbits closely resemble those of acute myocarditis in humans. lesions are distributed throughout the ventricles and consist of necrotic fibers surrounded by macrophages and lymphocytes. adjacent areas of the myocardium appear normal. the endocardium and pericardium are spared. later, necrotic fibers are replaced with connective tissue or calcification, and the number of macrophages and lymphocytes are reduced [1, 25] . the most extensively studied models for virus-induced myocarditis and congestive heart failure are in mice inoculated with enteroviruses (encephalomyocarditis virus or coxsackie b virus) [1, 5, 21] . there are also model systems for influenza virus-induced metabolic alterations in the heart [29] , reovirus-induced myocarditis in mice [3o], and parvovirus-induced myocarditis in dogs [31] . in rabbits, poxvirus infections may also result in degeneration and necrosis of myocytes and myocarditis [32] . rabbit coronavirus infection is similar to the encephalomyocarditis virus model. early in that infection, animals are probably dying from acute heart failure caused by a complete atrioventricular block [15, 33] . in the subacute stage of infection, animals die from myocarditis-induced congestive heart failure [13, 15] . the most notable difference between the two models is that myocarditis, myocyte necrosis, and calcification appear to be much more extensive after encephalomyocarditis virus infection [14, 15] . in the case of encephalomyocarditis and coxsackie b virus infections, maximum inflammation and cardiac necrosis occur after the clearance of virus from the heart, suggesting that direct viral cytotoxicity to myocytes is oflimited importance [i] . rather, the preponderance of data suggest that cardiac damage is immune-mediated [12, 13, [34] [35] [36] [37] [38] [39] . the pathogenic mechanisms for myocyte injury after rbcv infection are unclear. early in infection, significant myocyte damage correlates with high viral titers in the heart and occurs before the presence of significant inflammatory infiltrates. heterophils and macrophages that are occasionally present early in infection probably represent a nonspecific response to necrotic cell injury. these data suggest that myocardial injury may initially result from direct viral infection, similar to findings reported early in canine parvovirus infection [31] . we have described a model system for virus-induced myocarditis and congestive heart failure in rabbits. these data provide the underlying foundation for future studies examining the mechanism of rbcv-induced heart disease in rabbits. viral myocarditis abelmann who viral myocarditis and its sequelae 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dilatedtype cardiomyopathy due to coxsackievirus b3 rabbit cardiomyopathy associated with a virus antigenically related to human coronavirus strain 229e pathogenicity and persistence of pleural effusion disease virus isolates in rabbits pleural effusion disease in rabbits. properties of the aetiological agent dilated (congestive) cardiomyopathy: a syndrome of severe cardiac dysfunction with remarkably few morphological features of myocardial damage animal models of congestive heart failure and congestive (dilated) cardiomyopathy due to viral myocarditis in mice antibodies to coxsackie b virus in congestive cardiomyopathy pathogenetic observations on pleural effusion disease in rabbits effect of cortisone administration on host-parasite relationships in early experimental syphilis myocarditis, a histopathologic definition and classification pleural effusion disease in rabbits. clinical and post mortem observations myocarditis and cardiomyopathy after arbovirus infections (dengue and chikungunya fever) detection of enterovirus rna in myocardial biopsies from patients with myocarditis and cardiomyopathy using gene amplification by polymerase chain reaction sequential metabolic alterations in the myocardium during influenza and tularemia in mice the reovirus m i gene, encoding a viral core protein, is associated with the myocardic phenotype of a reovirus variant experimental viral myocarditis: parvoviral infection of neonatal pups susceptibility of the heart of the rabbit to specific infection in viral diseases electrocardiographic findings in experimental myocarditis in dba/2 mice: complete atrioventricular block in the acute stage, low voltage of the qrs complex in the subacute stage and arrhythmias in the chronic stage coxsackievirus b3 myocarditis. identification of different pathogenic mechanisms in dba/2 and balb/c mice coxsackievirus b-3 myocarditis in balb/c mice. evidence for autoimmunity to myocyte antigens differences in cytolytic t cell response ofbalb/c mice infected with myocarditic and nonmyocarditic strains of coxsackievirus group b, type 3 autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to coxsackievirus b3-induced myocarditis coxsackievirus-b'i-induced myocarditis: virus receptor antibodies modulate myocarditis monoclonal antibody to coxsackievirus b4 reacts with myocardium we thank gillian harris and edna lennon for excellent secretarial assistance and james e. hall, sheila peel, mary c. schaad, and robert e. johnston for editorial comments. key: cord-259004-plst2wno authors: van elden, leontine j. r.; anton m., anton m.; van alphen, floris; hendriksen, karin a. w.; hoepelman, andy i. m.; van kraaij, marian g. j.; oosterheert, jan-jelrik; schipper, pauline; schuurman, rob; nijhuis, monique title: frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction date: 2004-02-17 journal: j infect dis doi: 10.1086/381207 sha: doc_id: 259004 cord_uid: plst2wno during the past years, human coronaviruses (hcovs) have been increasingly identified as pathogens associated with more-severe respiratory tract infection (rti). diagnostic tests for hcovs are not frequently used in the routine setting. it is likely that, as a result, the precise role that hcovs play in rtis is greatly underestimated. we describe a rapid, sensitive, and highly specific quantitative real-time reverse-transcriptase polymerase chain reaction (rt-pcr) for the detection of hcov that can easily be implemented in the routine diagnostic setting. hcov was detected in 28 (11%) of the 261 clinical specimens obtained from patients presenting with symptoms of rti ranging from common cold to severe pneumonia. only 1 (0.4%) of the 243 control specimens obtained from patients without symptoms of rti showed the presence of hcov. we conclude that hcovs can be frequently detected in patients presenting with rti. real-time rt-pcr provides a tool for large-scale epidemiological studies to further clarify the role that coronavirus infection plays in rti in humans. during the past years, human coronaviruses (hcovs) have been increasingly identified as pathogens associated with more-severe respiratory tract infection (rti). diagnostic tests for hcovs are not frequently used in the routine setting. it is likely that, as a result, the precise role that hcovs play in rtis is greatly underestimated. we describe a rapid, sensitive, and highly specific quantitative real-time reverse-transcriptase polymerase chain reaction (rt-pcr) for the detection of hcov that can easily be implemented in the routine diagnostic setting. hcov was detected in 28 (11%) of the 261 clinical specimens obtained from patients presenting with symptoms of rti ranging from common cold to severe pneumonia. only 1 (0.4%) of the 243 control specimens obtained from patients without symptoms of rti showed the presence of hcov. we conclude that hcovs can be frequently detected in patients presenting with rti. real-time rt-pcr provides a tool for large-scale epidemiological studies to further clarify the role that coronavirus infection plays in rti in humans. coronaviruses are enveloped rna viruses that can cause disease in humans and animals. the human coronaviruses (hcovs) were first identified in 1962. they belong to the family coronaviridae, genus coronavirus, and the 2 human strains, hcov 229e and oc43, are divided into 2 antigenic groups. hcovs are recognized as the second-most frequent cause of the common cold syndrome [1] . during the past years, hcovs have more often been believed to be responsible for severe upper and lower respiratory-tract infection (rti). they have occasionally been identified as a cause of pneumonia in older adults, infants, and immunocompromised patients [2] [3] [4] [5] . also, in otherwise healthy adults-for example, in military recruits-clusters of infections have been reported as a cause of pneumonia [6] . moreover, in a recent outbreak of hcov in normandy, the clinical manifestation ranged from mild symptoms to pneumonia [7] . recently, a heightened interest in the coronavirus has been documented, because a previously unknown type that does not resemble the known hcovs is believed to be responsible for the outbreaks of severe acute respiratory syndrome (sars) in hong kong and toronto [8] [9] [10] [11] . these studies indicate that coronaviruses are increasingly identified as a pathogen causing severe respiratory illnesses and that there is a need for reliable and rapid identification of coronaviruses. the diagnosis of hcov infections is hampered, in part, by the difficulty to replicate in-cell cultures, whereas serologic testing is time-consuming and therefore has little clinical significance. as a consequence, efforts have been made to develop more-sensitive molecular detection methods, such as reverse-transcriptase polymerase chain reaction (rt-pcr) and nested rt-pcr [12, 13] . these methods have been shown to be very valuable for the detection of hcov in different populations of patients, such as children with otitis media, patients with multiple sclerosis, immunocompromised patients with pneumonia, and frail elderly persons with symptoms of rti [3, 5, 8, 9, 14, 15] . although they are highly sensitive and specific, the current rt-pcr and nested rt-pcr methods are less suitable for routine laboratory detection because they are prone to contamination and still require time-consuming sample handling and post-pcr analysis. here, we describe the detection of hcov in a variety of clinical specimens obtained from patients presenting with rtis ranging from common cold to severe pneumonia, using a novel, sensitive, and highly specific taqman-based realtime pcr. in addition, we tested a multiplex-format real-time rt-pcr assay for the detection of hcovs and the novel coronavirus that has been identified in patients with sars. hcov 229e and oc43 were provided by the laboratory for virology, national institute for public health and the environment (rivm; bilthoven, the netherlands), and were propagated on 2 human embryonic lung cell lines (mrc5 and hel). cells and supernatants were harvested after 24, 48, and 72 h, respectively, and were frozen at ϫ70њc. after rna extraction of each stock, series of 10-fold serial dilutions were used to determine, by use of an in-house nested pcr, which propagated stock contained the most virus particles. the stocks, 1 of each strain, that contained the most virus particles were used for further experiments to evaluate the taqman-based real-time pcr. a panel of various respiratory viruses-including influenza virus a/pr/8/34, influenza virus b/lee/40, parainfluenza viruses 1-4 (american type culture collection), and reference strains of rhinovirus 1a, rhinovirus 14, rhinovirus 16, echovirus 12, coxsackie virus a9, respiratory synctial virus (rsv) a long strain, rsv b 9320, and sars-associated coronavirus-were used to determine the specificity of the taqman-based real-time pcr. clinical specimens. clinical specimens were obtained at the hospital's virology laboratory and consisted of the following: (1) nasal wash (nw) specimens and combined nose and throat swabs (ntss) from patients presenting with symptoms of upper or lower rtis and (2) bronchoalveolar lavage (bal) specimens and ntss from adult patients admitted to the hospital with pneumonia. ntss from healthy volunteers and ntss obtained at set time points from patients without symptoms of rti who participated in a prospective 6-month follow-up study to assess the role of respiratory viruses following bone marrow transplantation were used as control specimens. each specimen was transported in 5 ml of virus transport medium. nw specimens, ntss, and bal specimens were vortexed for 10 s and centrifuged at 2000 g for 15 min. one milliliter of the supernatant was used directly for routine virus culture of other respiratory viruses (influenza viruses, rsv, parainfluenza viruses, picornaviruses, and adenovirus). the remaining material was stored at ϫ70њc until further processing. viral rna isolation and cdna synthesis. rna extraction was performed by use of the magnapure lc total nucleic acid kit (roche diagnostics), as described elsewhere [16] . the rna was then either eluted in 100 ml of 40 ng/ml polya rna before performing a 1-tube rt-pcr or eluted in 100 ml of elution buffer and directly used for cdna synthesis. the reverse transcription and cdna synthesis were both performed as described elsewhere [17] , and the products were stored at ϫ70њc until further use. in-house nested pcr. an in-house nested pcr was performed for hcov 229e and oc43. first-round amplification primers and nested primers were derived from the literature [12] and targeted the nucleocapsid (n) gene, with 1 minor modification: in contrast to the published sequence, we omitted an excess t on position 13 from the nested antisense primer. a 1tube rt-pcr followed by a second (nested) amplification was applied, as described elsewhere [16] , by use of a pe 9600 thermocycler (perkin elmer). pcr products were visualized on an ethidium bromide-stained agarose gel by use of uv illumination. a 5-ml 100-bp marker was used to control fragment lengths. taqman-based real-time pcr. type-specific primers and probes for hcov 229e and oc43 were selected by use of primer express software (pe applied biosystems) and were based on the genomic regions of high conservation of the n gene. the forward and reverse primers (n229e-1, n229e-2, noc43-1, and noc43-2) and probes (n229e-p and noc43-p) that were used are shown in table 1. the primers and probes for hcov 229e and oc43 were tested for possible interactions, to make sure that they could be used in combination. after optimization of the primer and probe concentrations, specimens were assayed in duplicate in a 25-ml reaction mixture containing 5 ml of and oc43 forward primers, respectively, 150 nmol/l and 450 nmol/l hcov 229e and oc43 reverse primers, respectively, 50 nmol/l hcov 229e probe, and 100 nmol/l hcov oc43 probe. the fluorogenic probes that can be labeled with different fluorogenic dyes were both labeled with a 5 reporter dye, 6carboxy-fluorescein, and with a 3 quencher dye, 6-carboxytetramethyl-rhodamine. amplification and detection were performed by use of the abi prism 7700 sequence-detection system by use of the following conditions: 2 min at 50њc to acquire optimal amperase ung activity and 10 min at 95њc to activate amplitaq gold dna polymerase, followed by 45 cycles of 15 s at 95њc and 1 min at 60њc. the primers and probes for the sars-associated coronavirus were used, targeting the polymerase gene, as recently described elsewhere [18] .the primers and probes for hcov 229e and oc43 and the sars-associated coronavirus were tested for possible interactions, to make sure that they could be used in combination in a multiplex assay. virus quantification. to estimate the quantity of the virus load, virus particles were expressed as relative units (ru). above a threshold cycle of 36, the quantitative value of rna copies can no longer be considered to be accurate. therefore, every value above a threshold cycle of 36 and below the detection limit threshold cycle of 45 was assumed to be 2. every amplification cycle represents a 2-fold increase in viral rna copies. the virus load was expressed as a 2-fold increase per cycle, relative to a baseline value of 2 copies at a threshold cycle of 36 (ru p 2 36-threshold cycle ). use of limiting-dilution series showed that, for hcov, the in-house nested pcr and the real-time rt-pcr have similar sensitivity. to compare the sensitivity of the in-house nested pcr with that of the real-time rt-pcr, on clinical specimens, for hcov, a total of 86 nw specimens obtained from asthmatic and otherwise healthy subjects with symptoms of upper and/or lower rti were analyzed for hcov by both the in-house nested pcr and the real-time rt-pcr. as shown in table 2, 14 of 86 specimens were found to be positive by real-time rt-pcr, compared with 10 of 86 specimens tested by the in-house nested pcr. the real-time rt-pcr performed better on the specimens containing a low virus load, compared with the nested pcr (figure 1). for hcov, the real-time rt-pcr was highly specific: none of the other respiratory viruses (rhinovirus 1a, rhinovirus 14, the real-time rt-pcr for hcov could successfully be combined with a real-time rt-pcr for sars-associated coronavirus. use of limiting-dilution series using a single (primers and probes for the sars-associated coronavirus only) and multiplex (combination of primers and probes for the hcov and sarsassociated coronavirus) format showed similar sensitivity in the detection of sars-associated coronavirus rna and hcovs. detection in clinical specimens and control specimens. to evaluate the real-time rt-pcr for hcov, we analyzed a total of 261 clinical specimens obtained at the hospital's virology laboratory: (1) 86 nw specimens and 151 ntss were obtained from patients presenting with symptoms of upper and/or lower rti, and (2) 11 bal specimens and 13 ntss were obtained from patients admitted to the hospital with pneumonia. moreover, 243 control ntss were evaluated from bone marrow-transplant recipients without symptoms of rti. in total, 28 (11%) of 261 clinical specimens tested positive for hcov. hcov was detected in the bal specimens from 2 (18.2%) of 11 patients. in addition, 2 (15.4%) of 13 ntss obtained from patients admitted to the hospital with pneumonia tested positive for hcov (table 2) . in contrast, hcov rna was detected in only 1 (0.4%) of 243 ntss that were obtained at set time points from patients without obvious symptoms of rti (table 2) . five patients with symptoms of upper rti and whose specimens tested positive for coronavirus by real-time rt-pcr were followed during the course of their infections. a total of 24 nw specimens were obtained just after the presentation of common cold symptoms, at several time points up to 60 days. the virus load was expressed as ru (ru p 2 36-threshold cycle ). as shown in figure 2, we were able to detect and quantify hcov in nw specimens up to 7 days after the initial presentation of common cold symptoms in 3 patients, and up to 14 days in 1 patient. in 1 patient (patient 5), the virus load was below the level of reliable quantitation. our findings have demonstrated that hcov is frequently detected in clinical specimens obtained at the hospital's virology laboratory from patients presenting with rti. the novel realtime rt-pcr assay allows rapid and specific detection of hcovs in patients with various presentations of rti. since there is increasing evidence suggesting that either the known or newly identified hcovs might be involved in moresevere disease, there is a need for more-rapid and more-reliable diagnostic tools. at present, a great deal of attention has been directed toward patients with sars. a novel coronavirus that has been identified in the majority of patients is the primary cause of sars [10, 11, [18] [19] [20] . genetic characterization of this novel coronavirus shows considerable differences between it and hcovs 229e and oc43 [18] . the real-time rt-pcr described here can detect the novel sars-associated coronavirus when used in a multiplex format. however, it has yet to be determined whether this is a favorable format, since the clinical presentation of sars differs from the assumed clinical presentation of hcov infection. however, for example, advanced age and underlying disease have also been associated with a more severe presentation of hcov infection [8, 9] . also, in case reports, hcov has been associated with pneumonia after autologous bone marrow transplantation, and we recently identified hcov by use of nested pcr in the bal specimen of a severely immunocompromised patient with pneumonia [3, 5] . interestingly, in the present study, we detected hcov by realtime rt-pcr in the bal specimens from 2 patients presenting with severe pneumonia and in the ntss from patients admitted to the hospital with pneumonia, which again suggests that hcov may be the cause of severe disease in some patients. by use of molecular detection methods, nicholson et al. [21] have already shown that 26% of upper rti in elderly people living at home are due to hcovs, and the identification of a recent community outbreak of hcov oc43 in france was facilitated by the use of rt-pcr [7] [8] [9] [10] [11] . although they are valuable in a research setting, these methods are less suitable for routine laboratory detection, because they still require timeconsuming sample handling and post-pcr analysis and are consequently prone to contamination. besides being rapid, the real-time rt-pcr assay has the advantage of a standardized protocol that can easily be applied to the detection of other respiratory viruses: the rt-pcr can be performed under uniform amplification conditions, thereby using target-specific primer and probe sets. another disadvantage is that most studies using rt-pcr for the detection of hcov lack proper control groups to evaluate the clinical value of a positive result by rt-pcr. to gain insight into the relevance of a positive result by this assay, we followed 5 symptomatic patients during the course of coronavirus infection and also obtained specimens from asymptomatic individuals. the follow-up of the 5 symptomatic patients showed that hcov rna could be detected by real-time rt-pcr up to 14 days after infection. moreover, we tested specimens obtained from patients without obvious signs or symptoms of rti. none of the specimens obtained from healthy individuals contained hcov rna. at 1 time point, just after the bone marrow transplantation, we detected hcov in an nts from a bone marrow-transplant recipient without obvious upper and/ or lower rti. it might be that the patient was suffering from a minor cold and that these symptoms remained unnoticed. from these results we have concluded that an hcov-positive finding by real-time rt-pcr in a specimen obtained from a symptomatic patient has diagnostic significance. diagnostic tests for hcovs are not frequently used in the routine setting. serologic testing methods do not allow rapid identification of virus, and, although both hcov 229e and oc43 can be propagated on specialized cells, the approach lacks sensitivity, is time-consuming, and often requires the expertise of a specialist. in addition, virus isolation is often considered to be redundant and without clinical consequence, since hcovs are thought to be mainly associated with the common cold syndrome. it is likely that, as a result, the precise role that coronaviruses play in rtis is greatly underestimated because of the lack of practical diagnostic tools. we realize that the specimens analyzed in the present study, which were obtained at the hospital's virology laboratory, probably represent specimens from a selected group of patients in which a respiratory virus is considered to be a possible pathogen on clinical grounds. the results, however, indicate that hcov is frequently detected and that the novel real-time rt-pcr assay provides a tool for large-scale epidemiological studies to further 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elderly people living in the community: comparative, prospective, population based study of disease burden we thank h.w. doerr (institute of medical virology, johann wolfgang goethe university, frankfurt am main, germany) for the gift of severe acute respiratory syndrome-associated coronavirus. key: cord-281158-vjh9z7l4 authors: storch, gregory a title: respiratory viruses in babies: important insights from down under date: 2018-02-01 journal: j infect dis doi: 10.1093/infdis/jix600 sha: doc_id: 281158 cord_uid: vjh9z7l4 nan for those of us interested in respiratory viruses, there is a lot to like about the study by sarna et al that appears in this issue of the journal of infectious diseases [1] . the study analyzes data from a birth cohort established as part of the observational research in childhood infectious diseases (orchid) project, based in brisbane, australia [2] . impressive study attributes include large size, community base, enrollment from birth, scheduled frequent longitudinal sampling with or without illness, high percentage of specimen acquisition rate, even enrollment of subjects throughout the year to account for virus seasonality, and testing of samples with an extensive panel of real-time polymerase chain reaction (pcr) assays. participating babies had anterior nasal and "dirty nappy" swabs obtained at birth and weekly thereafter by parents trained by research staff. parents also kept daily symptom diaries listing predefined symptoms. the present manuscript reports only on the respiratory swabs. this massive undertaking yielded analyzable data on 9594 samples from 152 participants, corresponding to 163 098 individual virus-specific pcr queries! this intensive prospective study of respiratory viruses in infants finds that human rhinovirus (hrv) is by far the most frequent virus detected in the infant respiratory tract. this single-stranded, positive-sense rna virus is increasingly recognized as an important human pathogen, with a strong relationship to childhood asthma [3] . significantly, sarna et al found that hrv was detected in an impressive 20.0% of all samples tested, accounting for 77.3% of all virus-positive swabs. by 2 years of age, 98% of participating infants had experienced hrv-c, 94% hrv-a, and 56% hrv-b. although impressive, this large proportional load of hrv should not be surprising, as it has also been shown in previous community-based studies using molecular tests, including an earlier study by these authors [4] and several others [5] [6] [7] [8] . similarly, laboratories using multiplex respiratory virus panels that include sensitive rhinovirus detection capability usually find rhinovirus/enterovirus to be the most common positive result. the us food and drug administration (fda)-cleared multiplex respiratory panels do not distinguish between rhinoviruses and enteroviruses, but apart from outbreak situations, such as occurred in 2014 with enterovirus d-68 [9] , most of the positive results from rhinovirus/enterovirus assays reflect hrv infection. it has become clear that in relation to respiratory viruses, hrv is the elephant in the room. in comparison to the universal occurrence of hrv, infections with other respiratory viruses were less frequent. rsv and parainfluenza viruses were each detected in 58% of subjects, influenza a in 8%, influenza b in 3%, human metapneumovirus in 21%, human coronaviruses in 72%, adenovirus in 51%, human polyomaviruses in 77%, and human bocavirus in 75%. the 58% occurrence of rsv is surprisingly low, as common understanding is that almost all infants experience rsv within the first 2 years of life [10] . possible explanations for the discrepancy include inadequate sample collection (samples were collected by parents), seasonal variation in the occurrence of rsv, and complete reliance on molecular assays. in some studies, serology has indicated some infections are not detected by pcr [11] . the authors point out the difference between their estimate and that of the houston family study [12] , which is a basis for the concept of universal rsv infection early in life and which relied heavily on serology [12] . however, they also cite a number of more recent studies that support their finding of less than universal infection. these results suggest that we may need to fine-tune our understanding of the frequency of rsv infection early in life. the focus of the sarna et al study was first respiratory virus infections, and the frequency of first hrv infections in young infants is noteworthy. of the 152 infants followed, 81% experienced a first infection with hrv by 6 months of age, compared to 8.5% for rsv, and 0.7%-9.4% for the other respiratory viruses. influenza a and b infections were infrequent, with only 0.8% and 1.4% experiencing first influenza a and b, respectively, by 6 months of age. the curves mapping virus occurrence by age show that all viruses other than hrv were relatively unusual in the first 6 months of life, with upward inflection points evident at approximately 6 months of age, consistent with diminishing maternal immunity. notably, the hrv occurrence curve is different, with rapid increase through the first 6 months and no obvious inflection point. the shape of this curve suggests that maternal immunity is not preventing hrv infections, although it is possible that it is mitigating clinical manifestations. this finding has important implications for hrv control strategies, and calls for mechanistic studies to further define the differences between the behavior of hrv and other respiratory viruses. with mounting undeniable evidence for the frequent occurrence of hrv infection in the first 2 years of life, it is important to assess the impact of these infections, and sarna et al provide relevant data. based on symptom diaries kept by parents, virus-positive episodes were characterized as symptomatic or asymptomatic and, if symptomatic, as involving the upper or lower respiratory tract. if we direct attention to the episodes in which only a single virus was detected (data shown in supplementary table 2 of the sarna et al manuscript) , we see that first hrv detections were less likely than first infections with the other rna viruses to correspond to symptomatic episodes (52% compared to 70%-85% for the other rna viruses), but were comparably likely compared to the dna viruses for which 46%-59% of first detection episodes corresponded to symptomatic infections. likewise, only 6% of first hrv infections were classified as lower respiratory infections, compared with 20%-46% for the other rna viruses and 10%-15% for the dna viruses. only 13% of the first hrv detections were associated with a medical visit, compared with 25%-42% for the other rna viruses and 8%-28% for the dna viruses. it should be noted that direct comparison of severity of hrv infections to the other respiratory viruses is complicated by the fact that a much higher proportion of first episode hrv infections occurred in infants <6 months of age, and data are not provided (and may not have been available because of the infrequency of infection with the non-hrv respiratory viruses during that age period) that allow us to ascertain whether an age effect is present. nevertheless, it is clear that most of the first hrv infections were mild, in spite of the fact that they were occurring predominantly in infants <6 months of age, an age when the immune system is not fully developed and some viral infections can disseminate. however, as the authors correctly point out, the mildness of the clinical illness associated with first hrv detections may not be the whole story. they cite a recent study by wolsk et al [13] showing that hrv infection in the first 4 weeks of life, even if asymptomatic, may program immune memory with an exaggerated t-helper 2 mucosal immune response and impaired antiviral responses. the implication is that hrv infection in young infants, even if asymptomatic, might promote the development of asthma later in life. this interaction may be viewed as hrv serving as an educator (or miseducator) of the immune system by virtue of early-in-life virus-host interactions. this concept fits well with an emerging view that one of the key functions of the infant's microbiome is education of the immune system [14] . the orchid study has made an important contribution by directing our attention to the extremely frequent interaction between hrv and the immature immune system, the implications of which clearly merit further study. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. potential conflicts of interest. author confirmed no potential conflicts. the author have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. timing of first respiratory virus detection in infants: a community-based birth cohort study observational research in childhood infectious diseases (orchid): a dynamic birth cohort study understanding the association of human rhinovirus with asthma community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study swiss paediatric respiratory research group. viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study community surveillance of respiratory viruses among families in the utah better identification of • editorial commentary germs-longitudinal viral epidemiology (big-love) study etiology of acute respiratory infections in infants: a prospective birth cohort study the emergence of enterovirus d68 report of the committee on infectious diseases serology enhances molecular diagnosis of respiratory virus infections other than influenza in children and adults hospitalized with community-acquired pneumonia risk of primary infection and reinfection with respiratory syncytial virus picornavirus-induced airway mucosa immune profile in asymptomatic neonates host-microbiota interactions and adaptive immunity key: cord-275863-qos9vu3r authors: dejnirattisai, wanwisa; webb, andrew i.; chan, vera; jumnainsong, amonrat; davidson, andrew; mongkolsapaya, juthathip; screaton, gavin title: lectin switching during dengue virus infection date: 2011-06-15 journal: j infect dis doi: 10.1093/infdis/jir173 sha: doc_id: 275863 cord_uid: qos9vu3r dengue virus receptors are relatively poorly characterized, but there has been recent interest in 2 c-type lectin molecules, dendritic cell–specific intercellular adhesion molecule 3 (icam-3)–grabbing nonintegrin (dc-sign) and its close homologue liver/lymph node–specific icam-3–grabbing integrin (l-sign), which can both bind dengue and promote infection. in this report we have studied the interaction of dengue viruses produced in insect cells, tumor cell lines, and primary human dendritic cells (dcs) with dc-sign and l-sign. virus produced in primary dcs is unable to interact with dc-sign but remains infectious for l-sign–expressing cells. skin-resident dcs may thus be a site of initial infection by insect-produced virus, but dcs will likely not participate in large-scale virus replication during dengue infection. these results reveal that differential glycosylation of dengue virus envelope protein is highly dependent on cell state and suggest that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution. the global prevalence of the dengue virus (denv) has grown dramatically in recent decades, and it is now endemic in .100 countries, with some 2.5 billion people at risk of infection. dengue is an arthropod-borne flavivirus that can be subdivided into the 4 major serotypes (den-1-den-4). most dengue infections either are asymptomatic or lead to a self-limiting febrile illness, dengue fever. in some cases the illness is more severe, leading to dengue hemorrhagic fever (dhf) with severe plasma leakage and bleeding that can be life threatening. dhf is more common in individuals undergoing secondary heterologous dengue infection than those suffering primary infections. there has been considerable work to understand the mechanism of severe disease, but the increased frequency during secondary infection and the occurrence of severe symptoms at a time when virus loads are falling sharply imply that it likely results from immunopathology driven by the acquired immune responses, rather than from direct viral cytopathology [1] [2] [3] [4] . levels of a number of cytokines, such as interferon c and tumor necrosis factor a [5, 6] , have been shown to correlate with disease severity, and a storm of inflammatory cytokine secretion has been proposed to lead to the vascular leak characteristic of dhf. recent reports have identified monocytes as a major cell target of viral replication, and heparin sulfates, dc-sign, mannose receptor, and other glycoproteins have been proposed as cellular receptors for denv [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . dc-sign polymorphism has been shown to be associated with the disease severity [19] . clec5a has also recently been described as a proinflammatory receptor for denv that contributes to lethal disease in a mouse model [20] . as dengue virus circulates between 2 hosts, humans and insects, it has to be adapted to replicate and infect both species. the majority of studies of dengue infection and tropism use viruses produced in insect cell lines such as c6/36 or mammalian tumor cell lines such as vero cells. infection of humans occurs in a stepwise fashion: initial infection of cells with insect virus, followed by sequential infection of cells by mammalian-produced virus. we were interested to determine whether there were any differences in the tropism of viruses produced in primary nontransformed human cells. the dengue-2 strain, 16681, was grown in c6/36 cells, vero cells, and monocyte-derived dendritic cells (dcs). cell-free supernatants were used either neat or after concentration by ultracentrifugation at 45,000 rpm for 4 h at 4°c, and the virus pellet was resuspended in 1.5% fetal bovine serum (fbs)/ leibovitz l-15. to concentrate large volumes of low-titer denv supernatant, denv were precipitated with 10% polyethylene glycol 8000 (sigma) before ultracentrifugation. u937 were maintained in 10% fbs/roswell park memorial institute medium (rpmi). nih/3t3, and dc-sign and l-sign nih/3t3 cell lines were obtained through the aids research and reference reagent program (division of aids, national institute of allergy and infectious diseases, from drs thomas d. martin and vineet n. kewal ramani) and maintained in 10% fbs/ dulbecco's modified eagle's medium. all media were supplemented with 2 mmol/l l-glutamine, 100 u/ml penicillin, and 100 u/ml streptomycin. the viral titers were determined by a focus-forming assay. briefly, virus was serially diluted and incubated with vero cells for 2 hours at 37°c. the monolayers were then overlaid with 1.5% carboxymethylcellulose and incubated at 37°c for 2 days. virus foci were stained with anti-e antibody followed by peroxidase-conjugated anti-mouse immunoglobulin (ig) and visualized by the addition of 3,3#-diaminobenzidine tetrahydrochloride substrate. dcs were cultured as described elsewhere [21] . human cd14 1 monocytes were cultured in rpmi 1640 with 20 ng/ml rhugm-csf (first link) and 25 ng/ml rhuil-4 (ebioscience). the appropriate phenotype of immature dc (ie, lacking cd14 1 but expressing major histocompatibility complex class i and class ii and dc-sign) was confirmed. cells were infected with denv at a multiplicity of infection (moi) of 1 for 24 h. cells were washed twice in facs wash (fw; phosphate-buffered saline [pbs] containing .5% bovine serum albumin, 2% fbs, 1% human serum, .01% nan 3 ). cells were fixed with 4% paraformaldehyde/pbs for 10 minutes and permeabilized with .5% saponin in fw for 10 minutes. cells were then incubated with anti-ns1 mab or with anti-e mab followed by anti-mouse igg/pe (dakocytomation) in .5% saponin/fw. cells were resuspended in fw and analyzed by facscan (becton dickinson). data were analyzed by using flowjo software (tree star). 3t3 and 293t were incubated with 20 u/ml heparin for 20 minutes at room temperature before being infected with denv. cells were incubated with denv produced from different cell types at equal amounts of e protein (measured by enzymelinked immunosorbent assay) for 2 h at room temperature. after washing with ice-cold fw, cells were fixed with 4% paraformaldehyde/pbs, and surface-bound virus was detected with an anti-e mab and anti-mouse igg/pe followed by facs analysis. the pcdna3-ll-sign plasmids expressing 7 (n7) and 5 (n5) tandem neck region repeats or a mixture of n7 and n5 plasmids (ratio 1:1) were transfected into 293t cells using the fugene6 (roche). l-sign expression was verified with l-sign-specific antibody (clone 120604, r&d systems). denv supernatant was precleared with protein a-agarose for one h at 4°c. bead-free supernatant was incubated with 10 lg of 4g2 at 4°c for 2 h followed by protein a-agarose for 1 h. the beads were washed with .05% tween/pbs 3 times and eluted with nonreducing loading buffer. the sample was run on nonreducing 10% sodium dodecyl sulfate (sds) polyacryramide gels and electroblotted onto nitrocellulose membrane (amersham). glycan types on denv proteins were determined using the dig glycan differentiation kit (roche). endoglycosidase h (endo h) or n-glycosidase f (pngase f; new england biolabs) was performed as described elsewhere [22] . digested proteins were separated by 10% sds polyacryramide gels and analyzed by western blot. e protein was detected by anti-e mab followed by peroxidase-conjugated anti-mouse igg ab. following the bite from an infected mosquito, the host first encounters virus produced in the mosquito, and following this initial inoculation subsequent rounds of infection are driven by virus produced by host cells. to study these 2 distinct stages of pathogenesis, we compared viruses from c6/36 (insect cells) and virus produced from primary human monocyte-derived dendritic cells. viral supernatants were titered using a focus-forming assay on vero cells, and equal amounts of titered virus were used to infect vero, 293t, or dcs at an moi of 1. the percentage of infected cells was monitored by cytofluorometry staining of the intracellular nonstructural dengue antigen ns1, which is produced only following productive infection. insect-derived virus was equally competent at infecting the 3 cell types with high efficiency ( figure 1a-c) . surprisingly, dc-produced virus was not able to reinfect dcs but was nevertheless fully competent at infecting 293t and vero cells. the lack of infectivity of dc-produced virus on dcs was also shown using a different mab, 4g2, which reacts with an epitope on dengue envelope protein ( figure 1c , lower panel). a time course of infection was performed where dcs or vero cells were infected with virus produced in c6/36, vero, or dcs, and infection was monitored by facs or using a focus-forming assay to measure infectious virus harvested from the supernatants of infected cells ( figure 1d -e). at 24, 48, and 72 h the dc-produced virus showed a much reduced ability to infect dcs when compared with virus produced in c6/36 or vero cells. the experiments we have described above used the dengue serotype 2 strain 16681. to see whether these results could be generalized, we checked infection of both dcs and vero cells with dengue serotype 2 strain new guinea c, serotype 1 strain hawaii, serotype 3 strain h87, and serotype 4 strain h241. infection efficiency was measured by facs at 24 and 48 hours following infection with virus produced in either c6/36 cells or dcs (figure 2a-b) . in all cases dc-produced virus was much less infectious for dcs than insect-produced virus, whereas infection of vero cells was roughly equal. it is known that mature dcs are relatively resistant to dengue infection, so to rule out the possibility of any dc-produced cofactors that might induce maturation or any other form of resistance to infection we performed a mixing experiment whereby dc-produced and c6/36-produced viruses were used to infect dcs. in these experiments using mixed viruses the dcs were again susceptible to infection, presumably by the c6/36produced virus fraction ( figure 2c ). the lack of reinfection of dcs by dc-produced virus may reflect a difference in the surface binding interaction of dc-versus insect-produced viruses to dcs. to test this vero cells and dcs were incubated with denv produced from the different sources, and binding to the cells was then assessed by staining with the anti-e mab 4g2. virus produced in c6/36 cells could bind to dcs, while binding of dc virus back onto dcs was almost completely absent ( figure 2d ). conversely, both dc-and insect-produced viruses were able to bind to vero cells. to formally prove that the loss of infection of dcs was a result of the loss of affinity of dc-produced virus for dc-sign, we went on to test infection on 3t3 cells expressing dc-sign and included in these assays the related c-type lectin l-sign ( figure 3a ), which has also been reported to be a receptor for dengue virus. insect-derived virus could efficiently infect both dc-sign-and l-sign-expressing cells when compared with control 3t3 cells ( figure 3b ). similar to the lack of observable infection of dcs, the dc-derived virus showed a much lower level of infection on dc-sign-expressing 3t3 cells, with ,2% infection. surprisingly, however, this virus progeny was still able to infect cells expressing l-sign, with up to 13% infection observed. previous reports with dengue virus have suggested that the virus shows equal tropism for both dc-sign and l-sign. these reports were from virus produced in tumor cell lines and not from primary cells. we tested the infectivity of virus produced in vero cells, and in agreement with these previous reports dengue produced in this tumor cell line was able to efficiently infect 3t3 cells via either dc-sign or l-sign ( figure 3c ). finally, we tested the binding of insect-and dc-produced virus to the 3t3 transfectants ( figure 3d ). in agreement with the dc binding experiments dc-produced virus was unable to bind to dc-sign-expressing dcs, whereas both insect-and dc-derived viruses could bind to l-signexpressing cells. to gain insight into the glycosylation profiles of denv produced in c6/36, vero, and dc, purified virus was tested against a panel of lectins with differing carbohydrate-binding specificities by western blot ( figure 4a ). among the 5 plant lectins tested, gna is the only one that selectively interacts with high-and pauci-mannose-type n-glycans. sna and maa bind to sialic acid terminally linked to galactose by a2,6 linkage and a2,3 linkage, respectively, which may occur on complex-type n-glycans. dsa recognizes repeating n-acetyllactosamine (galactosyl b1,4 n-acetylglucosamine) sequences; these may also occur on complex-type n-glycans. pna, unlike dsa, binds preferentially to b1,3-linked terminal galactose, such as galactosyl b1,3 n-acetylgalactosamine, a sequence commonly occurring on o-glycosylated proteins and gangliosides. virus produced in c6/36 cells bound exclusively to gna, implying that it contained predominantly high-or pauci-mannose n-glycans consistent with the glycosylation patterns seen in insect cells. vero-produced virus was bound by 3 of the lectins tested-gna, sna, and dsa-suggesting that the virus contained a variety of high-mannose and complex-or hybrid-type n-glycans with or without a2,6-linked sialic acids, possibly on polynacetyllactosamine outer chains. the dc-produced virus was bound only by sna and dsa, indicating that there was a lack of high-mannose or hybrid n-glycans and that only complex-type n-glycans were present. all of the lectin binding signals occurred at the same position on the gels as e protein in the region of 60 kda; we did not see any signal at 19 kda where prm would be expected to migrate. however, this could be due to a low level of prm on these viruses. finally, we assessed the n-linked glycan on the envelope protein by digestion with either endo h, which will remove high-mannose simple glycans containing 3 or more terminal mannose residues, or pngase f, which removes all simple or complex n-linked glycan ( figure 4b ). dc-produced virus showed a mobility shift of around 3 or 6 kda corresponding to the addition of 1 or 2 complex carbohydrates at positions 67 and 153. this was completely resistant to digestion with endo h, indicating the presence of complex low-mannose carbohydrate. for both insect-and vero-produced virus, envelope protein migrating at 2 or 4 kda higher than the pngase f-digested material indicated, as with dc-produced virus, that envelope had either 1 or 2 added sugars. following endo h digestion a complex pattern of bands was revealed, indicating that some of the added sugars were endo h sensitive and therefore contained .3 terminal mannose residues, whereas others were endo h resistant and contained more heavily processed structures, which is in agreement with a recent report [23] . we conclude from these experiments that in c6/36, vero cells, and dcs both n-linked glycosylation sites can be used but that a single site is used in 50% of cases. envelope from dc-produced virus contains complex, highly processed endo h-resistant carbohydrate. however, in both c6/36 and vero, a proportion of the sugar is high mannose and therefore will allow interaction with dc-sign, which is consistent with the results from lectin blotting shown above. l-sign contains a tandem repeat in its neck region, nterminal to the carbohydrate recognition domain, which can be of variable length between 3 and 9 repeats. l-sign exists as tetramers, and previous studies have suggested that heterogeneity in the tandem repeat region of the l-sign neck region may contribute to severe acute respiratory syndrome (sars) susceptibility by altering the viral env-receptor affinity [24] . to determine whether heterogeneous l-sign neck lengths affect denv infection levels, we examined the effects of a single length repeat and compared it with cells expressing 2 different-length l-sign alleles simultaneously. we examined this by transient transfection, and 293t cells were used for these assays as they can be transiently transfected to a high level. 293t were transfected with l-sign containing 5 or 7 repeats singly or together in equal amounts. l-sign expression was confirmed by surface staining and flow cytometry. equal expression of alternate-length l-sign constructs was also confirmed by western blotting. transfectants were then infected with dengue virus, and infection was monitored by intracellular staining together with an antibody specific to l-sign to reveal transfected cells. all transfectants were equally infected with no reduction in cells expressing both l-sign alleles, suggesting that heterotetrameric l-sign was as effective as homotetrameric l-sign at promoting infection ( figure 5 ). finally, although dc-produced virus cannot reinfect dcs efficiently, we were interested to determine whether dc-produced virus was still able to infect cells by antibody-dependent enhancement (ade), which would allow it to replicate by infection of fc receptor-expressing cells. c6/36-and dc-derived viruses were incubated with increasing levels of pooled convalescent dengue immune serum and subsequently used to infect u937, a monocyte cell line that expresses the fc receptor and which shows relatively low infectivity without the presence of enhancing antibodies. viruses produced in both dcs and insect cells were susceptible to enhancement, over the same range of antibody concentrations, showing that dc-produced virus could exploit ade to replicate in individuals undergoing a secondary dengue infection ( figure 6a ). in a final series of experiments we investigated whether dc-produced virus could be induced to infect primary human dcs by ade ( figure 6b ). dc-produced virus could be enhanced to infection by .20-fold, whereas the already high level of infection of dcs by insectproduced virus was not enhanced further by dengue serum. during natural dengue infection, mosquito saliva containing denv particles is injected into the skin during a blood meal. skin-resident immature dcs have been proposed to be the primary site of infection for insect-derived denv [25] . the primary receptor on dcs for dengue virus is believed to be dc-sign. dc-sign binds n-linked high-mannose oligosaccharides, including glycans with terminal fucose residues that include the blood group lewis x and lewis a eptitopes [26] . l-sign, like dc-sign, binds to intercellular adhesion molecule 3(icam-3) and is thought to establish cellular interactions with icam-3-expressing t cells. l-sign is able to capture a variety of viruses ( [27] [28] [29] [30] [31] . dc-sign and l-sign preferentially bind pyranose sugars, in particular mannose. a study comparing l-sign-and dc-sign-specific ligands using glycan arrays revealed a restricted repertoire of glycan ligands for l-sign, namely, the high-mannose-type n-glycans. in contrast, dc-sign bound to fucosylated glycans in addition to highmannose-type n-glycans [32] . the dengue e protein has 2 potential n-linked glycosylation sites at positions 67 and 153. cryo-electron microscopy reconstructions of dengue virus complexed to soluble dc-sign show interaction with the glycan at position 67 [33] . there have been several reports on the n-linked glycosylation of dengue envelope. some reports suggest the use of both sites, whereas others suggest that only one is used [23, [34] [35] [36] . we show that in c6/36, vero cells, and dcs either one or both sites are used for the dengue 2 serotype 16681. although both glycosylation sites can play important roles in infectivity and viral replication, functional studies have confirmed the importance of asn-67 for infection of dc-sign-expressing cells [33] . wnv e protein contains a single n-linked glycosylation site at position 154 that is absent from some virus strains [37] . both dengue and wnv contain a single n-linked site in prm, and although prm is cleaved by furin during viral maturation, a substantial fraction of uncleaved prm is found on some dengue viruses, particularly that produced in insect cells, and such partially cleaved viruses can still be infectious. for wnv the n-linked site on prm can also mediate interaction with l-sign, and in common with glycan at position 154 this also showed differential specificity for l-sign when expressed in mammalian cell lines, perhaps mediated by the presence of n-acetylglucosamine-terminated structures [29] . the difference in the binding specificities of dengue viruses produced in vero versus primary dendritic cells was somewhat surprising and likely related to the expression of high-mannose moieties in vero. a number of tumor cell lines express high-mannose sugars, and we have previously described the generation of a monoclonal antibody that recognizes a variety of tumor cell types and which binds to high-mannose moieties [38] . the differential affinity of dc-sign for ligands expressed by tumor cell lines versus primary cells has been observed before and led some to speculate that dc-sign may participate in tumor surveillance [39] . there appears to be a further added level of complexity as the glycosylation profiles may vary in a given cell line depending on the exact position of the n-linked site within the polypeptide chain [29] . in humans, l-sign expression is restricted to endothelial cells beneath the subcapsular sinus in lymph nodes [40] , sinusoidal endothelial cells in the liver [27, 41] , alveolar and endothelial cells in the lung [42] , and capillaries in the villous lamina propria of terminal ileum and peyer patches [43] . dengue antigens have been demonstrated in the sinusoidal tissue of the liver and vascular endothelium of the lung and spleen and may provide an explanation for the unique pathology observed in these organs [44, 45] . the accumulation of antigen in l-signexpressing tissue may also result in an increase in localized transinfection in a similar way that l-sign is thought to be responsible for the capture of hcv from the blood and transmission to hepatocytes or in the case of hiv, to cd4 1 t cells [28, 41] . we conclude that skin-resident dcs are a likely target for initial infection by dengue virus injected by the infecting mosquito; however, subsequent dissemination of the virus to monocytes and other cell types will no longer use dc-sign as a primary receptor and may rely in part on a shift to l-sign as the primary lectin receptor. the differential glycosylation of the denv e protein during replication in primary mammalian cells suggests that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution. cross-reacting antibodies enhance dengue virus infection in humans immunodominant t-cell responses to dengue virus ns3 are associated with dhf immunopathological mechanisms in dengue and dengue hemorrhagic fever original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever multiplex cytokine profile from dengue patients: mip-1beta and ifn-gamma as predictive factors for severity high levels of stnfr p75 and tnf alpha in dengue-infected patients monocytes, but not t or b cells, are the principal target cells for dengue virus (dv) infection among human peripheral blood mononuclear cells phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate dc-sign (cd209) mediates dengue virus infection of human dendritic cells the mannose receptor mediates dengue virus infection of macrophages dengue 1 virus binding to human hepatoma hepg2 and simian vero cell surfaces differs dengue virus entry into liver (hepg2) cells is independent of hsp90 and hsp70 heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells identification of grp 78 (bip) as a liver cell expressed receptor element for dengue virus serotype 2 bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a cd14-dependent mechanism dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals dendritic-cell-specific icam3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses a variant in the cd209 promoter is associated with severity of dengue disease clec5a is critical for denguevirus-induced lethal disease a complex interplay among virus, dendritic cells, t cells, and cytokines in dengue virus infections histidine 39 in the dengue virus type 2 m protein has an important role in virus assembly n-linked glycans on dengue viruses grown in mammalian and insect cells homozygous l-sign (clec4m) plays a protective role in sars coronavirus infection human skin langerhans cells are targets of dengue virus infection the dendritic cell-specific c-type lectin dc-sign is a receptor for schistosoma mansoni egg antigens and recognizes the glycan antigen lewis x a dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv-1 infection l-sign (cd209l) and dc-sign (cd209) mediate transinfection of liver cells by hepatitis c virus the location of asparagine-linked glycans on west nile virions controls their interactions with cd209 (dendritic cell-specific icam-3 grabbing nonintegrin) human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus structural basis for distinct ligandbinding and targeting properties of the receptors dc-sign and dc-signr cryo-em reconstruction of dengue virus in complex with the carbohydrate recognition domain of dc-sign the envelope glycoproteins of dengue 1 and dengue 2 viruses grown in mosquito cells differ in their utilization of potential glycosylation sites both e protein glycans adversely affect dengue virus infectivity but are beneficial for virion release essential role of dengue virus envelope protein n glycosylation at asparagine-67 during viral propagation envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 west nile virus strains enhanced immune recognition of cryptic glycan markers in human tumors the b7 homolog butyrophilin btn2a1 is a novel ligand for dc-sign dynamic populations of dendritic cell-specific icam-3 grabbing nonintegrin-positive immature dendritic cells and liver/lymph node-specific icam-3 grabbing nonintegrin-positive endothelial cells in the outer zones of the paracortex of human lymph nodes dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus expression of dc-sign by dendritic cells of intestinal and genital mucosae in humans and rhesus macaques lethal antibody enhancement of dengue disease in mice is prevented by fc modification localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization we thank t. feizi and yan liu for advice and critically reading the manuscript. key: cord-285087-i3nz5bvs authors: heimdal, inger; moe, nina; krokstad, sidsel; christensen, andreas; skanke, lars høsøien; nordbø, svein arne; døllner, henrik title: human coronavirus in hospitalized children with respiratory tract infections: a 9-year population-based study from norway date: 2019-04-15 journal: j infect dis doi: 10.1093/infdis/jiy646 sha: doc_id: 285087 cord_uid: i3nz5bvs background: the burden of human coronavirus (hcov)-associated respiratory tract infections (rtis) in hospitalized children is poorly defined. we studied the occurrence and hospitalization rates of hcov over 9 years. methods: children from sør-trøndelag county, norway, hospitalized with rtis and asymptomatic controls, were prospectively enrolled from 2006 to 2015. nasopharyngeal aspirates were analyzed with semiquantitative polymerase chain reaction (pcr) tests for hcov subtypes oc43, 229e, nl63, and hku1, and 13 other respiratory pathogens. results: hcov was present in 9.1% (313/3458) of all rti episodes: 46.6% oc43, 32.3% nl63, 16.0% hku1, and 5.8% 229e. hospitalization rates for hcov-positive children with lower rtis were 1.5 and 2.8 per 1000 <5 and <1 years of age, respectively. the detection rate among controls was 10.2% (38/373). codetections occurred in 68.1% of the patients and 68.4% of the controls. in a logistic regression analysis, high hcov genomic loads (cycle threshold <28 in pcr analysis) were associated with rtis (odds ratio = 3.12, p = .016) adjusted for relevant factors. conclusions: hcovs occurred in 1 of 10 hospitalized children with rtis and asymptomatic controls. a high hcov genomic load was associated with rti. hcovs are associated with a substantial burden of rtis in need of hospitalization. human coronaviruses (hcov) are commonly detected in nasopharyngeal aspirates (npas) from children with respiratory tract infections (rtis). they were first described in the 1960s as agents of the common cold [1] [2] [3] . recently, hcov has received renewed interest, due to both more sensitive diagnostic methods and increased attention to hcov after the sars outbreak in 2002, resulting in the identification of new hcov subtypes [4] [5] [6] [7] . six species of hcov infect humans: oc43, 229e, sars, nl63, hku1, and mers. while sars and mers are feared for their potential for severe illness and pandemics [8] , the other subtypes have traditionally been associated with milder upper rtis (urtis). in children, however, hcov may also cause lower respiratory tract infections (lrtis) in need of hospitalization [9] [10] [11] [12] , but only a few population-based reports have precisely estimated the risk of hcov-associated hospitalizations [13] [14] [15] [16] . oc43, nl63, 229e, and hku1 are distributed worldwide [17] [18] [19] , and their detection frequency varies [15, [19] [20] [21] . some studies found similar [21] [22] [23] , or even lower [24] , detection rates of hcov among hospitalized children compared to controls. in addition, long-term studies of hcov are rare and most previous studies lack the inclusion of asymptomatic controls. hence, the significance of hcov detections in children with lrtis, the seasonality, and the overall burden of hcov in hospitalized children remains poorly defined. to address this lack of information, we used data from a 9-year prospective population-based survey of children admitted to st olavs hospital in norway. our primary aim was to determine the occurrence of hcov detections in children hospitalized with rti and the hospitalization incidence rates for hcov-associated lrtis in children. to help evaluate the role of hcov in rtis, we compared the presence and genomic loads of hcovs between hospitalized children with rtis and an asymptomatic control group. the study was conducted at the children's department at st olavs hospital in trondheim, norway. the department of pediatrics is the sole pediatric reference center for approximately 59 000 children in sør-trøndelag county. signs of rtis. most patients were enrolled during their stay at the hospital, and some were retrospectively included after hospital discharge. children hospitalized <24 hours were further defined as outpatients. exclusion criteria were: (1) age over 16 years, (2) hospital-acquired rtis, including newborns not discharged from the hospital, (3) ongoing cytostatic and/or immune-suppressive treatment, and (4) a non-rti primary infectious diagnosis. the same child could be included more than once with different rti episodes. children admitted to elective surgery from 2007 to 2015 were recruited monthly as the control group. their caregivers were asked to confirm that they were asymptomatic for rtis the previous 2 weeks. in addition, children undergoing ear, nose, or throat surgery were not included in the control group. caregivers and older children (>12 years) received both oral and written information about the study during their stay at the hospital. written consent to participate was collected from most caregivers. invitation letters were sent to the children and their caregivers after discharge if nasopharyngeal aspirates (npas) had been taken for clinical purposes but they had not been asked to participate in the study due to practical challenges. no response after 2 weeks was regarded as passive consent. all children with rti were examined, diagnosed, and treated by physicians in accordance with the hospital's routines. a physician or member of the study group recorded relevant study information before discharge. for children included after discharge, the data were collected from medical records. participants were divided in 2 main groups: urtis and lrtis. urtis included a diagnosis of rhinosinusitis, pharyngitis, tonsillitis, otitis media, and acute laryngitis without signs of lrtis. a lrti was defined as the presence of dyspnea, signs of lower airway obstruction (wheezing, retractions), and/or a chest roentgenogram with positive results such as infiltrates, atelectasis, and/or air trapping [25] . npas were routinely collected from all children, and placed into a universal virus transport medium without antibiotics. a total of 94% of all npas were sampled during the first 2 days of hospitalization. clinical laboratory technicians also performed in-house taqman real-time polymerase chain reaction (rt-pcr) tests to detect respiratory pathogens [9] . we analyzed for 4 subtypes of hcov: oc43, nl63, 229e, and hku1. thirteen other viruses were also routinely tested for: human adenovirus (hadv), human bocavirus (hbov), human enterovirus (hev), human parechovirus (hpev), human metapneumovirus (hmpv), influenza virus a and b (flua/b), parainfluenza virus (piv) types 1-4, respiratory syncytial virus (rsv), and human rhinovirus (hrv). semiquantitative results were reported based on the cycle threshold value (ct value), with a high genomic load defined as a ct value <28, a medium load defined as a ct value of 28 to <35, and a low load defined as a ct value 35-40. lastly, a ct value >40 was regarded as a negative test. we defined an epidemiologic year from the beginning of august to the end of july in the following year. the annual hospitalization (incidence) rates were estimated based on: (1) hcov detection rates for children hospitalized ≥24 hours with a lrti from our survey, (2) statistics on a lrti diagnosis in need of hospitalization from the hospital's patient administrative system (pas), and (3) population data for sør-trøndelag county provided by statistics norway. in the pas, a lrti was defined as a main diagnosis (icd-10 code) of pneumonia (j10.0, j11.0, j12.0-j12.9, and j13-j15), bronchitis (j20), bronchiolitis (j21), unspecified lrti (j22), and/or asthma exacerbation (j45-j46). we were not able to exclude children hospitalized <24 hours in the pas. data were described with mean, median, interquartile range, or percentages, as appropriate. categorical data were analyzed with a pearson x 2 test or a fisher exact test, and reported with odds ratios (or). continuous and not normally distributed data (age) were tested with a mann-whitney u test. to determine the relationship between hcov and rtis, we conducted unadjusted analyses on relevant predefined variables, including genomic load, season, codetection of severe rti-causing viruses, age, gender, and high-risk condition such as chronic disease and/or premature birth. a multivariate logistic regression was performed on the significant variables extracted from the unadjusted analyses to help determine the independent association of each variable with rtis. the strength of the associations was reported with ors and 95% confidence intervals. for all tests, a p value < .05 was considered statistically significant, and all analyses were performed using ibm spss statistics 24 or sigmaplot 14.0 software. the study was approved by the regional committees for medical during a 9-year period from november 2006 to july 2015, we included 3458 episodes of rtis in hospitalized children and 373 controls. hcovs were detected in 9.1% (313 of 3458) of the episodes in the patient group, and were the fifth most common viruses after hrv (58.2%), rsv (29.3%), hev (11.3%), and piv type 1-4 (9.1%). a total of 39.3% (123 of 313) of the children with a positive hcov infection were outpatients. the detection rate of hcov in the control group was 10.2% (38 of 373), and hcovs were the fourth most commonly detected viruses after hrv (46.9%), hev (24.7%), and piv type 1-4 (10.7%). the prevalence of hcov was equal in the two groups, although detection rates of hcovs in cases and controls differed when stratified by age (supplementary table 1 ). children with rtis were younger (p = .001) and more likely to have a high-risk condition such as chronic diseases and/or premature birth (p = .036) ( table 1) . among the hcov-positive patients, 46.6% (146 of 313) were positive for oc43, 32.3% (101 of 313) for nl63, 16.0% (50 of 313) for hku1, and 5.8% (18 of 313) for 229e. two children were infected with both nl63 and 229e. in the control group, 31.6% (12 of 38) were positive for oc43, 36.8% (14 of 38) for nl63, 18.4% (7 of 38) for hku1, and 15.8% (6 of 38) for 229e. one control was positive for both nl63 and 229e. hcov was detected among patients in all 9 years. the average detection rate was 34.8 per season, with a range from 18 in 2013/2014 to 60 in 2006/2007 ( figure 1) . overall, the majority of hcov detections (71.9%) occurred during the period from november through march, with an average of 51 detections each month. from april to october, the average monthly detection rate was 8.3. during the study period, the highest number of detections per month were in december, and there was only 1 detection in august. the detection pattern of hcovs varied with subtypes and seasons. oc43 was the most frequently detected hcov, and we found a high number of oc43 detections every second season. this was also true for nl63, with the exception of the 2008/2009 season. in high-detection seasons (excluding 2008/2009), the average rate was 26 detections per year for oc43 and 20 for nl63. in low-detection years, the average detection rate was 6 detections per year for oc43 and 4 for nl63 (excluding 2008/2009). during the 9-year period, oc43 was detected in all months of the year, but not in every month in all years. this was not true for nl63, which was never detected from august through october. oc43 was detected before nl63 in each season. hku1 appeared with high numbers every second season, when detection rates for oc43 and nl63 were low. in the high-detection seasons, hku1 had an average detection rate of 12 per season, and 88% of hku1 detections appeared in the months from november to february. 229e was seldom detected, with an average of only 3 detections per year. among hcov-positive patients, 31.9% had a single hcov infection, 41.9% had 2 viruses (including those with both oc43 and 229e), and 26.2% had ≥3 viruses detected (table 2) . for the control group, the corresponding figures were 31.6% with a single hcov detection, 15.8% with 2 viruses detected, and 52.6% with ≥3 viruses detected (supplementary table 2 ). hence, it was not more likely for a single hcov to be detected in the patient group compared to the control group (or = 1.02, p = .96). the most common codetections in the patient group were hrv (24.9%), rsv (23.3%), and hev (16.6%) ( table 2 ). in total, 36.7% had codetection of viruses known to cause severe rtis in need of hospitalization (rsv, hmpv, piv type 1-3, and flua/b). in the control group, the most common codetected viruses were hrv (42.1%), hev (34.2%), and pif 1-4 (21.1%), while the detection of viruses known to cause severe rtis were rare in this group (supplementary table 2 ). hospitalized children with rti and hcov detection were more likely to have a codetection with a severe rti-causing virus (rsv, hmpv, piv type 1-3, and flua/b) than the control group (or = 3.4, p < .004). a total of 43.5% of the children with rtis had a high hcov genomic load, 30.7% had a medium load, and 25.9% had a low load. for the controls, the corresponding numbers were 21.1%, 36.8%, and 42.1%, respectively. hcov-positive children with rtis more often had a high genomic load compared to asymptomatic controls (or = 2.59, p = .010) ( table 3) . among children with rtis, a high genomic load was detected in 67.0% with a single hcov detection, 24.3% with codetections of severe rti-causing viruses, and 41.8% with codetections of other viruses (p < .001). patients with a sole hcov detection were more likely to have a high genomic load of hcov compared to patients with codetections of severe rti-causing viruses (or = 6.3, p < .001). in contrast, there were no significant differences in genomic load between controls with single detections and controls with codetections (or = 1.11, p = .722). we investigated the relationship between hcov detection and rtis in consideration of other factors in a multiple regression model. a high genomic load of hcov was independently associated with rtis (or = 3.12, p = .016), and adjusted for the codetection of severe rti-causing viruses, age, gender, and high-risk conditions (table 3) . a total of 60.7% (190 of 313) of the hcov-positive children with rtis were hospitalized for more than 24 hours. in this group, 73.7% were diagnosed with a lrti. from november 2006 to december 2015, the average yearly hospitalization rate of children with hcov and lrtis was 1.5 per 1000 children younger than 5 years, and 2.8 per 1000 children younger than 1 year ( table 4 ). the yearly hospitalization rates ranged from 0.5 to 3.2 per 1000 children younger than 5 years and 0.9 to 5.5 per 1000 children younger than 1 year (table 4 ). it is difficult to determine the real burden of hcov due to influencing factors such as codetections and genomic loads estimations. we therefore also calculated separate hospitalization rates for the detection of oc43 or nl63, including single infections and the detection of high genomic loads (supplementary table 3 ). for children younger than 5 years, approximately one-third of the oc43 and nl63 detections had a high genomic load, with one-fifth single infections. thus, the hospitalization rate for children younger than 5 years with a high genomic load of oc43 was 3.0 per 10 000, and 1.6 for a high genomic load of nl63. hcov occurred in approximately 1 of 10 hospitalized children with rtis and controls during the 9-year period. the 4 investigated hcov subtypes had different detection rates and different seasonal distributions. although more than two-thirds in both groups were codetected with other viruses, our data support that hcov leads to a substantial burden of rtis in need of hospitalization. the average hospitalization rate of children with lrtis and hcov detection was 1.5 per 1000 children <5 years old, and 2.8 per 1000 children <1 year old. almost half of all hcov detections were oc43 and one-third were nl63, whereas hku1 and 229e appeared infrequently . all hcov subtypes were primarily detected in winter, from november through march, with some detections throughout the year. our data verify findings from studies in both the us and europe, which report the highest detection rates in the winter and spring seasons [10, 17, 18, [26] [27] [28] [29] . others have reported that in asia, hcov may peak during all seasons of the year [19, 22, 30, 31] . consequently, the seasonal circulation of hcovs is possibly dissimilar in temperate and tropical regions [30] . in our study, oc43 and nl63 appeared in winter epidemics with clear biennial peaks, whereas hku1 appeared in alternating winter outbreaks with low oc43 and nl63 activity. hku1 was rarely detected outside outbreaks, and 229e only appeared sporadically. dijkman et al diagnosed hcov infections by serology, and observed a similar pattern [20] . on this basis, the authors hypothesized that cross-reacting antibodies may be induced during oc43 and nl63 infections, thereby explaining why outbreaks of other hcov subtypes seldom occur at the same time, and the lower detection rates of hku1 and 229e [20] . using pcr, the average detection rate of hcov in hospitalized children with rtis in the present study was 9.1%, varying with a factor of 3.3 between seasons. previous studies of a shorter duration have reported hcov detection rates with larger variations from 1% to 15% [15, [19] [20] [21] . we also found that the control group had a detection rate of similar magnitude (10.2%), as has been reported before [21, 23] . this finding may question whether hcovs are the causal pathogen of rtis when hcovs are detected in nasopharyngeal aspirates. another prominent finding was the high viral codetection rates in more than two-thirds of the infected children, as well as in the controls. previously, somewhat lower codetection rates have been reported [10, 17, 18, 22, 30] , but our finding might simply reflect the high number of virus types analyzed in our study. in the current study, children with rtis were more likely to have a codetection of rsv and other viruses known for their potential to cause severe rtis, whereas controls more often had a codetection of less-pathogenic viruses. this finding might also question whether hcov caused the rtis in this study. however, we made 3 observations on the basis of genomic loads estimations, which support that hcov causes rtis among hospitalized children. first, assuming that a high genomic load more than a low genomic load usually indicates an active infection, we found that children with rtis were more likely to have a high hcov genomic load than the controls. second, children with a single hcov infection were more likely to have a higher genomic load (67%) compared to those with a codetection of rhinovirus and other less-pathogenic viruses (41.8%), and those with codetections of rsv and other viruses with the potential to cause severe rtis (24.3%). van der hoek et al have published similar findings for hcov subtype nl63 [16] . finally, a high genomic load of hcov was independently associated with rtis in multiple logistic regression analyses, adjusted for severe viral codetections and other confounder variables. in the control group, hcov genomic load did not differ between those with single and codetections. on the basis of this observation, we suggest that detections of low levels of nucleic acids from hcov in the controls are more likely to be traces from previous infections rather than an expression of ongoing asymptomatic infections, for which we would have expected higher hcov loads to be present. unfortunately, serologic analyses were not available and cultures are not helpful, as hcov does not grow in conventional cell lines. the average hospitalization rate of children with lrtis and hcov detection was 1.5 per 1000 children <5 years old. children <1 year had a higher rate of 2.8 per 1000 children. these figures may be an overestimation due to the possible contribution of rsv and other codetected viruses, but because the exact causal contribution of each codetected virus is difficult to ascertain, we decided to report incidence rates based on the total number of hcov-positive samples. our incidence rates were calculated only for lrtis, because urtis are underreported in our hospital patient administrative system. previously, only a few studies have reported hospitalization rates in hcov-infected children, and these data are difficult to compare with ours [13] [14] [15] [16] . on the basis of calculations in our dataset, we have recently published a somewhat higher hospitalization rate for hmpv compared to hcov (1.9 lrtis per 1000 children <5 years old), and rsv appeared 7 times more often (10.5 lrtis per 1000 children <5 years old) [25] . to give a more precise estimation of the burden of hcov, we also reported on the lrti hospitalization rates for detections with high genomic loads and single detections of oc43 and nl63. however, these figures may be underestimations because the presence of other viruses or a low hcov load cannot exclude a possible causal contribution by hcovs. for children younger than 5 years, the hospitalization rates for a high genomic load of oc43 and nl63 infections were 3.0 and 1.6 per 10 000 children, respectively. in a comparable study over 5 consecutive years, van der hoek et al reported a somewhat higher hospitalization rate for nl63 (2.2 per 10 000 children <3 years old), even when they defined a nl63 infection as a single nl63 detection with high genomic loads (>10 000 copies/ml in nasopharyngeal aspirate) [16] . the strengths of the present study are the population basis and prospective enrollment of children with infections from the same county in norway over a 9-year period. it is also an advantage that during the entire study period we have used the same pcr tests for a high number of virus types, and the use of semiquantitative genomic loads estimations. although the cross-sectional design is not optimal, the inclusion of asymptomatic controls allowed us to study causal relations between hcov and infection. the nasal swabs from the control group were sampled during anesthesia, which might have resulted in higher viral detection rates. age and gender differed among cases and controls, but was controlled for in the analyses. our 9-year population-based study shows that the hcov subtypes oc43, nl63, 229e, and hku1 appear in hospitalized norwegian children with characteristic outbreak patterns, primarily in the winter. although detection rates were equal between children with rtis and controls, our data support that hcovs contribute to respiratory tract infections in hospitalized children. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel 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in queensland children with acute respiratory tract illnesses in 2004 we acknowledge the contributions of dr anne-gro wesenberg rognlien, oslo university hospital, oslo, norway, research nurses ragnhild widerø, stine saus, wenche håhjem, barbro medås, siv anita myhre, and dr per eirik haereid †, all at the department of pediatrics, and the biomedical scientists at the department of medical microbiology, st olavs university hospital.disclaimer. the financing institutions had no role in the design or conduct of the study, in the collection, management, analysis or interpretation of the data; or in the preparation of the manuscript. all findings are the result of independent contributions of the authors. the decision to publish the data was made solely by the authors, who are fully responsible for all contents of the manuscript.financial support. this work was supported by the central norway regional health authority (grant number 96987/2008); trondheim university hospital (grant number 13/8985-119); and the student research program at the norwegian university of science and technology. key: cord-288072-42sx52tn authors: monto, arnold s.; lim, sook k. title: the tecumseh study of respiratory illness. vi. frequency of and relationship between outbreaks of coronavims infection date: 1974-03-17 journal: j infect dis doi: 10.1093/infdis/129.3.271 sha: doc_id: 288072 cord_uid: 42sx52tn specimens of blood collected in tecumseh, michigan over a four-year period were studied for rise in antibody titer against coronavirus oc43. peaks of infection were found in the winter and spring of 1966, 1968, and 1969; at other times, infections occurred sporadically. all age groups were involved, especially the very young. rises in titer by cf and by hai tests frequently did not occur together in the same individual. agreement between the two tests was better in 1966 and 1969 than in the other years. a portion of the paired specimens showing rises in cf and/ or hai titer was tested by neutralization. rises in neutralizing antibody were usually found in pairs collected in 1966 and 1969 but not in those collected in 1967 and 1968. the infecting viruses in 1966 and 1969 thus appeared more closely related to oc43 than did those in 1967 and 1968. coronaviruses are significantly involved in respiratory infections of man, especially in those occurring in the colder months of the year [1] [2] [3] [4] . however, neither the total number of different coronaviruses nor their relative importance is known. understanding of the behavior of these viruses has been hampered greatly by the difficulties in propagation encountered in the laboratory. only one coronavirus, 229e, can be isolated in cell culture, and this is difficult [5] . a major contribution has been the adaptation of viruses originally isolated in organ culture to animals or cell cultures [6, 7] . several serologic tests have thus been made possible, and infections can now be identified by rise in antibody titer [8, 9] . as part of the study of respiratory infections in tecumseh, michigan, specimens of blood were collected on a regular basis from families under surveillance [10] . by testing of specimens collected in early 1967, a large-scale outbreak of infection with coronavirus 229e was detected [11] . all segments of the population were involved, and infection was significantly associated with respi-ratory illness. specimens collected over four years have now been tested for infection with coronavirus oc43. the present report describes the occurrence of coronavirus infections during this period as determined by different serologic techniques. population studied and processing of specimens. details of the surveillance methods used in tecumseh have been described [12] . families for study were selected randomly from those with children whose parents were under 46 years of age. each family was followed for one year. specimens were obtained for isolation of virus when a respiratory illness was reported within two days of onset [13] . samples of blood were collected at time of recruitment, six months later, and at the end of the year of surveillance [10] . because recruitment of families was staggered, specimens of blood were collected during all months of the year. blood was drawn by venipuncture from individuals of approximately five years of age and older, and the specimens of serum were stored at -20 c. the specimens were collected from younger children on filter paper disks by finger stick. at least four disks were obtained at one time, and they were dried and stored at 4 c until use. for cf and hal tests, two disks were placed in 0.5 ml of veronal-buflered saline, and the blood was eluted for 18 hr at 4 c. thereafter, the fluid was completely expressed from the disks and inactivated at 56 c for 30 min. the eluate was centrifuged for 1 hr at 650 g, and the supernatant was considered to represent a 1:4 dilution. the specimens of serum were inactivated at 56 c for 30 min before dilution for cf, hal, or neutralization tests. a ntigens used. oc43 antigen was prepared from a mouse brain-adapted strain obtained through the courtesy of mr. h. s. kaye, center for disease control. the seed was propagated by intracerebral inoculation of suckling mice. antigen was prepared as a 10% suspension, in phosphatebuffered saline for hal tests, and in tryptose-phosphate broth for neutralization tests. for cf tests, antigen was prepared as a 20% suspension in veronal-buffered saline. the identity of each pool of virus was confirmed by cf with a homologous and a hyperimmune antiserum to murine hepatitis virus. the seed of 229e virus was originally obtained from dr. dorothy hamre and was passed serially in wi-38 cell culture. antigen for cf tests was prepared by inoculation of wi-38 bottles or tubes with undiluted virus. after incubation for 34-38 hr at 33 c the cultures were frozen and thawed three times, fluids were centrifuged, and the supernatant was used as antigen [11] . serologic tests. cf and hal tests were performed on all specimens of serum collected from members of 269 families. these families were selected randomly from within each of the four years of study so that all periods would be represented. the standard cf test was performed by a microplate technique with sheep erythrocytes, 4 units of antigen, and 1.8 units of complement. the hal test was performed in microplates by the method of kaye et al. [8] . sera were serially diluted in phosphate buffered saline from the initial 1: 4 dilution, and 4 units of antigen were added. after incubation for 30 min at 24 c 0.4% chick erythrocytes were added. the test was read after incubation for an additional 1 hr at 24 c. for both cf and hal, all sera from the same individual were tested simultaneously. a significant rise in titer was considered to have occurred if antibody appeared in a 1: 8 dilution when prior specimens did not have demonstrable antibody at a 1: 4 dilution. a fourfold rise from a previous antibody titer was also considered significant. approximately 40% of serum pairs demonstrating a significant rise in antibody titer by cf and hal were also tested by neutralization [9] . the neutralization tests were performed in cultures of bs-c-1 cells. cells were grown in eagle's minimal essential medium (mem) made in hanks' salts with 10% fetal bovine serum. before use, the cultures were washed three times with balanced salt solution, and they were maintained on serum-free mem in earle's salts. virus was used at a dose of 100-320 tcid50/0.1 ml. after inactivation at 56 c for 30 min the sera were serially diluted. to 0.3 ml of diluted sera, 0.3 ml of the appropriate dilution of virus was added, and the mixture was incubated at 24 c for 1 hr. thereafter 0.2 ml of each mixture of serum and virus was inoculated into each of two cell-culture tubes. the tubes were placed in a roller drum and incubated at 33 c. after four days, rat erythrocytes were washed three times, and 0.2 ml of a 0.4% solution was added to each cell-culture tube. tubes were examined microscopically after 45 min at 24 c, and end points were determined based on the presence of virus-specific hemadsorption. all specimens of serum collected from members of 269 families were studied by cf and hal for rise in titer of antibody to coronavirus oc43. these specimens had been collected from november 1965 to june 1969. in nearly all cases, three specimens were obtained from each individual during each period of surveillance. to determine the temporal occurrence of infection with oc43related viruses, the three specimens collected from each person were divided into two component pairs, each spanning a six-month period. the time of each pair was identified by the month of collection of the second serum of that pair. results of the serologic tests are shown in figure 1. rises in cf and hal titers frequently were independent of each other. therefore, each rise was counted without regard to the presence or absence of the other and is indicated as a separate curve in the figure. bars indicate the percentage of total specimens tested in which the rises in titer by cf and hal occurred together. it has been reported that the principal periods of transmission of coronavirus are in the winter and early spring [2, 3] . this pattern could be seen when the cf and hal curves for oc43 were examined. frequent infections were detected by cf and hal in sera collected during the sixas one means of exploring the divergence in serologic results. results are given in table 1. the overall rate of infection for each age group was calculated by determining the total number of individuals who showed a rise in antibody titer by cf and/or hal. the proportion of these infected individuals who had a rise in cf antibody was then calculated as was the proportion of infected individuals with a rise in hal antibody. since the intent was determination of the relative frequency of rise in titer detected by each test, persons with increases in both cf and hal titer were counted in both categories. as shown in table 1, age did not appear to be a factor in determining the type of response. at all ages below 40 years, rises in titer were more frequently detected by hal than by cf, but the difference was often not great. only among individuals 40 years and older was this pattern reversed slightly. the total rates of infection (table 1) fell gradually as age increased, but not as precipitously as with other respiratory pathogens ' [10, 13] . a high annual rate of infection was observed among all adults studied. study of specificity of antibody response. agreement between the hal and cf tests was better in the first part of 1966 and in early 1969 than at other times, including the peak of infections in 1968. it seemed possible that agents similar to oc43 might have been responsible for the 1966 and 1969 outbreaks and more distantly related agents for activity at other times. such a hypothesis could be tested by evaluation of the specificity of observed rises in titer through use of the recently described neutralization test [9] . approximately thereafter the rates dropped, showed a suggestion of an increase in march-april 1967, the period of the 229e outbreak [11] , and then remained low until march-april 1968. this peak of infection was detected by both serologic tests with the rate by cf twice that by hal. after this point the rates dropped only to peak in 1969, again in march-april. during this outbreak rates of incidence were very high, with considerably more infections detected by hal than by cf. during the four years studied, 156 paired specimens showed a rise in titer by cf and/or hal; of these, only 37 (23.7%) had a rise in titer by both techniques. this divergence in serologic results, which has been noted in previous studies of oc43 virus [4] , was not uniformly spread over the full period. as shown by the relative height of the bars in figure 1 , agreement between the two serologic tests was an infrequent occurrence except in may-june 1966 and again in 1969. during these periods the percentage of positive paired specimens in which the rises occurred together was 39.2 %, but in the rest of the four years it was 9.8%. even during march-april 1968, when a peak of infections occurred, there was still little correlation between the two tests. age-specific occurrence of infection. results of past investigations have suggested that age may be a factor in producing differences in sensitivity of the cf and hal tests; cf was found to be most sensitive when the population studied was composed of adults [9] , and hal was most sensitive when the population was made up exclusively of children [4] . the ages of individuals who exhibited the rises in titer were therefore examined, showed rises in antibody by cf and hal were tested by neutralization. they were selected randomly from within individual respiratory years so that all periods would be represented; the respiratory year has been defined as extending from september to the following august and is identified by the date in which most of the year fell. results are given in table 2. period of collection was the most important predictor of presence or absence of rise in titer of neutralizing antibody. since the 1966 and 1969 periods, on the one hand, and the 1967 and 1968 periods, on the other, resembled each other, they have been combined. in serum pairs in which the second specimen was collected in 1967 and 1968, and in which there was a rise in cf titer alone, no rises in titers of neutralizing antibody were found. in the pairs from this period with a rise in hal antibody, there was an occasional rise in titer of neutralizing antibody, and among the few pairs showing an increase in titer by both cf and hal, 28.6% had a rise in titer by neutralization. the situation with the 1966 and 1969 specimens was quite different. the specimens with rises in cf or hal titer both showed 66.7% agreement with the neutralization test. all specimens with a combined cf and hal increase also had a rise in titer by neutralization. thus, the 1966 and 1969 period was distinct from the 1967 and 1968 period in having not only more frequent combined cf and hal rises in antibody titer, but also because all rises in antibody titer were more likely associated with rises in neutralization titer. these findings confirm that the 1966 and 1969 outbreaks were caused by viruses more closely related to oc43 than the viruses involved in infections during the 1967 and 1968 period. during 1967, a large-scale outbreak of 229e monto and lim virus was detected in tecumseh [11] . it is possible that some of the rises in titer of antibody to oc43 during 1967 might represent cross-reactions from an actual infection with 229e. therefore, the 21 sera with hal or cf rises in titer of oc43 antibody in 1967 were tested by cf against a 229e antigen. of those tested, only two (9.5%) showed a rise in titer. since this cf test is sensitive when used with sera collected close in time to infection, it is probable that the agent involved in producing the rises in oc43 titer during 1967 was other than 229e. study of the 1969 outbreak. the outbreak detected serologically in late 1968 and the first part of 1969 not only was large scale, but was also probably caused by an agent related to oc43. to define better the occurrence of infection during this period, serologic data from the 106 families whose second and third specimens of blood were collected during the period from august 1968 to june 1969 were examined separately. to complete the study of these families the blood specimens collected on filter paper disks from the younger children were also tested by cf and hal against oc43 antibody. persons were identified as infected if they had a significant increase in antibody titer by cf or hal test, since during the period both were equally likely to agree with the neutralization test. the results, shown in table 3, calculated on the basis of person-years of observation, indicate the great frequency of infection with the agent. those under five years of age showed the highest rates of infection, and the rates fell slightly as the age of children increased. among adults, the 30-39-year age group had the highest rates. the very high rate of infection in adults was similar to that observed in the commutable 3 . age-specific occurrence of infection with coronavitus oc43 during 1968-1969 as determined by cf and hal tests. * number of persons with rise in antibody titer by cf alone, hal alone, or both cf and hal. nity during the 229e outbreak of 1967 [11] . howevert the marked involvement of children under age five was not noted at that time nor has it been appreciated in other studies of coronavirus infections. relative frequency of cf and hal response among these small children was like that of the rest of the population during this period t with approximately two and a half times as many rises in titer detected by hal compared with those detected by cf. familial aggregation of infection was frequent, as has been observed with other respiratory viruses [10] . as many as four infections were observed in a single family. during the 229e outbreak in 1967, in spite of the high rate of infection and the fact that infection was significantly associated with illness, no peak in incidence of illness could be identified [11] . similarly, in the present outbreak no peak of incidence of illness could be identified in february or march 1969, the likely period of peak dissemination of virus. investigations on the behavior in populations of respiratory pathogens ideally use as methods both isolation of infecting viruses and testing of sera. an isolate can be used as antigen in the serologic procedures and rises in antibody titer related directly to the activity of that virus. unfortunately, with most of the coronaviruses isolation is difficult [14, 15] . seroepidemiology is thus the only technique available for study of infections caused by these agents in large numbers of people. the relation between the virus chosen for antigen and the virus actually infecting the population can thus be determined only by inference. it is known that rises in titer of heterologous antibody do occur [16] [17] [18] ; however, the exact nature and frequency of these cross-reactions have not been determined. only limited numbers of natural infections have been studied by both isolation and serology, a situation in which the critical observations on antibody response should be possible; thus basic gaps in essential knowledge exist. use of parallel cf and hal procedures was based on the possibility that heterologous crossreactions might be encountered; it seemed more likely that there would be agreement between the two tests when the infecting organism was closely related to oc43. disagreement could be a result 275 of either lack of close relation between the infecting organism and oc43 or the fact that one of the serologic tests was more sensitive than the other. however, in terms of differences in agreement from year to year, the former factor was operative. the viruses in circulation in 1966 and 1969 were clearly more closely related to oc43 than those in circulation in 1968, a difference that would not have been detected had both cf and hal not been used. neutralization tests confirmed the difference, and indicated that in the individual case during a period of prevalence of agents related to oc43, even if cf and hal do not agree, it is likely that an infection with the agent actually did occur. it may also be speculated that some minor differences existed between the 1966 and 1969 viruses, since cf was more frequently positive in the former period and hal in the latter. the annual periodic pattern of coronavirus infections in general is clear from the tecumseh data when the 1967 229e outbreak is included, and is in agreement with the findings of others [2, 3, 8] . the outbreaks take place in the period of late winter through early spring, with sporadic infections at other times of the year. a longer cycle for a specific virus type can also be deduced from the data. the period between the oc43-like outbreaks was three years. periods of high incidence of 229e infections have also been reported to recur on a two-or three-year cycle [19] . the repetitive activity of specific coronaviruses and the fact that they cause large-scale outbreaks of infection is in marked contrast to the situation with the rhinoviruses. cycling of these agents occurs but not on any sort of a regular or predictable basis. however, the number of infections caused at anyone time is low, and many agents take part in a single peak of illness [20] . the fact that coronaviruses individually seem to cycle more regularly and cause many infections suggests that there are a reasonably small number of these agents in existence. this has positive implications in terms of control. however, reinfection with coronaviruses is a very frequent event. in the present study, 81.5% of the infections studied by the neutralization tests occurred despite prior neutralizing antibody. others have also noted that antibody is usually present before a rise in titer and have thus called into question the protective value of this circulating neutralizing antibody [9, 19] . the occurrence of reinfections explains, in part, the age-specific patterns observed with oc43 infection in tecumseh. antibody is acquired early in life, as shown by high incidence of infection in children less than five years of age. subsequently, reinfection is frequent in older children and in adults. this frequency of infection in adults may explain the relation of coronaviruses to exacerbations of chronic bronchitis [21] . the proportion of the oc43 infections detected serologically that were expressed as clinical illness is difficult to assess. kaye et al. found that 47.3% of infections detected in children by hal were accompanied by illness [4] . if such a ratio prevailed in tecumseh in the 1969 outbreak, it would mean that 12.1 % of all individuals experienced a symptomatic infection during the course of a single season. clearly the coronaviruses make a major contribution to the overall burden of respiratory illnesses. although illnesses caused appear to be mild, their frequency and possible relation to chronic respiratory disease makes further understanding of their behavior essential. isolation from man of "avian infectious bronchitis virus-like" viruses (coronaviruses) similar to 229e virus, with some epidemiological observations coronavirus infections in working adults: eight-year study with 229e and oc 43 seroepidemiologic studies of coronavirus infection in adults and children seroepidemiologic survey of coronavirus (strain oc 43) related infections in a children's population a new virus isolated from the human respiratory tract growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease the adaptation of two human corona-monto and lim virus strains (oc38 and oc43) to growth in cell monolayers some characteristics of hemagglutination of certain strains of "ibvlike" virus hemadsorption by coronavirus strain oc43 the tecumseh study of respiratory illness. iii. incidence and periodicity of respiratory syncytial virus and mycoplasma pneumoniae infections community-wide outbreak of infection with a 229e-like coronavirus in tecumseh, michigan the tecumseh study of respiratory illness. i. plan of study and observations on syndromes of acute respiratory disease the tecumseh study of respiratory illness. ii. patterns of occurrence of infection with respiratory pathogens, 1965-1969 recovery in tracheal organ cultures of novel viruses from patients with respiratory disease cultivation of a novel type of common-cold virus in organ cultures coronavirus antibody titers in sera of healthy adults and experimentally infected volunteers antigenic relationships among the coronaviruses of man and between human and animal coronaviruses antigenic relationships amongst coronaviruses virologic studies of acute respiratory disease in young adults. v. coronavirus 229e infections during six years of surveillance the tecumseh study of respiratory illness. iv. prevalence of rhinovirus serotypes, 1966-1969 coronavirus infections in exacerbations of chronic bronchitis key: cord-012509-887xlllb authors: roy-ghanta, sumita; van der most, robbert; li, ping; vaughn, david w. title: responses to a(h1n1)pdm09 influenza vaccines in participants previously vaccinated with seasonal influenza vaccine: a randomized, observer-blind, controlled study date: 2014-11-01 journal: j infect dis doi: 10.1093/infdis/jiu284 sha: doc_id: 12509 cord_uid: 887xlllb background. prior receipt of a trivalent seasonal influenza vaccine (tiv) can affect hemagglutination inhibition (hi) antibody responses to pandemic influenza vaccines. we investigated the effect of tiv priming on humoral responses to as03-adjuvanted and nonadjuvanted a(h1n1)pdm09 vaccines, the role of as03 on cell-mediated immune (cmi) responses, and vaccine safety. methods. healthy adults (aged 19–40 years) were randomized 1:1:1:1 to receive tiv or saline followed 4 months later by 2 doses, 3 weeks apart, of adjuvanted or nonadjuvanted a(h1n1)pdm09 vaccine and followed up to study end (day 507). preand postvaccination responses of hi and neutralizing antibody, cd4(+)/cd8(+) t cells, memory b cells, and plasmablasts were assessed. results. ninety-nine of the 133 participants enrolled completed the study. no vaccine-related serious adverse events were recorded. in tiv-primed participants, a(h1n1)pdm09-specific antibody and cd4(+) t-cell and memory b-cell responses to the pandemic vaccine tended to be diminished. vaccine adjuvantation led to increased responses of vaccine-homologous and -heterologous hi and neutralizing antibodies and cd4(+) t cells, homologous memory b cells, and plasmablasts. conclusions. in healthy adults, prior tiv administration decreased humoral and cmi responses to a(h1n1)pdm09 vaccine. adjuvantation of a(h1n1)pdm09 antigen helped to overcome immune interference between the influenza vaccines. no safety concerns were observed. registration. clinical trials.gov identifier nct00707967. from the recognized onset of an h1n1 pandemic in 2009 until its end in august 2010, the swine-origin a/ california/7/2009 [a(h1n1)pdm09] virus caused more than 600 000 laboratory-confirmed infections and 18 449 deaths [1, 2] , though estimates suggest a death toll of 15 times that amount [3] . in response, a number of a(h1n1)pdm09 vaccines were developed [4] [5] [6] , several of which were formulated with an adjuvant to enhance immunogenicity and reduce the antigen dose [7] [8] [9] . influenza a(h1n1)pdm09 vaccine clinical studies have shown that the prevaccination serostatus of the studied population is an important determinant for the vaccine's immunogenicity [5, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . specifically, results from a number of trials have suggested that prior receipt of a trivalent seasonal influenza vaccine (tiv) or another nonadjuvanted influenza vaccine can affect the hemagglutination inhibition (hi) antibody responses to adjuvanted [8, [10] [11] [12] [13] [14] and nonadjuvanted [5, 9, [15] [16] [17] [18] [19] a(h1n1)pdm09 or h5n1 [20, 21] vaccines. several authors [16, 22] have suggested that this effect of immune interference is akin to the original antigenic sin mechanism [23] , in which sequential exposure to closely related viruses can lead to diminished antibody responses to the novel antigens in the strain of the last exposure. however, the relevance of this concept for sequential influenza vaccinations is subject to debate [24, 25] . in mice, it has been shown that such immune interference effects may be circumvented by adjuvantation of the priming vaccine [26] . this is in agreement with recent clinical studies that show that adjuvants can enhance cd4 + t-cell responses [9, 14, 27] . increased cd4 + t-cell responses, in turn, could promote bcell adaptation to antigens that are less related to the specificities in the memory b-cell pool, such as those present in a pandemic influenza vaccine [28, 29] . thus, cd4 + t-cell responses could play a role in shaping an immune response that is better prepared to respond to the subsequent vaccination. indeed, clinical studies have shown that adjuvant system 03 (as03) [30] enhanced cd4 + t-cell and humoral responses to a(h1n1) pdm09 [9, 14] and h5n1 [27] antigens. our aim in this study was to investigate the effect of priming with tiv on subsequent hi and neutralizing antibody (nab) responses to adjuvanted and nonadjuvanted a(h1n1)pdm09 vaccines. we also evaluated the role of as03 on the frequency and/or phenotypes of cell-mediated immune (cmi) responses in terms of t cells, memory b cells, and plasmablasts, as well as vaccine safety. we selected a study population (aged 19-40 years) for whom potential immunosenescence effects would not be expected. this phase 1/2 randomized observer-blind study (clinicaltrials. gov; nct01059617) was conducted from february 2010 through january 2011 at 3 study centers in the united states, after approval by an independent local ethics committee. the study was undertaken in accordance with the helsinki declaration and good clinical practices. all participants provided written consent before entering the study. using an internet-based algorithm, eligible participants were randomized 1:1:1:1 to 4 parallel groups to receive either tiv followed by 2 doses of adjuvanted (group a) or nonadjuvanted (group b) a(h1n1)pdm09 vaccine, or placebo (saline) followed by 2 doses of adjuvanted (group c) or nonadjuvanted (group d) a(h1n1)pdm09 vaccine ( figure 1 ). vaccine doses were administered on day 0 (tiv/placebo; dose 1), followed 4 months later by 2 doses of a(h1n1)pdm09 vaccine (doses 2 and 3) given at 3-week intervals (days 122 and 143). participants were followed for 364 days after dose 3 (day 507). blood samples for immunogenicity and safety evaluations were taken before vaccination (days 0, 122, and 143); 1, 2, and 3 weeks post dose 1 (days 7, 14, and 21); 3, 7, and 14 days post dose 2 (days 125, 129, and 136); and 1, 2, 3, and 23 weeks post dose 3 (days 150, 157, 164, and 304, respectively). the primary objective was to describe the homologous and heterologous hi antibody responses following adjuvanted or nonadjuvanted a(h1n1)pdm09 vaccination of individuals vaccinated previously with tiv or placebo. secondary objectives were to describe the homologous and heterologous nab and cmi responses to the pandemic vaccine virus, as well as vaccine safety across the study period. healthy participants (male and female) aged 19-40 years were enrolled. exclusion criteria were as follows: previous administration of any influenza vaccine including any a/california/7/ 2009 (h1n1)v-like virus vaccine, administration of any vaccine within 30 days before first vaccination, use of investigational or nonregistered products, confirmed or suspected immunosuppression, receipt of immunoglobulins or blood products within 9 months preceding the study, and known or suspected allergy to any of the vaccine constituents. the a(h1n1)pdm09 vaccine was a monovalent split-virion influenza vaccine that contained ha of a/california/7/2009 (h1n1) nymc x-179a mixed with either pbs to contain 15 µg ha/0.5-ml dose (groups b and d) or with as03 a to contain 3.75 µg ha/0.5-ml dose (groups a and c). as03 a (henceforth referred to as as03) is an oil-in-water emulsion-based adjuvant system that contains 11.86 mg tocopherol/dose. vaccines and placebo were administered intramuscularly (deltoid) in the nondominant arm (doses 1 and 2) or dominant arm (dose 3). immunogenicity evaluations were performed at each bloodsampling time point with the exception of visit days 14 and 125 for nab responses and visit day 125 for hi responses. neutralization and hi assays were performed as described previously [12, 31] . hi responses were assessed using seropositivity rates ( percentages of participants with titer ≥1:10), seroconversion rates (scrs; percentage of participants with prevaccination titer <1:10 and postvaccination titer ≥1:40 or prevaccination titer >1:10 and ≥4-fold increase in postvaccination titer, with day 122 considered as prevaccination for day 304), seroprotection rates (sprs; percentage of participants with titer ≥1:40), and geometric mean fold rises (gmfrs; geometric mean of the within-participant ratios of postvaccination reciprocal titer to prevaccination reciprocal titer at days 0 or 122). assessment of hi responses was based on the european medicines agency, committee for medicinal products for human use (chmp) guidance targets for pandemic influenza vaccines in adults [32] (point estimates, scr >40%; spr >70%; gmfr >2.5) and the center for biologics evaluation and research (cber) licensure criteria [33] [lower limits of 95% confidence interval (ci) ≥40% for scr and ≥70% for spr]. seropositivity rate for nab responses was the percentage of participants with titer ≥1:28. t-cell frequencies were evaluated using intracellular cytokine staining (ics) and flow cytometry, as described previously [9] . b-cell frequencies were assessed by memory b-cell enzymelinked immunosorbent spot (elispot), as described previously [27] . frequencies of plasmablasts (cd27 + /cd38 + b cells) were evaluated following a previously described method [24] and expressed as percentages of cd3 − /cd20 lo /cd19 + b cells. solicited local and general adverse events (aes) were recorded during the 7-day period post vaccination (days 0-6, 122-128, 143-149); unsolicited aes during the 84-day, 42-day, and 21day periods following doses 1, 2 and 3, respectively; and selected hematological and biochemical clinical laboratory parameter abnormalities on days 0, 21, 122, 164, and 304. the occurrence of/relationship to vaccination of medically attended adverse events (maes), potential immune-mediated diseases (pimds), serious aes (saes), and withdrawals due to aes were assessed up to day 507. descriptive analyses were performed. immunogenicity analyses were performed on the according-to-protocol cohorts for immunogenicity (atp-i) at days 164 and 304. geometric mean titers (gmts), scrs, sprs, and gmfrs for hi antibodies and gmts and scrs for nab responses were tabulated with 95% cis. safety analysis was based on the total vaccinated cohort (tvc). incidences of solicited and unsolicited aes after each vaccination were tabulated with 95% cis. overall, 171 participants were screened, of whom 133 (77.8%) were vaccinated and included in the tvc. ninety-nine patients completed the study at day 507 ( figure 1 ). withdrawals from the study (24 participants) were mainly due to loss to followup. demographics were comparable across groups. in the tvc, the median age was 29 years (range, 19-40 as per protocol), the male-to-female ratio was 45.9%:54.1%, and the majority of participants were of white-caucasian/european (70.7%) or african (27.1%) heritages. after tiv or placebo administration (dose 1; days 0-122), gmts of a(h1n1)pdm09-specific hi responses in both groups remained low (ranges, 12.5-19.9 and 13.3-13.9, respectively), and chmp or cber criteria were not met (supplementary table 1) . at day 122, the day of the first a(h1n1)pdm09 vaccination (dose 2), a(h1n1)pdm09-specific hi antibody responses (gmts) ranged from 12.5 to 16.1 across groups (table 1 and figure 2a ). two weeks later (day 136), gmts had increased substantially (range gmfrs, 24-40 across groups) and, when comparing the adjuvanted groups a and c as well as the nonadjuvanted groups b and d, tended to be lower after previous administration of tiv (gmts 492, 603, 304, and 386, respectively). there was also a tendency for higher gmts in the adjuvanted groups relative to nonadjuvanted groups. all participants were a(h1n1)pdm09 seropositive from day 136 onward (14 days following first dose of a(h1n1)pdm09 vaccine [dose 2]). at day 164, 3 weeks after the second a(h1n1)pdm09 vaccination (dose 3), gmts had further increased in the adjuvanted groups and remained relatively constant in the nonadjuvanted groups. gmts in the saline-primed groups still tended to exceed those in the tiv-primed groups for both pandemic vaccine formulations, with this trend being greater for the adjuvanted groups a and c (651 vs 799) than for the nonadjuvanted groups b and d (259 vs 333). moreover, gmts were higher following adjuvanted vaccine relative to nonadjuvanted vaccine, with greater differences between the saline-primed groups c and d. similar trends were observed in scrs, sprs, and gmfrs. scrs were 84.8%-100% across groups, sprs 100% in all groups, and gmfrs ranged from 20.8 (group b) to 57.4 (group c), thus fulfilling chmp and cber criteria. between day 164 and day 304, gmts decreased 3-to 4-fold but remained, along with scrs, sprs, and seropositivity rates, above baseline (day 122) values in all groups; at day 304 scrs and sprs still met chmp criteria. nab responses followed trends that were similar to trends for hi responses, with a tendency for higher responses in salineprimed groups relative to tiv-primed groups (a < c and b < d) and in adjuvanted groups relative to nonadjuvanted groups (a > b and c > d). differences between groups were generally smaller than observed for hi responses. table 1) . after a(h1n1)pdm09 vaccine administration (doses 2 and 3; days 129-304), gmts in the tiv-primed groups remained at levels comparable to prevaccination levels (day 122; supplementary table 2 and figure 2b ). in the saline-primed groups, gmts increased slightly but remained lower than in the tivprimed groups. irrespective of the priming, gmts in the adjuvanted groups were comparable to those in the nonadjuvanted groups. at day 164, scrs met the chmp criterion in both tivprimed groups a and b but not in the saline-primed groups c and d (76.2%, 54.5%, 24%, and 7.4%, respectively), while they met the cber criterion only in group a. sprs met the chmp criterion in groups a-c at day 164 and day 304 (range, 72%-95.2%) but not in group d (51.9% on both days), while the cber criterion was only met in both tiv-primed groups. the chmp criterion for gmfr was met at day 164 in all groups except group d. data shown are of the according-to-protocol cohort for immunogenicity (atp-i cohort) at day 164, except for day 304, which are for the atp-i cohort at day 304. groups a and b received trivalent seasonal influenza vaccine at day 0 and either adjuvanted (group a) or nonadjuvanted (group b) pandemic vaccine at day 122 and day 143. groups c and d received saline at day 0 and adjuvanted (group c) or nonadjuvanted (group d) pandemic vaccine at day 122 and day 143. seroconversion rate: n/%, = number/percentage of seroconverted participants. seroconversion: titer ≥40 1/dil after vaccination (for initially seronegative participants at day 0) or a titer after vaccination ≥4-fold the prevaccination titer (for initially seropositive participants at day 0). seropositivity: n/%, number/percentage of participants with titer within the specified range. seroprotection rate: n/%, number/percentage of participants with titer within the specified range. geometric mean fold rise: geometric mean of the within-participant ratios of the post-vaccination reciprocal hi titer to the day 122 reciprocal hi titer before pandemic vaccination. abbreviations: ci, confidence interval; na, not available. as compared with hi responses, nab responses followed parallel trends ( figure 2b) . we evaluated the roles of adjuvant and tiv priming in inducing b-cell responses. first, frequencies of a(h1n1)pdm09specific memory b cells were measured by elispot ( figure 3a ). low frequencies were observed prior to vaccination (day 0) in all groups. some a(h1n1)pdm09-specific memory b-cell responses were induced by the tiv. following vaccination with a(h1n1)pdm09 vaccines, stronger memory b-cell responses were observed with adjuvanted vaccine relative to nonadjuvanted vaccine (groups: a > b and c > d). moreover, tiv vaccination prior to adjuvanted a(h1n1)pdm09 vaccination had a negative effect on the memory b-cell frequencies (group: c > a). we also evaluated the frequencies of plasmablasts (defined as cd27 + /cd38 + b cells) as a percentage of b cells (cd20 lo / cd19 + /cd3 − ; days 0-129; figure 3b ). it has previously been shown that plasmablasts can be detected 7 days after tiv vaccination and that a large proportion of cd27 + /cd38 + plasmablasts are specific for influenza vaccine antigens [24] . evaluation of cd27 + /cd38 + plasmablast frequencies 1 week after dose 1 revealed increased frequencies in both tiv groups but not in the saline groups. one week post a(h1n1) pdm09 vaccination (day 129), plasmablast responses, defined by increased frequencies of cd27 + /cd38 + b cells, were observed in both saline-primed groups but not in the tivprimed groups, with the strongest increase in the (adjuvanted) group c. in order to evaluate the effects of the adjuvant on the frequency and quality ( polyfunctionality) of a(h1n1)pdm09-specific and tiv strain-specific t-cell responses, we assessed the vaccineinduced responses of antigen-specific cd4 + t cells expressing at least 2 immune markers (among cd40l, interleukin [il]-2, tumor necrosis factor-alpha [tnf-α], and interferon-gamma [ifn-γ]) by ics after in vitro stimulation of peripheral blood mononuclear cells. low responses of a(h1n1)pdm09-specific immune markerexpressing cells were seen after tiv administration but not after administration of saline ( figure 4a ). after pandemic vaccination, a(h1n1)pdm09-specific cd4 + t-cell responses tended to be lower in the tiv-primed group a than in the salineprimed group c among the adjuvanted groups and were of comparable magnitudes for saline and tiv-primed groups among nonadjuvanted groups. responses to the adjuvanted vaccine tended to be higher and more persistent, regardless of previous administrations of tiv or saline (groups: a > b and c > d). cd4 + t-cell responses specific for the tiv strains after pandemic vaccination followed similar patterns, although with smaller differences between groups (shown for a/brisbane/59/2007; figure 4b ). cytokine expression profiles of responding cd4 + t cells were assessed by evaluating the frequencies of antigen-specific cells producing at least 1 marker (supplementary figure 1; shown for a(h1n1)pdm09). different cytokines were produced, with more cells expressing il-2 and/or cd40l than tnf-α and/or ifn-γ. the strongest responses were measured in the adjuvanted groups a and c. there were no detectable cd8 + t-cell responses to the a(h1n1)pdm09 strain or any of the tiv strains (data not shown). following dose 1, 65.7% and 28.8% of the recipients of tiv and saline, respectively, reported any symptom. among local symptoms, injection site pain was most frequently reported and was more common in the tiv group than in the saline group (53% vs 7.8%; figure 5 ). among general symptoms, headache, joint pain, and muscle aches were also more common among tiv recipients, and grade 3 aes were only reported in the tiv group (1 each of fatigue, headache, muscle aches, and joint pain). after a(h1n1)pdm09 vaccination (doses 2/3), injection site pain was also the most frequently reported local ae and occurred at a higher rate in the adjuvanted groups a and c than in the nonadjuvanted groups b and d (76.0%, 82.8%, 43.8%, and 37.0%, respectively), while no clear trend was observed for grade 3 pain. fatigue, muscle aches, and headache were the most frequently reported general aes. grade 3 general aes occurred at a frequency of ≤10.3% of participants' aes. after dose 1, unsolicited aes occurred at similar frequencies in tiv and placebo recipients; 22.4% and 21.2% of participants, respectively, reported at least 1 unsolicited ae. grade 3 back pain was reported once by a placebo recipient. nine percent of all participants (10.4% and 7.6% in tiv and saline groups, respectively) reported at least 1 mae. after a(h1n1)pdm09 vaccination (doses 2 and 3), frequencies of unsolicited aes were comparable among groups ( table 2) . nausea and upper respiratory tract infections were the most common events (≤2 participants/group). two grade 3 aes (diabetes mellitus and uterine leiomyoma) were reported in group a. from the participants of groups a-d, 27.3%, 32.4%, 15.2%, and 12.1%, respectively, experienced at least 1 mae up to day 507, none of which were considered related to vaccination, and 9.1%, 2.9%, 3.0%, and 3.0%, respectively, experienced at least 1 sae, none of which were fatal or considered related to vaccination. saes (1 report each) included hepatitis c 7 days after dose 1, which was unresolved at day 507; uterine fibroid that developed within 1 month after dose 3; worsening of aortic aneurysm 37 days after dose 3 (all in group a); elevated liverfunction test 6 months after dose 3 (group b); moderate clostridium difficile infection 63 days after dose 3 (group c); and cholelithiasis 5 months after dose 3 (group d). there were no reports of pimds or major clinical laboratory abnormalities, and there were no withdrawals due to saes or aes. this study assessed the effect of prior vaccination with a tiv on the immune response to the a(h1n1)pdm09 vaccine in young, healthy adult volunteers lacking a history of previous influenza vaccination. secondary objectives were to assess the role of the adjuvant on cmi and humoral responses and to evaluate vaccine safety. although no formal statistical comparison between groups was made, our results indicate that there was a trend for diminished a(h1n1)pdm09-specific humoral and cd4 + t-cell responses following vaccination with the pandemic vaccine in participants who had previously received tiv. also, results showed that adjuvantation of the a(h1n1)pdm09 vaccine led to increased responses of vaccine-homologous and -heterologous hi antibodies, nab and cd4 + t cells, and homologous memory b cells and plasmablasts. receipt of tiv a few weeks to many months prior to a(h1n1)pdm09 or h5n1 vaccines has previously been shown to affect hi responses [5, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] , irrespective of whether these vaccines were adjuvanted or not. one explanation could be the behavior of the memory b-cell pool after vaccination. seasonal vaccination has been shown to lead to rapid expansion of plasmablasts that produce vaccine antigen-specific antibodies [24] . the b-cell response to the tiv could be shaped by the epitopes present on the tiv strains. the resulting b-cell memory pool could limit the capacity of the b-cell compartment to adapt to antigenically more distant vaccines, such as a(h1n1)pdm09 vaccine antigens administered subsequently. thus, a b-cell repertoire that is "fixed" by tiv could limit adaptability of this response. cd4 + t cells can have a role in helping memory b cells by stimulating somatic hypermutation, thereby facilitating adaptability of the b-cell response [34] . indeed, not only frequencies of specific cd4 + t cells but also hi responses and memory b-cell frequencies were enhanced after the first and second doses of adjuvanted vaccine; this is in line with observations in previous h5n1 and/or a(h1n1)pdm09 vaccine studies [9, 27] . moreover, the epitope breadth and binding affinity of the antibodies to pandemic influenza vaccines were previously shown to be improved by mf59, another oil-inwater-based adjuvant [28, 29] . in a previous a(h1n1)pdm09 vaccine study, cd4 + t-cell frequencies after the first dose of pandemic vaccine correlated with hi titers measured 3 weeks later [9] . although the presence of such correlation was not assessed in our study, we speculate that after the first dose of adjuvanted vaccine, stimulation of cd4 + t-cell responses may have resulted in increased "help" for b cells, resulting in better adaptation of b cells and, subsequently, in increased hi titers to the variant ha in the pandemic vaccine. in short, after the first dose of pandemic vaccine, the adjuvant may have promoted b-cell adaptation to the more distant a(h1n1)pdm09 antigen and helped to shape t-and b-cell pools to better respond to the subsequent vaccination. overall, the reactogenicity and safety data for the tiv recipients were consistent with data for those who received a comparable tiv [35] . injection site pain was more common in the tiv group than in the placebo group after first vaccination and in recipients of adjuvanted vaccine relative to recipients of nonadjuvanted vaccine after a(h1n1)pdm09 vaccination. consistent with earlier studies with adjuvanted and nonadjuvanted a(h1n1)pdm09 vaccines [9, 11] , the current data do not suggest relevant safety concerns for any of the studied vaccines in the given study population. several retrospective epidemiological studies have described an association between vaccination with a different a(h1n1)pdm09 vaccine (pandemrix ™ ) and the later onset of narcolepsy in persons aged <21 years as well as in adults [36] . recent experiments revealed a potential molecular basis for the link, which lies in the ha amino acid sequence of h1n1 [37] . no narcolepsy cases were reported in the current study, though the current trial was not designed to detect rare events. trial limitations were the small sample size and relatively large number of withdrawals from the study. the study size selection was a consequence of the complexity of the laboratory assays for this descriptive study, while the number of withdrawals may have been related to the trial duration, weekly blood-sampling frequency over the course of 1 year, and/or the relatively large blood-sample volumes required to perform the detailed immunogenicity assessments. in healthy participants aged 19-40 years, prior vaccination with tiv decreased the humoral and cmi responses to the a(h1n1) pdm09 vaccine. adjuvantation of the pandemic vaccine helped to overcome the immune interference between influenza vaccines. no clinically relevant safety concerns were observed with either of the study vaccines. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. a single base-pair change in 2009 h1n1 hemagglutinin increases human receptor affinity and leads to efficient airborne viral transmission in ferrets world health organization. pandemic h1n1 (2009)-update 112. global alert and response weekly update estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study response to a monovalent 2009 influenza a (h1n1) vaccine immune response after a single vaccination against 2009 influenza a h1n1 in usa: a preliminary report of two randomised controlled phase 2 trials trial of 2009 influenza a (h1n1) monovalent mf59-adjuvanted vaccine stockpiling prepandemic influenza vaccines: a new cornerstone of pandemic preparedness plans as03 aadjuvanted influenza a (h1n1) 2009 vaccine for adults up to 85 years of age effect on cellular and humoral immune responses of the as03 adjuvant system in an a/h1n1/2009 influenza virus vaccine administered to adults during two randomized controlled trials predictors of immune response and reactogenicity to as03b-adjuvanted split virion and nonadjuvanted whole virion h1n1 (2009) pandemic influenza vaccines immunogenicity and safety in adults of one dose of influenza a h1n1v ) • 1429 2009 vaccine formulated with and without as03 a -adjuvant: preliminary report of an observer-blind, randomised trial safety and immunogenicity of an as03-adjuvanted a(h1n1)pmd09 vaccine administered simultaneously or sequentially with a seasonal trivalent vaccine in adults 61 years or older: data from two multicentre randomised trials immunogenicity, boostability, and sustainability of the immune response after vaccination against influenza a virus (h1n1) 2009 in a healthy population long-term persistence of humoral and cellular immune responses induced by an as03 a -adjuvanted h1n1 2009 influenza vaccine: an open-label, randomized study in adults aged 18-60 years and older effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the influenza pandemic h1n1 2009 vaccine: a randomized controlled trial optimal vaccination strategies for 2009 pandemic h1n1 and seasonal influenza vaccines in humans seroprevalence of pandemic h1n1 antibody among health care workers in hong kong following receipt of monovalent 2009 h1n1 influenza vaccine reduced antibody responses to the pandemic (h1n1) 2009 vaccine after recent seasonal influenza vaccination immunogenicity of a monovalent 2009 influenza a(h1n1) vaccine in infants and children: a randomized trial safety and immunogenicity of a prototype adjuvanted inactivated split-virus influenza a (h5n1) vaccine in infants and children priming with as03 a -adjuvanted h5n1 influenza vaccine improves the kinetics, magnitude and durability of the immune response after a heterologous booster vaccination: an open non-randomised extension of a double-blind randomised primary study original antigenic sin responses to influenza viruses disquisitions on original antigenic sin. i. evidence in man rapid cloning of high affinity human monoclonal antibodies against influenza virus amount and avidity of serum antibodies against native glycoproteins and denatured virus after repeated influenza whole-virus vaccination strategies to alleviate original antigenic sin responses to influenza viruses h5n1 influenza vaccine formulated with as03 a induces strong cross-reactive and polyfunctional cd4 t-cell responses vaccines with mf59 adjuvant expand the antibody repertoire to target protective sites of pandemic avian h5n1 influenza virus mf59 adjuvant enhances diversity and affinity of antibody-mediated immune response to pandemic influenza vaccines adjuvant system as03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity ten years of experience with the trivalent split-influenza vaccine guideline on influenza vaccine prepared from viruses with the potential to cause a pandemic and intended for use outside of the core dossier context (emea/ chmp/vwp/263499/2006). european agency for the evaluation of medicinal products (chmp) guidance for industry: clinical data needed to support the licensure of pandemic influenza vaccines the regulation and role of t follicular helper cells in immunity rapid licensure of a new, inactivated influenza vaccine in the united states pandemic influenza a h1n1 vaccines and narcolepsy: vaccine safety surveillance in action cd4 + t cell autoimmunity to hypocretin/orexin and cross-reactivity to a 2009 h1n1 influenza a epitope in narcolepsy acknowledgments. the authors are grateful for the vital contribution of the participating study volunteers, clinicians, nurses, and laboratory technicians at the study sites. the authors are indebted to the principal investigators jonathan wilson and anthony puopolo and to all teams of the glaxosmithkline (gsk) group of companies for their contributions, including global study managers wendy talbott and cherie barreca, catena lauria for clinical operations management, and us study manager stacy sanders. the authors also thank miguel madariaga for reviewing the protocol, karl walravens for laboratory support, and steven phay tran for cmi testing, chuck buscarino and janine linden for protocol writing assistance (all employees of gsk group of companies at the time of the study). dorothy slavin (gsk at the time of the study) was the clinical safety representative and rosalia calamera (gsk) and veronique grosjean (4 clinics on behalf of gsk) were responsible for database management. the authors also thank eddy denis and benoit le pioufle (keyrus on behalf of gsk) for their contributions to the statistical analyses and benoit vincart and philippe auquier (both gsk) for conducting the plasmablast measurements. finally, the authors thank ellen oe and shirin khalili (both xpe pharma&science on behalf of gsk) for writing assistance and publication management, respectively.authors contribution. all authors contributed substantially to the conception, design, analysis, and interpretation of the data presented. all authors had full access to the data and were involved in critical revision of the manuscript for important intellectual content. the corresponding author was responsible for manuscript submission.trademarks. pandemrix ™ and flulaval ™ are trademarks of the glaxo-smithkline group of companies.financial support. the study was funded by glaxosmithkline biologicals sa, which was involved in all stages of the study conduct and analysis (clinicaltrials.gov; nct00707967). glaxosmithkline biologicals sa also paid for all costs associated with the development and publishing of this manuscript.potential conflicts of interest. all authors are employees of the glaxo-smithkline group of companies. s. r.-g. holds gsk stock options and r. v.d m. and d. v. report receiving restricted shares of the company. r. v.d m. declares that us provisional patent applications have been filed in relation to some of the information discussed in this manuscript. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-258905-0hgdtalg authors: bond, katherine; nicholson, suellen; lim, seok ming; karapanagiotidis, theo; williams, eloise; johnson, douglas; hoang, tuyet; sia, cheryll; purcell, damian; mordant, francesca; lewin, sharon r; catton, mike; subbarao, kanta; howden, benjamin p; williamson, deborah a title: evaluation of serological tests for sars-cov-2: implications for serology testing in a low-prevalence setting date: 2020-08-06 journal: j infect dis doi: 10.1093/infdis/jiaa467 sha: doc_id: 258905 cord_uid: 0hgdtalg background: robust serological assays are essential for long-term control of the covid-19 pandemic. many recently released point-of-care (poct) serological assays have been distributed with little premarket validation. methods: performance characteristics for 5 poct lateral flow devices approved for use in australia were compared to a commercial enzyme immunoassay (elisa) and a recently described novel surrogate virus neutralization test (svnt). results: sensitivities for poct ranged from 51.8% (95% confidence interval [ci], 43.1%–60.4%) to 67.9% (95% ci, 59.4%–75.6%), and specificities from 95.6% (95% ci, 89.2%–98.8%) to 100.0% (95% ci, 96.1%–100.0%). elisa sensitivity for iga or igg detection was 67.9% (95% ci, 59.4%–75.6%), increasing to 93.8% (95% ci, 85.0%–98.3%) for samples >14 days post symptom onset. svnt sensitivity was 60.9% (95% ci, 53.2%–68.4%), rising to 91.2% (95% ci, 81.8%–96.7%) for samples >14 days post symptom onset, with specificity 94.4% (95% ci, 89.2%–97.5%). conclusions: performance characteristics for covid-19 serological assays were generally lower than those reported by manufacturers. timing of specimen collection relative to onset of illness or infection is crucial in reporting of performance characteristics for covid-19 serological assays. the optimal algorithm for implementing serological testing for covid-19 remains to be determined, particularly in low-prevalence settings. the coronavirus disease (covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a global public health emergency on an unprecedented scale. first reports in late december 2019 described a cluster of patients with pneumonia, linked to a live animal market in wuhan, china [1] [2] [3] . to date, laboratory testing has comprised detection of sars-cov-2 virus using reversetranscriptase pcr (rt-pcr) assays, predominantly from patients meeting specific epidemiological criteria. however, the immense scale of rt-pcr diagnostic testing has placed extraordinary demands on laboratories, with challenges relating to supply chains of swabs, laboratory reagents, and the human and financial resource required to support population-level testing. over the past 2 months, there has been rapid development of serological assays for covid-19 in a number of countries [4] . serological tests rely on detection of specific antiviral antibodies (immunoglobulin m [igm], immunoglobulin g [igg], immunoglobulin a [iga], or total antibody) in patient serum, plasma, or whole blood. the broad array of serological tests now available vary both in analytical performance and in their particular utility in the overall public health response to covid-19. the most publicized serological tests for covid-19 have been lateral flow immunoassays, also known as serological point of care tests (poct), which have been manufactured and deployed in several countries. most available poct involve detection of anti-sars-cov-2 igm or igg antibodies through binding to immobilized antigen (generally domains of the spike [s] protein) attached to colloidal gold, followed by detection of the conjugates by an anti-human igm or igg antibody. in addition, a control line is usually included in the assay, which helps determine whether the test result is valid. the relatively cheap and simple nature of lateral flow assays means that production is suited to scaling-up for increased testing capacity. in many countries, including the united states and australia, the rapid development and implementation of covid-19 diagnostics has meant that normally stringent regulatory criteria have not been applied to all tests, with limited published data supporting assay performance in clinical settings. here, in order to inform the deployment of poct in australia, we compared the performance characteristics of 5 commercially available poct with a commercially available enzyme-linked immunosorbent assay (elisa) and a recently described surrogate virus neutralization assay (svnt), using samples from (1) patients with rt-pcr-confirmed covid-19; (2) patients who were rt-pcr negative but presented with respiratory symptoms during the peak of the pandemic in australia, and (3) patients before the covid-19 pandemic. a testing panel was specifically developed to test poct devices for this study (supplementary material), consisting of 3 patient populations: (1) sera from 91 patients with sars-cov-2 detected by rt-pcr from upper and/or lower respiratory tract specimens; (2) sera from 36 patients with seasonal coronavirus infections or other acute infections (eg, dengue, cytomegalovirus, epstein-barr virus); and (3) serum from a random cohort (56 patients) of the australian population obtained in 2018. one of the devices was also tested against serum samples from 1217 patients who were sars-cov-2 rt-pcr negative but presented to a hospital emergency department between 6 february and 15 april 2020, spanning the initial peak of the covid-19 pandemic in australia. serum samples were obtained from a large academic hospital in melbourne, australia (royal melbourne hospital, rmh), or the victorian state reference laboratory for virology (victorian infectious diseases reference laboratory, vidrl). convalescent patients were followed up at home by the rmh@ home hospital in the home team. information on each cohort is provided in the supplementary material. cases were classified clinically as mild (not admitted to hospital), moderate (admitted to a hospital ward, but not the intensive care unit [icu]), or severe (admitted to icu). of the 91 cases, 71 were mild, 17 were moderate, and 3 were severe. sars-cov-2 rna was detected using the coronavirus typing assay (ausdiagnostics), a 2-step, heminested multiplex tandem pcr, with 7 coronavirus rna targets plus a proprietary artificial sequence as an internal control. all positive samples underwent additional confirmatory testing for sars-cov-2 at vidrl, using previously published primers [5] . serological poct devices were tested exactly as per the manufacturer's stated instructions for use, including use of plastic droppers and buffers provided in the kits. devices were provided through the australian government therapeutic goods administration, based on device availability at the time of the study (supplementary table 1 ). in brief, 10 μl of serum was added to the device, with addition of between 60 and 100 μl of the manufacturer's provided buffer. devices were incubated at room temperature according to the time period defined in the instructions for use (generally 10-15 minutes). all results were read as per the instructions for use. testing was performed by laboratory technicians, all of whom had undergone competency training in the use of lateral flow assays. testing of each sample in the serum panel was performed in duplicate, with a triplicate deciding test for discordant results. any faint line present at test termination was considered a positive result. results were recorded in a password-protected database available only to study investigators. all patient samples were deidentified. five elisa testing was performed using the euroimmun anti-sars-cov-2 elisa, a commercially available elisa (supplementary table 1 ). the assay involves semiquantitative detection of anti-sars-cov-2 iga or igg antibodies in serum through binding to a recombinant structural antigen (s1 domain of the spike protein) fixed to reagent wells. if test sera contain anti-sars-cov-2 antibodies, a second incubation step using enzyme-labelled anti-human iga or anti-human igg will catalyze a color reaction, detected by an optical density reader. semiquantitative results were reported as a ratio as per the manufacturer's instructions for use and interpreted as follows: to further assess antibody response, we used a recently described svnt, that detects circulating antibodies directed against the spike protein receptor binding domain (rbd) in an isotype-and species-independent manner, based on antibodymediated blockage of interaction between the ace2 receptor protein and the rbd [6] . in brief, 10 μl of test serum was diluted with 90 μl of sample dilution buffer and incubated with horseradish peroxidase conjugated sars-cov-2 rbd protein (hrp-rbd); the test solution was added to wells coated with fixed ace2 receptor. the degree to which test serum inhibited binding of the hrp-rbd to ace2 receptors, compared to control serum, was determined by optical density reading, with 20% inhibition and above considered a positive result. sera tested in the svnt included samples from 110 patients with rt-pcr-confirmed covid-19 and 142 samples from 142 control patients, of which 36 samples were from patients with seasonal coronavirus or other acute infections, and 106 samples were from a random cohort of the australian population obtained in 2016 and 2018 (supplementary material). a first round of testing on all samples followed the instructions for use; subsequently, samples within the 10% coefficient of variation (cv) range as stated in the instructions for use (inhibition cutoff of 18%-22%, n = 21) were repeated in duplicate to assess for interrun variation. an in-house microneutralization assay was performed at the university of melbourne. sars-cov-2 isolate cov/australia/ vic01/2020 [7] passaged in vero cells was stored at −80°c. serial 2-fold dilutions of heat-inactivated plasma were incubated with 100 tcid 50 (50% tissue culture infectious dose) of sars-cov-2 for 1 hour and residual virus infectivity was assessed in quadruplicate wells of vero cells; viral cytopathic effect was read on day 5. the neutralizing antibody titer was calculated using the reed/muench method as previously described [8, 9] . all statistical analyses were conducted using r (version 3.6.3) or graphpad prism (version 8.4.2). binomial 95% confidence intervals (ci) were calculated for all proportions. differences in nonnormally distributed numerical data were calculated using the wilcoxon rank sum test. receiver operating characteristic (roc) area under the curve (auc) analysis was performed in graphpad prism (version 8.4.2). ethical approval for this project was obtained from the melbourne health human research ethics committee (rmh hrec qa2020052). the overall sensitivity for either iga or igg detection was 67.9% (95% ci, 59.4%-75.6%) and specificity was 72.8% (95% ci, 62.6%-81.6%) ( table 1 ). the sensitivity for iga or igg detection increased to 93.8% (95% ci, 85.0%-98.3%) when only samples collected >14 days post symptom onset were considered (table 2) , and a significant rise in signal/cutoff ratio was observed for both iga and igg over time (p < .001) ( figure 1 ). roc auc analysis was performed for both iga and igg. overall, the iga roc auc was 0.74 (95% ci, 0.69-0.81) and the igg roc auc was 0.66 (95% ci, 0.59-0.72) (supplementary with seasonal coronavirus (2 hku1, 1 nl63, and 1 oc43) were positive for iga. neither of the 2 samples with anti-mers-cov antibodies displayed cross-reactivity for sars-cov-2 iga or igg, but 1 sample with anti-sars-cov-1 antibodies had positive results for sars-cov-2 iga and igg (ratios 3.81 and 1.26, respectively; figure 2 ). we compared the sensitivity and specificity of 5 poct devices, using rt-pcr as our reference standard, and interpreting poct results as positive when either an igm or igg result was read as positive. overall, the sensitivities ranged from 51.8% (95% ci, 43.1%-60.4%) to 68.6% (95% ci, 60.1%-76.3%), and specificities from 95.6% (95% ci, 89.2%-98.8%) to 100.0% (95% ci, 96.1%-100.0%) ( table 1 and figure 3a and 3b). when only samples collected >14 days were considered, the sensitivities ranged from 78.5% (95% ci, 66.5%-87.7%) to 93.8% (95% ci, 85.0%-98.3%) ( table 2 ). using the onsite device (for which there was a surplus of kits), additional testing was conducted on 1217 samples from patients who presented with respiratory symptoms but tested rt-pcr negative for sars-cov-2. in total, 39/1217 (3.2%) samples tested positive for igm and/or igg. on further testing, 6/39 samples (15.4%) tested positive to iga and/or igg using the elisa assay, of which 1 was confirmed by svnt (inhibition 63.9%) when an inhibition cutoff of 20% was employed (see below). in addition, this sample was confirmed as positive using a whole-virus microneutralization assay. this patient presented 21 days following symptom onset, with significant epidemiological risk factors for sars-cov-2 acquisition, and likely represents a true infection. using the highest performance device characteristics (sensitivity 68.6% [wondfo] and specificity >99.9% [hightop] ) and lowest performance characteristics (sensitivity 51.8% [vivadiag] and specificity 95.6% [onsite]) as hypothetical best and worse scenarios, respectively, the performance of poct was assessed across a range of sars-cov-2 population prevalence estimates (0.1%, 1%, 5%, and 10%; table 3 and supplementary figure 1) . with the best performing poct characteristics at an estimated sars-cov-2 period prevalence in australia of 0.03%, the positive predictive value was only 17.1%. in total, 311 samples were also tested using the svnt assay. applying a 20% inhibition cutoff and using rt-pcr as the reference standard, the sensitivity of svnt was 62.7% (95% ci, 55.0%-70.0%); this increased to 91.2%% (95% ci, 81.8%-96.7%) when only samples collected >14 days post symptom onset were considered (table 1 and table 2 ). specificity was 94.4% (95% ci, 89.2%-97.5%), with cross-reaction observed for 8 samples (figure 4 and supplementary table 2 ). increasing the inhibition cutoff to 25%, or repeating samples with an initial inhibition score between 18% and 22% improved the specificity to 99.3% (95% ci, 96.1%-99.9%) with little change in sensitivity (table 1 and table 2 ). the % cv for the in-house control sample with respect to the percentage inhibition was 10.8% between runs and 5.8% within run. accurate laboratory testing is integral to the prevention and control of covid-19. the unprecedented scale of diagnostic testing has led to the rapid development and implementation of a large range of diagnostic assays for sars-cov-2, including serological tests. however, there are limited peer-reviewed data on the performance characteristics of serological tests, and in order to best inform the implementation of these assays, high-quality postmarket validation data are urgently needed to guide laboratories, public health agencies, and governments in the appropriate and responsible use of such tests [10] . in this study, we assessed the performance characteristics of 5 serological poct, a commercial elisa, and a commercial novel svnt against a large serum panel from a cohort of over 100 patients with rt-pcr-confirmed sars-cov-2. in keeping with other studies [11] , the sensitivity of all assays was low (<70%) when all sample collection time points were considered. however, as expected given the reported antibody response to sars-cov-2 infection, sensitivity increased considerably when samples collected >14 days post symptom onset were assessed [12] , with the majority achieving sensitivities over 90%. our findings provide further support for recent commentary suggesting that current serological assays have limited, if any, role in the diagnosis of acute covid-19, with rt-pcr remaining the gold standard for diagnosis in the acute setting [13, 14] . specificities for poct ranged from 92.4% to 100%; it is possible this may reflect differences in the antigens used in each assay, although specific information about the sars-cov-2 recombinant antigen used in the assay was not described for most poct. in keeping with previous reports [15, 16] , when both iga and igg components of the elisa were considered, specificity was low (72.8%), but considering igg alone, specificity increased to 97.8%. this study is one of the first to utilize a recently described svnt assay [6] . previous work describing the development of this assay reported a 95%-100% sensitivity and 100% specificity using cohorts in singapore and china [7] . in our cohort at >14 days post symptom onset the test achieved sensitivity of 91.2% (95% ci, 81.8%-96.7%) and specificity of 94.4% (95% ci, 89.2%-97.5%). although limited clinical data are available on the cohort used to develop and validate the assay [6] , it is possible that our relatively mild clinical cohort may generate lower antibody titers than a more severely unwell cohort, potentially influencing sensitivity of the assay [12, 17] . of note, the majority of our nonspecific (false positive) samples in the svnt assay recorded inhibition just over the 20% cutoff. however, specificity improved when either (1) a higher inhibition cutoff was used or (2) samples within an arbitrary range (based on the instructions for use reported % cv of the assay) were tested in triplicate. in our low-prevalence setting where the test is more likely to act as a confirmatory assay, raising the inhibition cutoff to 25% increased the specificity to 99.3% (95% ci, 96.1%-99.9%), thus improving clinical utility. alternatively, introduction of an equivocal range for the assay with repeat testing for samples within this range, would be another approach to mitigate potential assay variation. in contrast to acute diagnosis, there are settings where highquality serological assays will have utility, including (1) defining antibody prevalence in key populations such as frontline workers; (2) determining the extent of covid-19 infection within the community; (3) identifying individuals for further evaluation of therapeutic immunoglobulin donation; and (4) vaccine development and evaluation. for (3) and (4), it is essential to have a good quantification of the functional neutralizing antibodies among donors or vaccines and the poct and elisa assays do not provide an endpoint titer. however, in order to appropriately deploy serological testing, it is critical to understand the limitations of test performance in the epidemiological context in which tests are used. this is particularly important in a setting such as australia, which, based on the number of reported cases of covid-19 (8001 cases as of 2 july 2020), has an estimated covid-19 period prevalence of 0.03% [18] . as such, even with highly sensitive and specific serological tests, the majority of positive results are likely to represent false positives. when considering the use of serology to inform policies relating to relaxing of physical distancing interventions, specificity of the assay becomes critical. if the majority of those identified as immune are actually false-positive results, then the threshold to maintain immunity within the community will not be achieved [19] . analogous to hiv testing in low-prevalence settings [20] , serological testing for sars-cov-2 may require a 2-step approach, whereby a sensitive high-throughput screening assay is followed by a high-specificity assay for confirmation (eg, neutralization testing or western blot). this approach could facilitate seroepidemiological studies in low-prevalence settings, which are required to better understand the extent of covid-19 infection at a population level. ongoing questions remain, however, about the duration and type of antibody response to sars-cov-2, particularly around the protective effect of neutralizing antibodies against future reinfection [12] . accordingly, the concept of an "immunity passport" that facilitates return to workplaces or school should be interpreted with caution, and the world health organization currently recommends the use of poct immunodiagnostic assays in research settings only, and not for clinical decision making until further evidence is available [21] . a key strength of this study was our systematic collection of convalescent samples. by establishing a community collection platform, we tested over 50 patients who were more than 21 days post symptom onset. ideally, validation of serological assays should be performed against a testing panel that includes samples from (1) patients at acute and convalescent stages of infection (to assess sensitivity), and (2) patients with other human coronavirus infections (to assess specificity). given the range of serological assays now available, there is a critical need for standardized protocols, including reference standards, across laboratories when conducting evaluations of emerging serological assays. further, the relatively recent emergence of sars-cov-2 means there are limited data on the sensitivities of serological assays at 3-6 months post infection. future work should assess any potential drop in sensitivity at varying time points post infection. in summary, our data describe the performance characteristics of 5 poct devices, a commercially available elisa assay, and a newly developed svnt. overall, our findings are in keeping with recent position statements that note serological assays have limited, if any, role in the diagnosis of acute covid-19 infection. our data strongly suggest that current poct devices should not be used in the diagnosis of acute covid-19 or as the sole assay in population-level serosurveys. nevertheless, there are settings where high-quality serological assays will have clinical utility. the curated panel of samples assembled for this study is being expanded and provides a valuable repository for rapid validation of new serological assays as they become available. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. a new 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testing for covid-19: a report from the national covid scientific advisory panel a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease. medrxiv royal college of pathologists australasia. position statement: covid19 igg/igm rapid poct tests statement on point-of-care serology testing for sars-cov-2 severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients evaluation of nine commercial sars-cov-2 immunoassays neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications australian government department of health. coronavirus (covid-19) current situation and case numbers implications of test characteristics and population seroprevalence on "immune passport" strategies laboratory diagnosis of hiv in adults: a review of current methods world health organization. immunity passports in the context of covid-19: scientific brief key: cord-000842-kff3gig0 authors: nayak, jennifer l.; fitzgerald, theresa f.; richards, katherine a.; yang, hongmei; treanor, john j.; sant, andrea j. title: cd4(+) t-cell expansion predicts neutralizing antibody responses to monovalent, inactivated 2009 pandemic influenza a(h1n1) virus subtype h1n1 vaccine date: 2013-01-15 journal: j infect dis doi: 10.1093/infdis/jis684 sha: doc_id: 842 cord_uid: kff3gig0 background. the ability of influenza vaccines to elicit cd4(+) t cells and the relationship between induction of cd4(+) t cells and vaccine-induced neutralizing antibody responses has been controversial. the emergence of swine-origin 2009 pandemic influenza a virus subtype h1n1 (a[h1n1]pdm09) provided a unique opportunity to examine responses to an influenza vaccine composed of both novel and previously encountered antigens and to probe the relationship between b-cell and t-cell responses to vaccination. methods. we tracked cd4(+) t-cell and antibody responses of human subjects vaccinated with monovalent subunit a(h1n1)pdm09 vaccine. the specificity and magnitude of the cd4(+) t-cell response was evaluated using cytokine enzyme-linked immunosorbent spot assays in conjugation with peptide pools representing distinct influenza virus proteins. results. our studies revealed that vaccination induced readily detectable cd4(+) t cells specific for conserved portions of hemagglutinin (ha) and the internal viral proteins. interestingly, expansion of ha-specific cd4(+) t cells was most tightly correlated with the antibody response. conclusions. these results indicate that cd4(+) t-cell expansion may be a limiting factor in development of neutralizing antibody responses to pandemic influenza vaccines and suggest that approaches to facilitate cd4(+) t-cell recruitment may increase the neutralizing antibody produced in response to vaccines against novel influenza strains. influenza a viruses can evade protective immune responses through both gradual antigenic drift of viral surface proteins and sporadic reassortment that can result in antigenic shift. such reassortment occurred in 2009, when the novel, swine-origin pandemic influenza a virus subtype h1n1 (a[h1n1]pdm09) emerged and spread globally, resulting in the first influenza pandemic of the 21st century [1] [2] [3] [4] . characterization of immunity to this virus revealed little antigenic seroreactivity with contemporary seasonal influenza a virus subtype h1n1 (a[h1n1]) [5] , relatively few conserved b-cell epitopes within the hemagglutinin (ha) protein [6] , and little or no preexisting neutralizing antibody in unexposed children or adults aged <60 years [7] [8] [9] . in contrast, preexisting memory cd4 + t cells, including cells directed against epitopes within the ha protein, were detected in peripheral blood mononuclear cells (pbmcs) of subjects not previously exposed to a(h1n1)pdm09 [6, [10] [11] [12] [13] . thus, this pandemic offered a unique opportunity to study cd4 + tcell responses with an influenza virus strain that would simultaneously induce naive and memory cd4 + t cells without large amounts of coexisting b-cell-mediated immunity. individuals are repeatedly exposed to influenza virus antigens through both vaccination and infection, resulting in competition between memory and naive lymphocytes over time. how this competition will affect the specificity of cd4 + t-cell responses is unknown. while the typical seasonal trivalent inactivated vaccine (tiv) is enriched for ha, it also contains the more conserved internal viral proteins, such as nucleoprotein (np) and matrix protein (m1) [14, 15] . thus, both repeated infections and vaccinations may lead to a dominance of t cells specific for peptide epitopes conserved among virus strains. while recent studies examining the t-cell repertoire in humans have shown broad reactivity to diverse viral proteins [10, 16, 17] , few studies have examined the distribution of reactivity among conserved and novel epitopes. neutralizing antibody is considered the major correlate of protection following influenza vaccination [18, 19] , while the protective role of cd4 + t cells remains more poorly understood. whether cd4 + t-cell responses even develop after vaccination with tiv remains controversial [12, [20] [21] [22] [23] [24] [25] [26] [27] [28] . lack of consensus may be due to inadequate subject numbers, variable levels of baseline anti-influenza immunity in study populations, or the use of recall antigens that do not elicit the full repertoire of influenza-reactive cells. knowledge of the relationship between cd4 + t cells and the development of a neutralizing antibody response following administration of tiv is even more limited, with the few studies addressing this question failing to find a correlation between these parameters [12, 20] , except when an adjuvanted influenza a virus subtype h5n1 vaccine was used [29] . in this study, we used an experimental approach designed to maximally detect antigen-specific cd4 + t cells by using cytokine enzyme-linked immunosorbent spot (elispot) assays with pools of overlapping synthetic peptides as recall antigens to quantify responses to conserved and novel epitopes following vaccination of adults with monovalent inactivated a (h1n1)pdm09 vaccine. we also examined the relationship between cd4 + t-cell responses and neutralizing antibody titers. these analyses revealed a readily detectable increase in numbers of cd4 + t cells directed against conserved portions of ha and the np and m1 proteins. further, cd4 + t-cell expansion was correlated with the development of a neutralizing antibody response against this pandemic virus. forty-nine healthy subjects were enrolled in 2 age groups (18-32 years and ≥60 years) between march and october 2010. subjects with a history of previous laboratorydocumented infection or vaccination with a(h1n1)pdm09, vaccination with a/new jersey/76, egg allergy, immunosuppression, or active neoplastic disease were excluded. younger subjects were excluded if they had a baseline a/california/07/ 09 hemagglutinin inhibition (hai) titer of >10, but this was not an exclusion criterion for older adults because of greater levels of expected preexisting immunity. blood was obtained before and at days 7, 14, and 28 after administration of inactivated subunit a/california/07/09 monovalent vaccine (novartis, east hanover, nj). pbmcs were purified using accuspin tubes with histopaque-1077 cell separation media (sigma-aldrich, st. louis, mo) and were frozen at a controlled rate in fetal bovine serum (fbs) containing 10% dmso. all sera were tested using the microtiter technique for neutralization of an egg-grown virus derived from the 2009 monovalent live attenuated influenza vaccine (lot 500914p; medimmune, gaithersburg, md). sera were heat inactivated prior to testing, starting at a 1:10 dilution. viral growth was determined by enzyme-linked immunosorbent assay following fixation with acetone, using np-specific monoclonal antibodies (who reagent kit). the antibody titer was defined as the reciprocal of the highest dilution that resulted in 50% inhibition of signal as compared to control wells. an end point titer was determined on all sera with an initial neutralizing antibody titer of ≥1280. sera without detectable neutralization activity were assigned a titer of 5, and values >40 000 were assigned a titer of 40 000 for calculation purposes. hai testing was performed on all sera in microtiter format, using turkey red blood cells with 4 ha units of egg-grown virus derived from the 2009 monovalent a(h1n1)pdm09 live attenuated influenza vaccine (lot 500914p; medimmune, gaithersburg, md) as antigen. sera were treated with receptordestroying enzyme (denka seiken, tokyo, japan) and heat inactivated prior to testing, starting at a 1:10 dilution. the antibody titer was defined as the reciprocal of the highest dilution that resulted in inhibition of hemagglutination. cryopreserved pbmcs were thawed and then rested overnight at 37°c and 5% co 2 in roswell park memorial institute 1640 medium containing 10% fbs and gentamicin (life technologies, carlsbad, ca), with a typical yield of >85% viable cells after thawing. after rest, pbmcs were depleted of cd8 + and cd56 + cells, using macs positive selection with ld separation columns as per manufacturer's instructions (miltenyi biotec, auburn, ca). elispot assays were performed as previously described [10] , with 400 000 or 200 000 cd8-and cd56-depleted pbmcs cocultured with either peptide pool or tetanus toxoid (calbiochem, billerica, ma). plates were analyzed using an immunospot reader series 2a with immunospot software, version 3.2 (cellular technology, shaker heights, oh). results were normalized to peptide-specific spots per 10 6 cells after averaging values for duplicate wells and subtracting background. peptides unique to a/california/07/09 were synthesized in our facility by using an apex 396 system (aapptec, louisville, ky) as described previously [30] . other peptide sets were ob a/california/07/09 ha and na peptides containing <6 contiguous conserved amino acids as compared to recently circulating a(h1n1) strains were pooled into a ha/na "unique" pool (38 peptides; supplementary table 1) . a "ha-conserved" pool containing ≤1 divergent amino acid as compared to recently circulating a(h1n1) strains was also used (35 peptides; supplementary table 2 ). because the a(h1n1)pdm09 vaccine was on an a/puerto rico/8/34 backbone that was largely conserved as compared to a/new york/348/2003, all np and m1 peptides were combined into a "np/m1-conserved" pool (123 peptides). as a control, divergent influenza a virus subtype h3n2 (a[h3n2]) peptides with <6 contiguous conserved amino acids as compared to a/california/07/09 (132 peptides) were pooled. the "total" influenza-specific response was calculated as the sum of the ha/na unique, ha-conserved, and np/m1-specific cd4 + t-cell responses. t-cell responses against a(h1n1)pdm09 vaccine were compared between prevaccination and postvaccination time points, using the wilcoxon signed rank test. the mann-whitney u test was used to compare unpaired groups. correlations between groups were examined using spearman rank correlation. to further investigate whether cd4 + t-cell help was specificity dependent, 2 regression models were used to examine the joint effects of the np/m1-and ha-conserved pools on the microneutralization titer; details of this analysis are in the supplementary materials. statistical analyses were performed using sas, version 9.2, or graphpad prism 5. all p values < .05 were considered statistically significant. the university of rochester research subjects review board approved this study protocol, and human experimentation guidelines of the us department of health and human services and the university of rochester were followed. study procedures were in accordance with the ethical standards of the declaration of helsinki. all subjects provided written informed consent prior to study participation. forty-nine healthy adults received the inactivated a/california/07/09 monovalent vaccine between march and october 2010. seventeen subjects (35%) were aged 18-32 years (median age, 25 years), and 32 subjects (65%) were aged ≥60 years (median age, 68 years [range, 60-82 years]). twenty-five subjects (51%) were female. younger subjects were excluded if they had a prevaccination hai titer of >10. twelve older subjects had a baseline hai titer of >10; 9 older subjects had a baseline hai titer of ≥40. hai and microneutralization titers were determined at all visits. good correlation was seen between the maximum antibody titer as determined by hai and microneutralization assay ( figure 1 ; r = 0.91, p < .0001). however, as neutralizing antibodies were fully titrated only with the microneutralization assay, these data are reported here. the maximum microneutralization titer ranged between <10 and >40 000, with a geometric mean of 482. seroresponse (defined as a ≥4-fold increase in microneutralization titer) occurred in 41 subjects (84%). we initially addressed whether we could detect cd4 + t-cell reactivity following a(h1n1)pdm09 vaccination. cd4 + t-cell responses at all time points were quantified following restimulation with ha-conserved and np/m1 peptide pools. a pool containing a(h3n2) peptides not present in the vaccine and tetanus toxoid were included as controls. figure 2 shows the number of interferon γ (ifn-γ)-producing cd4 + t cells per 10 6 cd8-and cd56-depleted pbmcs in both the cohort including all subjects (a-c) and only those subjects with preexisting microneutralization titers of <40 (d-f). reactivity to the ha-conserved pool (figure 2a and 2d) and np/m1 pool ( figure 2b and 2e) was clearly detected following vaccination. when postvaccination responses were compared to baseline using the wilcoxon signed rank test, these responses were statistically significant at all time points. the readily detectable responses to np and m1 are likely the result of contaminating internal viral proteins within the vaccine, as has been demonstrated in previous studies [14, 15] and as we have confirmed in this vaccine, using a western blot for np (data not shown). no significant increases in responses to tetanus toxoid or the a(h3n2) divergent pool were observed (supplementary figure 1) . these results provide strong evidence that subunit vaccines elicit cd4 + t cells specific for influenza virus epitopes. in this study cohort, there was no effect of age or body mass index on either the maximum microneutralization titer achieved ( supplementary figure 2a and 2b ) or the overall expansion of cd4 + t cells (supplementary figure 2c and 2d) , although the preexisting immunity of older adults may have biased the results of the age analysis. the second issue addressed was whether responses to previously unencountered peptide epitopes would develop in the face of existing memory cd4 + t cells specific for conserved epitopes. to evaluate this, cd4 + t cells specific for ha and na peptides that were not conserved as compared to seasonal vaccines ("unique") were quantified before and after vaccination. we found a trend toward greater numbers of cd4 + t cells specific for this unique pool, but the increase did not reach statistical significance ( figure 2c ). when subjects with evidence of preexisting immunity (microneutralization titer, ≥40) were excluded, this trend became much less apparent ( figure 2f) . because of the potential usefulness of preexisting cd4 + t cells as a predictive biomarker [31] [32] [33] , we next examined whether prevaccination influenza virus-specific cd4 + t cells correlated with postvaccination cd4 + t-cell responses and, thus, potentially with the help available for antibody responses. we were not able to find a predictive relationship between total prevaccination influenza virus-reactive cd4 + t cells and postvaccination responses when calculated as a change from baseline ([peak valuebaseline value]; r = 0.11, p = .46). however, when the response magnitude was quantified as a fold-change [peak response/baseline value], an inverse correlation between these parameters was observed (r = −0.5, p = .0002), as has been previously reported [20, 25, 34] . if not all influenza virusreactive cd4 + t cells are recruited into the vaccine response, the inclusion of unstimulated cells in the denominator could artificially lessen the response estimate for subjects starting with higher baseline levels of immunity when calculating "foldchange." to avoid this potential pitfall, we chose to present the cd4 + t-cell response as a "change from baseline." cd4 + t-cell help is critical for production of high-affinity antibody responses, but it is not clear whether the cd4 + t-cell response magnitude in any way correlates with or limits the magnitude of the antibody response. to evaluate this, the maximal change in the "total" cd4 + response (sum of the ha/na unique, ha-conserved, and np/m1-specific cd4 + t-cell responses) was determined for each subject and plotted against the maximum or fold-increase in neutralizing antibody titer. as demonstrated in figure 3a and 3b, there was a highly significant correlation between cd4 + t-cell expansion and both the maximum (r = 0.53, p < .0001) and fold-increase in (r = 0.46, p = .0004) microneutralization titer. the above correlations were also statistically significant when the maximal fold-increase in cd4 + t cells was quantified and compared to the neutralizing antibody titer, when the antibody titer was quantified using hai, and when all subjects with an hai of ≥10 at baseline were excluded from the analysis (data not shown). in contrast, there was no detectable correlation between gains in neutralizing antibody titers and the number of influenza virus-reactive cd4 + t cells present before vaccination ( figure 3c and 3d) . we conclude from this that expansion of cd4 + t cells rather than the baseline number of influenza virus-reactive cells correlates with and predicts the development of a neutralizing antibody response following a (h1n1)pdm09 vaccination, a result that is consistent with the idea that cd4 + t-cell help may limit the antibody response to pandemic influenza vaccines. under physiologic conditions, antigen-specific b cells primarily internalize antigen via the immunoglobulin receptor and recruit cd4 + t cells via the major histocompatibility complex class ii restricted display of peptides from this immunoglobulin-mediated event [35, 36] . if influenza virus proteins within the vaccine are not aggregated, other viral proteins will not be internalized by ha-specific b cells. this will lead to the selective presentation of ha-derived epitopes by ha-specific b cells. in this scenario, b cells producing neutralizing antibody will only recruit help from ha-specific cd4 + t cells [37] . clearly, the sampling limitations in humans preclude direct examination of b-cell antigen presentation within the draining lymph node following intramuscular vaccination. thus, to evaluate the role of cd4 + t-cell specificity in neutralizing antibody production, we examined correlations between the neutralizing antibody response and expansion of ifn-γproducing cd4 + t cells specific for peptides within either the ha-conserved or np/m1 pools. figure 4 demonstrates that there was a significant correlation between neutralizing antibody response and expansion of both ha-specific ( figure 4a and 4b) and np/m1-specific ( figure 4c and 4d) cd4 + t cells, with the strongest correlation seen when ha reactivity was examined. figure 5 represents the subjects grouped by degree of cd4 + t-cell expansion. vaccine recipients with the largest cd4 + t-cell response increases consistently had the greatest gains in neutralizing antibody production. interestingly, subjects with a >100 spot increase in cd4 + t cells specific for peptides within the conserved ha pool had a higher geometric mean titer of and fold-increase in neutralizing antibody production ( figure 5a and 5c) when compared to the similar np/m1 group ( figure 5b and 5d) , suggesting that ha-specific b cells may display a limited repertoire of peptides derived primarily from ha and thus may preferentially recruit cognate help from ha-specific cd4 + t cells. however, even in this scenario, cd4 + t cells specific for proteins such as np and m1 that are stimulated in the lymph node may be able to promote antibody responses through noncognate interactions via the provision of cytokines [38] . to further distinguish the contribution of ha-specific and np/m1-specific cd4 + t cells to the neutralizing antibody response, multiple regression models were used. modeling indicated that the majority of the effect of np/m1-specifc cd4 + t-cell expansion on the neutralizing antibody response could be accounted for by simultaneous increases in the haconserved response. furthermore, increases in cd4 + t cells specific for peptides within the ha-conserved pool predicted the antibody titer better than np/m1 responses. however, this difference in prediction power did not reach statistical significance. the experiments presented here demonstrate that the monovalent inactivated a(h1n1)pdm09 influenza vaccine elicits readily detectable cd4 + t-cell responses and that the magnitude of these responses correlates with gains in neutralizing antibody. this positive relationship between cd4 + t cells and antibody responses was most apparent when looking at cd4 + t-cell responses directed against conserved peptide epitopes within the ha protein of the a/california/07/09 virus. on the basis of these results, we postulate that activation of cd4 + t cells following vaccination may be one of the limiting factors for neutralizing antibody production following pandemic influenza vaccination. as there is great interest in defining biomarkers that will predict success in vaccination, our studies evaluated whether prevaccination levels of influenza virus-specific cd4 + t-cell reactivity predicted the magnitude of the neutralizing antibody response. we did not observe any correlation between these parameters but instead found a correlation between cd4 + tcell expansion and the titer of neutralizing antibody produced. this suggests that, following vaccination, only a subset of influenza virus-reactive cells are able to be recruited into the draining lymph nodes and expand, leading the cells that ultimately reenter the circulating pool to be most indicative of the help available for the antibody response. the effect this has on cd4 + t-cell memory will be important to address in future studies. our results contrast with what was recently reported in the study by wilkinson et al [39] , in which greater baseline circulating numbers of influenza virus-specific cd4 + t cells were protective against development of severe disease in an influenza challenge model. one potential reason for this difference is that the study by wilkinson et al predominantly examined the role of effector cd4 + t cells that exerted their function prior to either viral clearance or the production of neutralizing antibody, possibly via cytolytic activity. this contrasts with our study, which considered the neutralizing antibody produced in response to vaccine challenge. additionally, more cd4 + t cells may be able to be recruited into the immune response following infection, because of a greater abundance and diversity of epitopes displayed by antigenpresenting cells. our ability to use peripheral blood cd4 + t-cell reactivity to predict future vaccine-induced b-cell responses is likely to require a more refined definition of epitopes recruited by vaccination and a better understanding of the subsets of cd4 + t cells that can participate in extrafollicular and germinal center responses to vaccine components. further, it is possible that the observed relationship between cd4 + t-cell and neutralizing antibody responses may be the result of an overall more robust vaccine response in some individuals. future efforts to selectively boost cd4 + t-cell responses will help to confirm the causal relationship between these parameters. an important issue we sought to evaluate in these studies was whether the influence of cd4 + t cells on neutralizing antibody responses to vaccination was related to antigen specificity. it is interesting that the strongest correlation observed was between expansion of ha-specific cd4 + t cells and the neutralizing antibody response, although the distinction between np/m1-and ha-specific expansion and the antibody response that we detected was modest. both concurrent expansion of ha-reactive cd4 + t cells and cells specific for the np/m1 pool and the inclusion of m1 peptides within the np pool, as m1 associates with the viral surface glycoproteins [40] and may be taken up with ha by b cells, may have lessened our ability to detect a potential linkage between b-cell and t-cell specificities. if ha-specific b cells do have preferential access to limited viral proteins, vaccine development efforts that focus the cd4 + t-cell response on ha-derived epitopes may improve the neutralizing antibody response following vaccination. one of the challenges to vaccination against pandemic influenza is that the viral protein composition of the next pandemic strain cannot be predicted [41, 42] . the failure to detect cd4 + t-cell responses to novel epitopes in the current study suggests either that these epitopes are poorly immunogenic or that naive cells fail to successfully compete with the more abundant and rapidly recruited memory cells. further studies involving individuals who are now primed with a(h1n1) pdm09 will help clarify the overall immunogenicity of these peptides and the effect of competing memory t cells on naive cd4 + t-cell expansion and response kinetics. for antibody responses to a(h1n1)pdm09, there may have been enough conserved ha epitopes to promote antibody responses, but for more distant viruses conserved ha epitopes may be quite limited, possibly resulting in a correspondingly low neutralizing antibody response. this could explain the disparity between the robust responses to a single dose of a(h1n1) pdm09 vaccine [43, 44] as compared to a(h5n1) vaccine, to which responses are modest [45, 46] . if future studies substantiate the link between ha-specific cd4 + t cells and anti-influenza virus neutralizing antibody production, efforts to enrich the cd4 + memory population with t cells specific for potentially cross-reactive ha epitopes by prepriming with peptidebased vaccines or novel ha constructs [47] may increase the recruitment of ha-specific cd4 + t cells on challenge with divergent ha proteins. such a strategy could promote a more broadly cross-reactive and rapid response to novel strains of influenza virus, increasing pandemic preparedness by providing stand-alone protection while allowing dose-sparing efforts to facilitate rapid deployment of limited vaccine stocks to the population in the event of a pandemic. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org/). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. emergence of a novel swine-origin influenza a virus (s-oiv) h1n1 virus in humans emergence of a novel swine-origin influenza a (h1n1) virus in humans diversity of influenza viruses in swine and the emergence of a novel human pandemic influenza a (h1n1) the first influenza pandemic of the new millennium antigenic and genetic characteristics of swine-origin 2009 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ligocyte. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-262840-fhfxnr76 authors: gorse, geoffrey j.; o’connor, theresa z.; hall, susan l.; vitale, joseph n.; nichol, kristin l. title: human coronavirus and acute respiratory illness in older adults with chronic obstructive pulmonary disease date: 2009-03-15 journal: j infect dis doi: 10.1086/597122 sha: doc_id: 262840 cord_uid: fhfxnr76 backgroundthe clinical features and incidence of human coronavirus (hcov) infections in chronically ill older adults need better definition methodshcov infection was determined on the basis of a 4-fold increase in serum antibody and the detection of hcov by reverse-transcription polymerase chain reaction. laboratory-documented influenza (ldi) was detected by serologic assay and culture. hcov illnesses were compared with other acute respiratory illnesses identified by active surveillance, during the 1998–99 winter respiratory-virus season, of 2215 patients with chronic obstructive pulmonary disease who were ⩾50 years old and who received influenza vaccines resultshcov-229e and hcov-oc43 were associated with 90 (14%) of 665 illnesses (hcov-229e in 22, hcov-oc43 in 67, and both in 1), ldi with 107 (16%) of 678 illnesses. in multivariate logistic regression analysis, myalgia was less likely with hcov infection than with ldi (or, 0.27 [95% confidence limit, 0.13–0.58]). a majority of these hcov and ldi illnesses exhibited each of 11 symptoms and signs of acute respiratory illness. spirometric results worsened most often with ldi, and many acute respiratory illnesses, regardless of etiology, were associated with hospitalization. a total of 8 illnesses were associated with hcov-nl63, 1 with hcov-hku1 conclusionsthe frequencies of hcov and ldi illnesses were similar. hcov illness was less severe than ldi illness, was accompanied by multiple respiratory and systemic symptoms, and was associated with hospitalization hcov-oc43-belong to groups i and ii, respectively. the emergence of severe acute respiratory syndrome (sars) [2, 3] necessitated a rethinking of the role that coronaviruses play as a potential cause of more-severe respiratory illness. even before the emergence of sars, as well thereafter, hcov infection has been viewed as a contributor to exacerbations of underlying chronic obstructive pulmonary disease (copd), asthma, congestive heart failure, and severe illnesses requiring emergency care and hospitalization of patients with chronic medical conditions [4 -16] . the renewed interest in coronaviruses has led to the identification of 2 new coronavirus strains, hcov-nl63 and hcov-hku1 [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] . hcov-nl63 is a group i strain that is more readily culturable from clinical specimens in tissue culture, whereas hcov-hku1 is a group ii coronavirus; both have been reported from geographically diverse regions but were first described in the netherlands and hong kong, respectively [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] , and they have been associated with a range of illnesses, from mild, febrile upper-respiratory-tract illnesses to severe illness, in hospitalized patients with bronchiolitis and pneumonia. reports describe these newer strains as occurring predominantly in children but also in adults [23] [24] [25] 34] . among hospitalized patients in hong kong who have acute respiratory-tract infections, coronaviruses-including (in order of decreasing frequency) hcov-oc43, hcov-nl63, hcov-hku1, and hcov-229e-have been detected, by reverse-transcription polymerase chain reaction (rt-pcr), in 2% of patients, who had an age range of 1 month-88 years [30] . kistler et al. [34] have reported the presence of hcov-nl63, hcov-hku1, and hcov-oc43 in adults with and without asthma who were evaluated early after the onset of common-cold symptoms. prototype strains of hcov groups i and ii have been adapted to tissue culture, but wild-type strains are not readily culturable and therefore are not amenable to isolation from clinical specimens. most earlier studies relied on serologic methods to detect hcov infection. more recently, rt-pcr has been used to detect hcov-229e and hcov-oc43 rna in respiratory secretions during acute infection. rt-pcr has confirmed that hcov-229e and hcov-oc43 can be associated with acute respiratory illnesses [15, 16] ; however, comparisons of concordance between serologic results, detection of the virus by rt-pcr, and correlation with clinical symptoms are needed. in department of veterans affairs cooperative study 448, older adults with copd were randomized to receive trivalent inactivated influenza-virus vaccine (tiv) intramuscularly either with or without intranasal live-attenuated, cold-adapted influenzavirus vaccine (laiv), for the 1998 -1999 winter respiratory-virus season and then were monitored prospectively for the occurrence of acute respiratory illness [35] . the present study (1) compares the clinical characteristics and significance of hcov-229e and hcov-oc43, the predominant hcov strains in the present study, with those of laboratory-documented influenza (ldi) and all other acute respiratory illnesses; (2) compares the frequencies of hcov-229e and hcov-oc43 infection detected by rt-pcr and serologic assay; and (3) characterizes illnesses that were found to be associated with either hcov-nl63 or hcov-hku1. patients 50 years of age who met spirometric criteria for copd were recruited at 20 va medical centers, as described elsewhere [35] . all patients gave written informed consent, and the study received approvals by responsible committees on human experimentation and followed procedures in accordance with the recommendations found in the helsinki declaration of 1975 as revised in 1983. pulmonary-function tests included forced expiratory volume in 1 s (fev 1 ), percentage of predicted fev 1 , and the ratio of fev 1 to forced vital capacity (fev 1 :fvc) [35] [36] [37] . spirometric criteria for copd were an fev 1 that was 80% of the predicted value and an fev 1 :fvc that was ͻ0.70. immunizations occurred from october 1998 through january 1999. all patients received tiv for the 1998 -1999 influenza season. patients were randomly assigned, in a 1:1 ratio, to receive intranasally either laiv (medimmune vaccines [formerly aviron]) (tc group) or placebo (tp group) [35] . from november 1998 through april 1999, the patients were evaluated when they had either 3 symptoms of acute respiratory illness or fever accompanied by 2 symptoms of acute respiratory illness. a nasal and oropharyngeal (nop) swab specimen and a serum specimen were obtained for detection of virus and antibody, respectively, and a second serum specimen was obtained 3-4 weeks after the onset of acute respiratory illness; all specimens were stored at ϫ70°c. ldi was defined as the sudden onset of respiratory illness plus (1) an nop-swab culture positive for wild-type influenza virus a or b and/or (2) a 4-fold increase in the end-point titer of serum hemagglutination inhibition (hai) antibodies to influenza a or b, measured as described elsewhere [35, 38, 39] . a change in titer of serum hai antibody was not used to diagnose an ldi-caused illness if the acute respiratory illness occurred ͻ28 days after vaccination for influenza. hcov-associated illness was defined as the sudden onset of respiratory illness plus (1) an nop-swab culture positive for either hcov-229e or hcov-oc43, by rt-pcr using the sars/coronaplex kit (prodesse), which detects sars coronavirus and hcov-oc43 and hcov-229e, and/or (2) a 4-fold increase in the end-point titer of serum antibody to hcov, by elisa. rna for rt-pcr assay was extracted from nop specimens according to the manufacturer's recommended procedure (qiaamp kit; qiagen). available nop specimens from illnesses that had a 4-fold rise in antibody to hcov-nl63 were cultured in llc-mk2 cells (a gift from lia van der hoek, university of amsterdam, the netherlands). rna extracted from supernatant fluids by use of the qiamp ultrasens virus kit (qiagen) was subjected to rt-pcr by use of the geneamp rna pcr kit (applied biosystems) and spike (s) gene nested primers for amplification of cdna. the hcov-nl63 s gene-specific outer primers comprised positions 22557-22582 (positive sense) and positions 23043-23063 (antisense), as described elsewhere [20] . the inner primers, designed by use of the netherlands hcov-nl63 isolate (genbank accession number ay567487) comprised positions 22519 -22538 (positive sense) and positions 22909 -22928 (antisense). specimens with viral bands confirmed by gel electrophoresis of the rt-pcr products were considered to be positive. rt-pcr products were purified by use of the qia quick pcr purification kit protocol (qiagen) and were sequenced, with the primers, by alpha biolaboratory. the hcov antigens used for the antibody elisa were produced as follows. hcov-229e and hcov-oc43 were obtained from american type culture collection (atcc) and were grown in mrc-5 and hct-8 -cell monolayers (atcc), respectively. hcov-nl63 was grown in llc-mk2-cell monolayers. virus-infected cells were frozen and thawed 3 times, the supernatant fluid was clarified of cell debris by centrifugation, the virus was concentrated by overnight centrifugation, and the virus pellet was resuspended in pbs. the concentrated virus was inactivated by psoralen compound (sigma), followed by irradiation by long-wavelength uv light, as described elsewhere [40, 41] . mock antigen was prepared, in the same way, from uninfected cells. his 6 -tagged recombinant n protein of hcov-hku1 was used as antigen in the elisa to detect antibody to hcov-hku1. expression vector pet-28b (ϩ) (novagen), encoding the n protein of hcov-hku1 cloned into the ecori and noti sites inframe and downstream from a series of 6 histidine residues, as described elsewhere [27, 28] , was a gift from k. y. yuen (university of hong kong, hong kong). the recombinant n protein was expressed by transformation of bl21 (de3) singles competent cells (novagen) by the plasmid and was purified by the ni 2ϩ -loaded hitrap chelating system (amersham biosciences), according to the manufacturer's instructions. in brief, transformed cells were disrupted, and the protein sample was prepared by isolation of inclusion bodies via sonication and washing. the protein sample was loaded onto the hitrap chelating hp column prepacked with precharged ni ϩ2 sepharose high performance. the protein then was purified and refolded by serial buffer washes of the column and by liquid chromatography in an fplc system (pharmacia lbk biotechnology), with elution of protein, which was collected in fractions. the fractions were analyzed for the presence of 53-kda protein by sds-page. protein concentration was determined by the bio-rad protein assay (bio-rad laboratories) and was adjusted to 3 g/ml in the elisa. mock antigen was produced from the same plasmid dna vector, but without the n protein gene sequence, by the same procedure. each viral antigen and its respective control were used to coat flat-bottom 96-well maxisorp immuno-plates (nalge nunc international). the sequence of reagents consisted of serum in a series of 8 2-fold dilutions to generate a broad dose-response curve, mouse anti-human igg (fc-specific) conjugated with horseradish peroxidase (accurate chemical and scientific), and peroxidase substrate (kpl). optical density (od) was measured at 405 nm by use of a tecan slt400 spectrophotometer (research triangle park, north carolina). the anti-hcov antibody titer in the elisa was calculated by the reference-line leastsquares-fit method [42] . the cutoff od was set at 0.3 for all viral antigens, a level that was at least twice the background od for the respective mock antigens, and corresponded to the linear range of od versus reciprocal-dilution curves. the first episodes of acute respiratory illness that occurred ͼ7 days after vaccination were included in the clinical analyses and to categorize patients in terms of "illness group." subsequent illnesses are not described, both because the earlier illness could affect the characteristics of the later illness and because the num-ber of later illnesses was small. the illness groups comprised those whose first episode of acute respiratory illness was associated with (1) either hcov-229e or hcov-oc43, (2) ldi, (3) ldi and either hcov-229e or hcov-oc43, or (4) neither ldi nor hcov-229e nor hcov-oc43. the respiratory illnesses associated with hcov-nl63 and hcov-hku1 are described individually. the severity of illness was assessed by use of the self-reported, 6 symptom-based, chronic lung disease severity index (cldsi), which was developed, as part of the veteran's health study [43] [44] [45] , to evaluate functional status and well-being and the effects that chronic lung disease has on quality of life. the cldsi score ranges from 6 (best) to 27 (most severe) [43] . hospitalizations during the study were identified on the basis of serious-adverseevent forms. hospitalization was considered to be temporally associated with an acute respiratory illness if it occurred either during the illness or ͻ1 month thereafter. descriptive statistics were used for the initial analysis of variables; 2 ϫ 2 2 tests or fisher exact tests were used to compare categorical characteristics of the illness groups, and the wilcoxon rank-sum test was used to compare continuous characteristics. p values are reported; as an arbitrary correction for multiple comparisons, p ͻ .01 was considered to be statistically significant. an fev 1 change between study visits was considered to be clinically significant if it was 15% [37, 46] . univariate logistic regression and 2 analysis were used to identify the univariate association of illness group with each of 11 clinical symptoms and signs of acute respiratory illness and the influenzavaccine group. multivariate analysis using stepwise logistic regression identified factors independently associated with hcov infections. p ͻ .10 was necessary for the factor to be included in the logistic model, and p ͻ .05 was necessary for it to be retained in the model. a total of 715 acute respiratory illnesses occurred among 2215 vaccinated patients; 678 of them were acute respiratory illnesses that were clinically assessable in 585 patients (503 patients had 1 acute respiratory illness and 82 had more than 1 respiratory illness). we tested for hcov-229e and hcov-oc43 infections in 679 illnesses among 586 patients, but only 665 among 575 patients that were tested for hcov-229e and hcov-oc43 infections were clinically assessable. overall, 90 (14%) of the 665 fully assessable illnesses were positive for either hcov-229e or hcov-oc43 infection (table 1) , and the number of illnesses associated with hcov-oc43 was ϳ3 times more than that associated with hcov-229e. of the 648 illnesses for which paired serum specimens were available, 74 (11%) had 4-fold changes in the titer of antibody to either hcov-229e or hcov-oc43 (table 1) . of the 504 illnesses assessed by rt-pcr, 50 (10%) were positive for either hcov-229e or hcov-oc43 (table 1) , and none were positive for sars-coronavirus. none of the 17 illnesses for which only an nop specimen was available were positive by rt-pcr. of the 161 illnesses for which only paired serum specimens were available, 18 (11%) were positive for hcov infection (1 for hcov-229e and 17 for hcov-oc43). of the 487 illnesses for which both nop-swab and paired serum specimens were tested, 72 (15%) were positive for either hcov-229e or hcov-oc43 (table 2). the proportion of these 487 illnesses that was found to be positive by antibody testing (56 [11%]) was not significantly different from that found to be positive by rt-pcr (50 [10%]), but only 34 (47%) of the 72 illnesses positive for either hcov-229e or hcov-oc43 were positive by both antibody testing and rt-pcr. no patient experienced more than 1 hcov-associated illness (figure 1). the hcov-229e-and hcov-oc43-associated illnesses occurred throughout the follow-up period at 19 of the 20 study sites. the majority of illnesses (50) associated with hcov-229e and -oc43 occurred during december 1998 and january 1999 (figure 1). until march 1999, the number of hcov-oc43associated illnesses was greater than the number of hcov-229e-associated illnesses (figure 1). our analysis of 585 first episodes of acute respiratory illnesses included 81 that were associated with hcov-229e and/or hcov-oc43 and 94 that were associated with ldi; 19 (20%) of the 94 ldi-associated illnesses were confirmed by culture, and the remainder were confirmed by serologic assay; 88 ldiassociated illnesses had 1 type of influenza virus (a/h3n2 in 59, a/h1n1 in 5, and b in 24), 3 had both type a and type b of influenza virus, and 3 had both a subtypes. a total of 12 of the first episodes of acute respiratory illness were associated with both ldi and either hcov-229e or hcov-oc43. demographic characteristics and health-care outcomes (hospitalization, prednisone use, and death) of the illness groups are listed in table 3. the baseline demographic characteristics of the group of 1630 patients who had no acute respiratory illness diagnosed during the study were similar to those of patients who did have such an illness diagnosed during the study (table 3) . in the hcov, ldi, and non-hcov/ non-ldi groups, prednisone use during the study, but not necessarily during the acute respiratory illness, increased from the levels at enrollment (table 3) ; the proportion of patients who used prednisone at some time during the study was smaller in the no-illness group and the ldi group than in the non-hcov/non-ldi group. the proportion of patients who were hospitalized during the study was significantly smaller in the no-illness group than in the non-hcov/non-ldi group (p ͻ .001) (table 3) . of the patients who were hospitalized, a majority were hospitalized either during or 1 month after acute respiratory illness that was associated with hcov infection and/or ldi (table 3) . all deaths in the hcov and ldi groups occurred ͼ1 month after the acute respiratory illness; of the 4 deaths in the non-hcov/non-ldi group, 2 occurred 1 month after the acute respiratory illness. of patients with illness only related to hcov, each of the 11 symptoms and signs of acute respiratory illness was found in ͼ50% of patients with hcov-related illness, and ͼ80% of these patients had new or increased cough, sputum production, nasal congestion, and fatigue and/or malaise (table 4). in stepwise logistic regression analysis, myalgia was found to be significantly less frequently associated with hcov illness (table 4) . patients in the ldi group were less likely to receive laivϩtiv vaccines. for each of the 11 signs and symptoms, the proportion of paa viral nucleic acid of either hcov-229e or hcov-oc43 was detected in the nop swab specimen. b 4-fold change in antibody titer to either hcov-229e or hcov-oc43, when acute and convalescent serum specimens were compared. c serologic assay indicated that 1 illness was positive for both hcov-229e and -oc43. tients in the hcov group was not significantly different from that in the non-hcov/non-ldi group (table 4) . compared with the mean fev 1 before the acute respiratory illness, both that at the visit for the first episode of acute respiratory illness and that 3-4 weeks later did not change in the hcov group; however, in both the ldi group and the non-hcov/non-ldi group, it was significantly lower (worse) at the visit for the first episode, improving 3-4 weeks later (table 5) . at the visit for the first episode, the proportion of the ldi group that had 15% worsening in fev 1 was significantly greater than both that of the hcov group and that of the non-hcov/non-ldi group. in the hcov, ldi, and non-hcov/non-ldi groups, the mean cldsi score increased (worsened) significantly at the visit for the first episode, improving 3-4 weeks later in the ldi and non-hcov/ non-ldi groups (table 5); the proportion of patients with a included among these 93 illnesses are 3 (2 hcov-229e and 1 hcov-oc43) that were not among the 665 illnesses that were assessable. for the patients infected with hcov-229e and hcov-oc43, the other respiratory illnesses, occurring before or after the hcov-associated illness, are also shown. of the patients with an illness associated with either hcov-229e or hcov-oc43, 1 had laboratory-documented influenza (ldi) before the hcov illness, 4 had an hcov illness before ldi, 13 had ldi concurrent with the hcov infection during the illness, 9 had a non-hcov/non-ldi illness before the hcov illness, and 12 had either an hcov-229e or hcov-oc43 illness before a non-hcov/non-ldi illness. each row represents 1 patient, and the reporting site is identified by the 2-letter code for us states and puerto rico (pr), to the left of the graph. of 20 sites, 19 in 13 states and pr reported illnesses associated with hcov (hcov-229e in 11 states and hcov-oc43 in 12 states and pr). hcov-associated illnesses were reported by 2 sites in florida, 2 in southern and 1 in northern california, 2 in texas, and 2 in virginia; study sites reporting the most hcov-229e and -oc43-associated illnesses were in virginia (15 illnesses), alabama (13 illnesses), minnesota (10 illnesses), missouri (9 illnesses), and texas (9 illnesses). the cumulative numbers of illnesses of hcov-associated illnesses, regardless of whether it was a first episode of acute respiratory illness and including those associated with hcov and those with both hcov and ldi, are graphed by calendar month (the cumulative number is that which occurred up to the beginning of the corresponding month); 32 hcov-associated illnesses (5 hcov-229e and 27 hcov-oc43) occurred by the end of 1998, and 61 occurred during 1999 (19 hcov-229e, 41 hcov-oc43, and 1 with both strains). the only period when hcov-229e-associated illnesses predominated was after february 1999 (10 hcov-229e and 7 hcov-oc43). 15% worsening in cldsi score at the visit for the first episode was largest in the ldi group. eight patients had a 4-fold rise in antibody titer to hcov-nl63. in these patients, 7 of the illnesses were first episodes of acute respiratory illness, and 4 were positive, by rt-pcr, for hcov-nl63. the sequences of the rt-pcr products were located between positions 22569 and 22882 and were very similar to that of hcov-nl63 (genbank accession number ay567487); there was 98%-100% similarity at the nucleotide level. one patient had a 4-fold rise in antibody titer to hcov-hku1 in association with the first episode, without concomitant evidence of infection by either hcov-229e or hcov-oc43 (table 6). all illnesses were associated with 5-10 symptoms of acute respiratory illness. of these 9 patients, 1 was hospitalized not in association with hcov, 3 received prednisone (including 1 who received it in association with hcov-hku1), and none died. hcov infections were clinically significant in this population of older patients with copd and other chronic illnesses. the frequency of hcov-229e and hcov-oc43 infections that were associated with acute respiratory illnesses was similar to that for ldi infection. we may have underestimated the number of hcov-associated illnesses; illnesses not meeting our criteria for evaluation were not assessed, and an end-of-study serum sample was not obtained, so we were unable to estimate the frequency of hcov infections in the 1630 patients who did not have an acute respiratory illness. hcov-associated illnesses were identified at diverse geographic sites within the continental united states and copd, chronic obstructive pulmonary disease; fev 1 :fvc, ratio of forced expiratory volume in 1 s to forced vital capacity; hcov, human coronavirus; ldi, laboratory-documented influenza; na, not applicable; ppfev 1 , percentage of predicted fev 1 . a includes both hcov-229e and hcov-oc43. b no acute respiratory illness met the criteria for evaluation in the study. c p ͻ .01 for the higher value in the ldi group vs. the non-hcov/non-ldi group; p ͻ .001 for the higher value in the no-illness group vs. the non-hcov/non-ldi group. d p ͻ .01 for the higher proportion of the group with both hcov and ldi , vs. each of the other groups. e p ͻ .01 for the higher proportion of the group with both hcov and ldi; p ͻ .001 for the non-hcov/non-ldi group vs. the ldi-only group. f p ͻ .01 for the higher proportion of the non-hcov/non-ldi group vs. the ldi-only group; p ͻ .001 for the no-illness group. g p ͻ .01 for the lower proportion of the no-illness group vs. the non-hcov/non-ldi group. h p ͻ .001 for higher mean number in the non-hcov/non-ldi group vs. the no-illness group i p ͻ .01, for the lower proportion of the non-hcov/non-ldi group vs. the no-illness group. likelihood of hcov-229e and/or hcov-oc43, or (95% ci) puerto rico. more than 80% of the hcov-associated infections were characterized by new or increased cough, sputum production, nasal congestion, and fatigue and/or malaise. in the multivariate logistic regression analysis, hcov-associated illness was statistically less likely than ldi to be associated with myalgia. hence, hcov infections were difficult to distinguish from ldi in this influenza-vaccinated population, whose influenzaassociated illnesses may have been less severe than those in an unvaccinated population. illnesses not associated with either hcov or ldi were not easily distinguishable, statistically, from hcov. in previous analyses, influenza was more likely to be associated with fever, myalgia, and a worsening in fev 1 than were noninfluenza acute respiratory illnesses [47, 48] . acute respiratory illness, no matter the cause, was associated with significant worsening in the cldsi score, but, on the basis of changes in fev 1 , hcov appeared to be less severe . serologic and rt-pcr assays were used as complementary tests for detection of infection by hcov-229e and hcov-oc43. more illnesses were positive for hcov by serologic than by rt-pcr criteria. delays in collection of nop specimens after the onset of symptoms of acute respiratory illness, as well as the long interval (ϳ7 years) between collection and assay of the samples, may have resulted in numbers of viral copies that were below the threshold for detection by the rt-pcr assay. hcov-oc43 and hcov-229e infections were associated with more illnesses in our older, chronically ill population than might have been expected on the basis of some previous reports [4 -6, 10, 11, 23, 26, 30] , although rates similar to those found by the present study have been reported. for instance, 22.9% of 70 virus-associated respiratory-tract infections in patients with copd that were reported by greenberg et al. were due to either hcov-229e or hcov-oc43 [8] . in a community-based study of elderly patients conducted during 1998 -1999, graat et al. [12] reported that 17% of 108 respiratory infections were due to hcov-229e and hcov-oc43 and that this frequency was second to that for rhinoviruses. as in the present study, hcov-oc43 also predominated over hcov-229e in a number of other studies [5, 6, 8, 15, 23, 30, 49] , whereas hcov-nl63 and hcov-hku1 predominated in 2 recent studies [26, 50] . we identified a small number of illnesses associated with the recently described hcov-nl63 and hcov-hku1. hcov-nl63 and hcov-hku1 cocirculated during the 1998 -1999 winter respiratoryvirus season at geographically diverse sites in the united states, and the spectrum of illness was similar to that for the hcov-229e and hcov-oc43 strains. respiratory infection due to hcov has been associated with hospitalization. glezen et al. [9] have reported that, in 26 (2.5%) of 1029 patients hospitalized because of acute respiratory conditions, the infections were related to either hcov-229e or hcov-oc43 and that 14 of these 26 patients were adults ͼ45 years of age. falsey et al. [11] have reported that 8 of 100 hospitalized elderly patients had concomitant hcov-229e or hcov-oc43 infection. in the present study, hospitalization was associated with respiratory infection in all illness groups, both during the month immediately after the visit for the first episode of acute respiratory illness and throughout the study follow-up. there was a lower rate of hospitalization in the group without an acute respiratory illness that qualified for evaluation by the a includes both hcov-229e and hcov-oc43. b p ͻ .001 for lower mean value during illness than before illness. c p ͻ .001 for lower mean value during illness than before illness. d p ͻ .001 for higher mean value 3-4 weeks after illness than during illness. e p ͻ .001 for higher mean value 3-4 weeks after illness than during illness. f p ͻ .001 for higher proportion in the ldi group than in the group with hcov-229e and/or -oc43 and the non-hcov/non-ldi group. g p ͻ .001 for higher mean value during illness than before illness, within each group. h p ͻ .001 for higher mean value during illness than 3-4 weeks after illness. i p ͻ .001 for higher proportion in the ldi group than in the non-hcov/non-ldi group. present study; during the study follow-up, the overall mean number of hospitalizations per patient in the hcov-infected group was more than double that in the no-illness group. in all illness groups, prednisone use increased during the study. although commonly thought to be associated with cold months [13, 18, 20, 21, 25] , hcov infection has been reported as also occurring during other times of the year [22, 23, 50] , and the seasonality of hcov in tropical and subtropical areas is not so restricted [26, 30] . the present study's follow-up period extended from october to april, and hcov infections were detected throughout that period, although they were most frequent during december and january. the results of the present study underscore the clinical importance of hcov infection in older 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rna viruses by psoralen derivatives psoralen inactivation of influenza and herpes simplex viruses and of virus-infected cells comparison of five calculation modes for antibody elisa procedures using pertussis serology as a model a symptom-based measure of the severity of chronic lung disease: results from the veterans health study patient-reported measures of health, the veterans health study measurement strategies designed and tested in the veterans health study interpretative strategies for lung function tests impact of a winter respiratory virus season on patients with copd and association with influenza vaccination recognizing influenza illness in influenza-vaccinated, older patients with chronic obstructive pulmonary disease erdman dd. human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays clinical disease in children associated with newly described coronavirus subtypes we thank gira b. patel and nancy krudwig, for laboratory technical assistance; carolyn novotny, for expert secretarial assistance, and yolanda blocker-hearn, for preparation of the figure. key: cord-011710-rz23ozxb authors: aoki, fred y.; hayden, frederick g. title: the beneficial effects of neuraminidase inhibitor drug therapy on severe patient outcomes during the 2009–2010 influenza a virus subtype h1n1 pandemic date: 2013-02-15 journal: j infect dis doi: 10.1093/infdis/jis727 sha: doc_id: 11710 cord_uid: rz23ozxb nan elsewhere in the pages of the journal, muthuri et al answer a question of substantial contemporary importance to clinicians and public health decision makers, namely, whether antiviral therapy for influenza can reduce severe outcomes of the disease in hospitalized patients [1] . in a welcome affirmation of the effectiveness of neuraminidase inhibitor (nai) treatment, they report that a meta-analysis of 90 observational studies involving 34 895 patients of whom 85% had laboratory-confirmed 2009 pandemic influenza a virus subtype h1n1 (a [h1n1]pdm09) infection revealed that antiviral therapy, principally oseltamivir, initiated within 48 hours of symptom onset reduced the likelihood of severe outcomes, namely admission to a critical care unit or death, by 49%-65%. the strength of the conclusions resides both in the methodologic rigor applied to the meta-analysis of the component studies and the large numbers of studies and patients analyzed. this finding confirms earlier reports of reduced mortality with oseltamivir therapy in those hospitalized with seasonal [2] or avian a(h5n1) [3, 4] influenza. the findings in the current report also complement observations from ecologic studies [5] . for example, japan, the country with highest per capita use of nais during the 2009 pandemic, also had the lowest case-fatality rate and remarkably no reported deaths in a(h1n1)pdm09-infected pregnant women [6, 7] . more recently, a countrybased analysis found that each 10% increase in oseltamivir supply (calculated in kilograms per 100 000 people) was associated with a 1.6% reduction in a (h1n1)pdm09 mortality [8] . while previous analyses and the current one have generally found greater effects with earlier compared with later therapy, it is important to note that multiple observational reports in those hospitalized with seasonal, a(h1n1)pdm09, or avian a(h5n1) influenza indicate that a treatment benefit can be demonstrated up to 5 days after symptom onset, including studies in high-risk groups such as pregnant women [2] [3] [4] [9] [10] [11] . it makes sense that even delayed antiviral intervention would benefit patients, when one considers the protracted duration of viral replication in many patients with serious influenza, sometimes despite oseltamivir administration [12, 13] , compared with its relatively short duration in outpatient adults with uncomplicated influenza. as pointed out by muthuri et al, the timing of nai initiation was examined carefully in only a few studies. delayed initiation often reflected late diagnosis or presentation to care and belated efforts at salvage. indeed, during the pandemic, misunderstanding the potential value of therapy initiated beyond 48 hours of illness unfortunately led many clinicians to not administer nais to those who might have benefited. thus, using 48 hours as a threshold for delayed therapy in hospitalized patients covers a diversity of reasons for late onset of therapy and may be less relevant than in outpatient settings. while time to treatment initiation is a key variable in assessing effectiveness, future analyses should also examine illness severity, cause for hospitalization (eg, influenza-associated pneumonia, exacerbations of underlying conditions, and presence of secondary bacterial infections), comorbidities, and virologic markers at the time of initiating therapy, preferably with propensity scoring that takes such factors into consideration. this current meta-analysis has advanced our understanding of the effectiveness of antiviral therapy for the management of pandemic influenza that began during the 1968-1969 global outbreak caused by the influenza a(h3n2) hong kong virus 44 years ago. antiviral therapy with amantadine was then first demonstrated to be more effective than placebo in accelerating the resolution of pandemic illness in otherwise healthy adults [14, 15] . in 1978, during the russian influenza a/ussr/77(h1n1) pandemic, comparable therapeutic effects of both adamantanes, amantadine and rimantadine, were again demonstrated using a placebo-controlled, randomized trial design [16] . however, the limited usefulness of the adamantane class of drugs for widespread use on a public health scale came to be recognized, given the rapid emergence of resistance during treatment and, in the case of amantadine, a narrow toxic to therapeutic ratio that necessitated individualized prescribing on the basis of renal function and weight, plus careful monitoring during therapy because of side effects. the development of nai therapy provided clinicians and public health decision makers with treatment options that were less limited by concerns about viral resistance, or, in the case of amantadine, safety. cumulated evidence on nai efficacy and safety for treatment of seasonal influenza and uncertainty about the potential severity of disease caused by a new virus precluded the use of the placebo-controlled, randomized study design to test the hypothesis that nai therapy could favorably impact severe outcomes during the a(h1n1)pdm09 pandemic. therefore, it fell to analysis of data from multiple observational studies to estimate the effect of nai therapy on outcomes of the 2009-2010 pandemic, as has been done in the current report. the limitations of the data and the conclusions drawn from them are thoughtfully discussed by muthuri et al. for example, their findings of an increased likelihood of pneumonia with nai use and that nai treatment versus no preadmission nai in subsequently hospitalized patients did not significantly reduce mortality highlight the key issue of confounding by indication. as pointed out by the authors and previously by others [11] , sicker patients are more likely to receive nai therapy, and untreated patients are likely to have had milder disease. thus, comparisons between nai-treated and untreated patients in the context of these observational studies are fraught with potential confounding and underestimate beneficial drug effects. in addition, one needs to consider the key questions not addressed in this analysis. because the scope of the studies considered was limited to hospitalized patients, it could not confirm earlier reports from studies of antiviral therapy for seasonal influenza that found early treatment to reduce the risks of influenza-associated complications and hospitalizations [2] . other key medical and public health outcomes, such as drug tolerability, antiviral resistance emergence and its relationship to effectiveness, the durations of supplemental oxygen therapy, hospital care, transitional facility care, and time to overall functional recovery, and the causes of mortality also remain to be addressed [17] . the current analysis, moreover, did not have sufficient numbers to assess the effectiveness of specific nais other than oseltamivir, so it remains unclear whether inhaled drugs such as zanamivir are as safe and efficacious as ingested or injected nais in hospitalized patients. of note, the optimal dose, duration, and even makeup of nai therapy in hospitalized influenza patients remains uncertain. available data suggest that more protracted administration is warranted in seriously ill or immunocompromised patients, while other careful studies using sequential sampling of patients given oseltamivir have indicated a need for more robust antiviral effects, especially in those with severe pneumonic disease [12, 13] . such virologic findings and the occurrence of deaths despite early therapy [18] highlight the importance of developing more potent antiviral regimens for such patients, particularly antiviral combinations. however, evidence indicates that some nai combinations, particularly oseltamivir plus zanamivir, may result in antagonistic antiviral effects and lesser clinical benefit, reminding clinicians about the need for detailed preclinical studies and special care when moving on to combination antiviral therapy [19, 20] . hopefully, new data to address some of these issues will come from, in part, the ongoing randomized, controlled trials of intravenous nais. although the current study provides clinicians and public health decision makers an answer to an important question at the apex of the hierarchy of the therapeutic effects of anti-influenza drug therapy, questions remain whose solution would further advance our ability to strategically use antiviral drugs to improve the management of influenza outbreaks, small and large. some of these remaining questions include whether antiviral therapy reduces transmission of influenza; whether therapy mitigates disease without reducing the immune responses to infection and, hence, future protection against drift virus variants; whether postexposure prophylaxis has advantages over early initiation of therapy; and which strategies are most effective at reducing risk of antiviral resistance development and transmission. the current report included studies conducted up to the declaration of the end of pandemic, in august 2010. however, it is important to emphasize that a(h1n1)pdm09 continues to circulate and cause serious illness and mortality. studies of mortality patterns in past pandemic periods found that individuals aged ≤65 years continued to experience excess mortality for many years after introduction of the pandemic strain [21] . consequently, wider use of nai therapy based on the recommendations of centers for disease control and prevention and the world health organization can mitigate these effects. of concern, studies have found reversion toward prepandemic prescribing patterns in united states [22] and elsewhere, with the consequence of worsened outcomes. in closing, the current report included patients treated in 29 different countries, reflecting the global scale of the pandemic and the widespread interest of researchers in the question of the beneficial effects of nai therapy on patient outcomes. however, all the studies included in the systematic review were observational designs, most were retrospective, and the methods used for data collection and reporting varied, so it is unsurprising that the meta-analysis found considerable heterogeneity across studies and risk of bias in reporting findings. furthermore, the authors were hampered by the lack of access to individual patient data. such circumstances highlight the critical need for clinical research networks, both domestic and international, that can collect samples and data in a systematic, prospective manner and conduct randomized trials of interventions. in this regard, a number of international funding organizations recently have supported the formation of the international severe acute respiratory and emerging infection consortium (isaric), a global federation of >30 existing clinical research networks, launched in december 2012 [23] . isaric is committed to undertaking pathogenesis and treatment studies both in response to emerging infectious disease events and in severe acute respiratory infections necessitating hospitalization including influenza during the interpandemic period. such initiatives need to be supported and sustained if we are to develop the key evidence needed to inform clinical management for current and future severe acute respiratory infection and emerging infectious disease threats. impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-10 influenza a (h1n1) pandemic: a systematic review and metaanalysis in hospitalized patients antivirals for treatment of influenza a systematic review and meta-analysis of observational studies strengthening observational evidence for antiviral effectiveness in influenza a (h5n1) determinants of viral effectiveness in influenza virus a subtype h5n1 impact of antiviral treatment and hospital admission delay on risk of death associated with 2009 a/h1n1 pandemic influenza in mexico transmission dynamics and impact of pandemic influenza a (h1n1) 2009 virus review of the pandemic (h1n1) 2009 among pregnant japanese women supply of neuraminidase inhibitors related to reduced influenza a (h1n1) mortality during the 2009-2010 h1n1 pandemic: an ecological study treatment with neuraminidase inhibitors for critically ill patients with influenza a (h1n1) pdm09 antiviral therapy and outcomes of patients with pneumonia caused by influenza a pandemic (h1n1) virus pandemic 2009 influenza a (h1n1) virus illness among pregnant women in the united states viral clearance and inflammatory response patterns in adults hospitalized for pandemic a (h1n1) virus pneumonia the natural viral load profile of patients with pandemic 2009 influenza a (h1n1) and the effect of oseltamivir treatment therapuetic effectiveness of amantadine hydrochloride in naturally occurring hong kong influenza-double blind studies amantadine therapy of epidemic influenza a (hong kong) successful treatment of naturally occurring influenza a/ussr/77 h1n1 end points for testing influenza antiviral treatments for patients at high risk of severe and life-threatening disease fatal cases of pandemic (h1n1) 2009 influenza despite their early antiviral treatment in japan triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant influenza virus strains in vitro efficacy of oseltamivir-zanamivir combination compared to each monotherapy for seasonal influenza: a randomized placebocontrolled trial pandemic versus epidemic influenza mortality: a pattern of changing age distribution reduced influenza antiviral treatment among children and adults hospitalized with laboratoryconfirmed influenza infection in the year after the 2009 pandemic international severe acute respiratory infection consortium web site potential conflicts of interest. f. g. h. has served as an unpaid consultant to multiple companies involved in influenza antiviral development (including roche, glaxosmithkline, bio-cryst, nexbio, toyama). the university of virginia received honoraria for his work in the neuraminidase inhibitor susceptibility network (nisn) from 2008 to 2011; nisn received financial support from roche and glaxosmith-kline. f. y. a. reports no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-304766-h9kuytuf authors: lei, hao; xu, modi; wang, xiao; xie, yu; du, xiangjun; chen, tao; yang, lei; wang, dayan; shu, yuelong title: non-pharmaceutical interventions used to control covid-19 reduced seasonal influenza transmission in china date: 2020-09-08 journal: j infect dis doi: 10.1093/infdis/jiaa570 sha: doc_id: 304766 cord_uid: h9kuytuf to suppress the ongoing covid-19 pandemic, the chinese government has implemented a set of non-pharmaceutical interventions (npis). because covid-19 and influenza have similar means of transmission, it is hypothesized that npis targeting covid-19 may also affect influenza transmission. in this study, the extent to which npis targeting covid-19 have affected seasonal influenza transmission was explored. indicators of seasonal influenza activity in the epidemiological year 2019/20 were compared with those in 2017/18 and 2018/19. results show that the incidence rate of seasonal influenza reduced by 64% in 2019/20 (p&0.001). these findings suggest that npis aimed at controlling covid-19 significantly reduced the seasonal influenza transmission in china. (105 words) in december 2019, a novel coronavirus named severe acute respiratory syndrome coronavirus-2 (sars-cov-2), led to a pandemic of coronavirus disease 2019 [0]. to suppress the covid-19 pandemic, from january 23-25, 2020, 30 provinces began a 1-level response and implemented a set of non-pharmaceutical interventions (npis), including not only the classical isolation of the confirmed/suspected cases and quarantine of their close contacts in special facilities, but also unprecedented measures like strict community containments with social distancing, such as the wuhan city travel ban to prevent the exportation of cases from wuhan and other priority areas of hubei province, extension of the spring festival holiday, suspension of traffic and transportation, closure of school and entertainment venues, banning of mass gathering activities, compulsory community use of facemasks in public areas, and information about the epidemic and prevention measures widely disseminated, public risk communications and health education strengthened, new hospital built to ensure that all cases could be treated [2] . both covid-19 and seasonal influenza are respiratory infections and mainly transmitted via respiratory droplets and contact routes [3] [4] [5] . in addition, northern china experiences influenza epidemics concentrated in winter-spring months, while southern china experiences a semi-annual cyclic pattern with clear peaks in both summer and winter. in both southern and northern china in the winter-spring months, the seasonal influenza epidemics always peak in january-february [6] , so months in which in 2020, the covid-19 pandemic overlapped with the flu season in the winter-spring months in china. it is postulated that the population-wide npis implemented to contain covid-19 would also have effects on seasonal influenza. two studies conducted in singapore and taiwan, china have reported a reduction in influenza activity during the covid-19 pandemic period [7, 8] . compared with these two regions, mainland china has significantly more covid-19 cases and has implemented stricter npis, including massive mobility restrictions, universal fever screen, use of big data and artificial intelligence to strengthen contact tracing and the management of priority populations [2] . this study is significant because it showcases a country that has experienced a relatively high covid-19 caseload and has implemented an intensive package of npis. by examining how npis targeting covid-19 affect the transmission of seasonal influenza epidemics in china, the study may help other countries to plan for the dual burden of covid-19 and influenza in the future. in this study, weekly reports of influenza cases from years 2017 to 2020 from the chinese national influenza center were used. the dataset provided the number of visits, the number of influenza like illness (ili) cases, and the number of specimens tested, the number of laboratory-confirmed influenza cases, in 554 sentinel hospital. the standard case definition of ili, is a body temperature 38 c with either cough or sore throat, in the absence of an alternative diagnosis [6] . please refer to shu et al. [9] for additional details about the chinese influenza surveillance system. since china is located in the northern hemisphere, an epidemiological annual cycle was defined as the period from october 1st (calendar week 40, epidemiological week 1 as noted in this study) to september 31st in the next year [6] , i.e. may 24, 2020, when this study stared. several indicators were defined to characterize influenza activity in china. first, the ili rate was defined as the number of ili cases divided by the number of visits. second, the influenza viral positive rate was defined as the number of laboratory-confirmed influenza cases divided by the number of specimens tested. third, the incidence rate was defined as the ili rate among the visiting patients in sentinel hospitals multiplied by the influenza viral positive rate, a count more precisely representing the influenza infections [10,] . the weekly incidence rate was then interpolated to daily incidence rate using splines [11] . changes in transmissibility were estimated over time using the effective reproductive number, rt. time-varying estimates of the daily effective reproductive number were made using the r package epiestim, assuming a mean serial interval of 2.85 days and a standard deviation of 0.93 days [12] . estimates of rt were conducted with r version 3.6.3 (r foundation for statistical computing). indictors of influenza activity in the year 2019/20 were compared with the average from the corresponding period in the two preceding epidemiological years. paired difference t-tests were performed using excel. compared with the epidemiological years 2017/18 and 2018/19, the number of outpatient visits was a lightly higher in the epidemiological year 2019/20 before npis implementations (p<0.001). from january 23, 2020, it would be reasonable to expect a rapid decrease of the outpatient visits due to the covid-19 pandemic indeed, results show that when compared with the same period during epidemiological year 2017/18-2018/19, the number of outpatient visits decreased by 56% in the 4 weeks following npis implementations (p<0.001) ( figure 1a ). however, because of the similarities in symptoms between covid-19 and influenza, the number of samples tested per week only decreased by 28% (p<0.001) ( figure 1b) , and there were no significant changes in ili rate (p=0.117) ( figure 1c ). in contrast, the influenza positive rate in samples in the epidemiological year 2019/20 decreased by 79% (p<0.001) ( figure 1d ). in the epidemiological year 2019/20, influenza incidence rates peaked on epidemiological week 13, and there was no significant difference with the mean influenza incidence rates in epidemiological year 2017/18 and 2018/19 (p=0.496) before npis implementation ( figure 2a ). when the npis were implemented to contain covid-19, the influenza incidence rates declined rapidly to below the average of the preceding two years, and it reached almost 0 within 7 weeks after the npis implementation (figure 2a) . the mean incidence rate reduced by 64% compared with the preceding two years (p<0.001). there was also a significant decrease in the daily effective reproductive number in epidemiological year 2019/20 in the 3-4 weeks after the npis were implemented to control the covid-19 pandemic compared with the preceding two years (p<0.001) ( figure 2b ). five or more weeks after the npis implementation, influenza activity reached a very low level (figure 2a ). some of the npis used to control covid-19,such as school closure [13] , community use of facemasks and hand hygiene [14] , have been shown to be effective against influenza epidemics. therefore, it's not surprising that these npis, when used to control covid-19, would also reduce the seasonal influenza transmission in china. however, what was unexpected from this study was the extent to which the npis reduced influenza transmission. the study showed that the mean incidence rate of influenza reduced by 64% in the epidemiological year 2019/20 after implementing after the implementation of npis to prevent covid-19. the reduction of 64% is significantly higher than the reported efficiency of single interventions used against influenza epidemics in the past, such as school closure (16-18% reduction of seasonal influenza cases) [13] and community use of facemasks (35% reduction of ili cases at most) [14] . this suggests that there may be a synergistic effect of deploying multiple npis at the same time. it also suggests that certain npis which have been uniquely utilized during the covid-19 pandemic, such as suspending public transport by bus and subway rail, might also be effective against seasonal influenza epidemics. healthcare avoidance during the covid-19 pandemic may be an important confounder for the results presented. however, it is unlikely that this confounding effect is significant for a number of reasons. first, in order to contain the covid-19 pandemic, the government encouraged people with ili to seek medical care in order to obtain a diagnosis., in addition, the influenza laboratories based on different levels of cdc ensured the appropriate influenza testing capacity for differential diagnosis with sars-cov-2. lastly, and the most important, healthcare avoidance did not explain the lower influenza positive rate in the tested samples ( figure 1d ). therefore, the evidence strongly suggests that the decreasing incidence rate of seasonal influenza in china was the result of the strict npis implemented in response to covid-19. there are two main limitations of this study. the first limitation is that there was an expected decrease in influenza transmission in february-march [6] , however, the decrease in 2019/20 is statistically significantly faster than previous years. the second limitation is the interpolation of daily incidence rates of influenza from the weekly data. the daily variation in influenza transmissibility might have been reduced because of this interpolation. the use of daily data of influenza, if available, would address this limitation. in conclusions, this study found a marked decline in seasonal influenza activity in china the novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) -china report of the who-china joint mission on coronavirus disease 2019 (covid-19) modes of transmission of virus causing covid-19: implications for ipc precaution recommendations routes of transmission of influenza a h1n1, sars cov, and norovirus in air cabin: comparative analyses transmission of influenza a in human beings characterization of regional influenza seasonality patterns in china and implications for vaccination strategies: spatio-temporal modeling of surveillance data decreased influenza incidence under covid-19 control measures collateral benefit of covid-19 control measures on influenza activity a ten-year china-us laboratory collaboration: improving response to influenza threats in china and the world real-time influenza forecasts during the 2012-2013 season mitigation of influenza b epidemic with school closures how generation intervals shape the relationship between growth rates and reproductive numbers estimating the impact of school closure on influenza transmission from sentinel data mask use, hand hygiene, and seasonal influenzalike illness among young adults: a randomized intervention trial the authors declare no conflict of interest. key: cord-313344-rqvi2ksc authors: farcas, gabriella a.; poutanen, susan m.; mazzulli, tony; willey, barbara m.; butany, jagdish; asa, sylvia l.; faure, peter; akhavan, poolak; low, donald e.; kain, kevin c. title: fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus date: 2005-01-15 journal: j infect dis doi: 10.1086/426870 sha: doc_id: 313344 cord_uid: rqvi2ksc severe acute respiratory syndrome (sars) is characterized by pulmonary compromise; however, patients often have evidence of other organ dysfunction that may reflect extrapulmonary dissemination of sars coronavirus (sars-cov). we report on the distribution and viral load of sars-cov in multiple organ samples from patients who died of sars during the toronto outbreak. sars-cov was detected in lung (100%), bowel (73%), liver (41%), and kidney (38%) in 19 patients who died of sars, with the highest viral loads observed in lung (1.0 × 10(10) copies/g) and bowel (2.7 × 10(9) copies/g). fatal sars was associated with multiorgan viral dissemination in a distribution that has implications for disease manifestation, viral shedding, and transmission. epidemic resulted in 251 probable cases and 44 deaths, making toronto the most affected center outside of asia [4] . coronaviruses are a diverse family of enveloped, positivestrand rna viruses that cause a wide spectrum of intestinal and respiratory diseases. for animal coronaviruses, data suggest that changes in the spike glycoprotein may contribute to differences in tissue tropism and virulence [5] . unlike the enteric and pneumonic illnesses associated with the animal coronaviruses, illnesses associated with previously recognized human coronaviruses (hcov-229e, hcov-oc43, and hcov-nl63) had been largely confined to the upper respiratory tract [6] . however, sars-cov appears to have arisen as a result of the zoonotic transmission of an animal coronavirus to humans, and little is known about its potential tissue tropism in humans [6] . although sars is primarily characterized by the presence of lower respiratory tract infection and subsequent pulmonary compromise, patients often have evidence of other organ dysfunction, including gastrointestinal symptoms and abnormal liver function [2, 7] , as well as splenic atrophy and lymphadenopathy [8] . this may reflect widespread immunopathology or the presence of extrapulmonary sars-cov dissemination and replication, as has been observed in other species infected with animal coronavirus [9] . the purpose of the present study was to investigate the presence of sars-cov, the degree of viral dissemination, and the viral loads in multiple organ samples from all patients who died of sars during the toronto outbreak (march to september 2003) and underwent a postmortem examination and compare the results with those found in patients who died of other causes during the outbreak. we demonstrate that, in fatal cases of sars, sars-cov is often disseminated to multiple organs, in patterns that have implications for clinical manifestations, viral shedding, and disease transmission. subjects and methods. all patients whose condition met the centers for disease control and prevention (cdc) and world health organization (who) case definition of probable sars in toronto, canada, and who underwent a postmortem examination during the period from the beginning of the outbreak in march to september 2003 were eligible for inclusion in this study. autopsies were performed on 21 of the 44 patients with deaths attributed to sars. fifteen autopsies were performed using a modified protocol for highly infectious cases, and 6 autopsies were restricted to biopsies of specified tissues. fifty-one patients who died of causes other than sars during the outbreak and who underwent postmortem examination were included as controls. causes of death in these individuals included congestive heart failure, cerebrovascular accidents, ath-erosclerotic heart disease, chronic obstructive pulmonary disease, invasive group a streptococcal infection, amiodorone pulmonary toxicity, and pulmonary fibrosis. clinical details were extracted, by use of hospital records, into standardized data extraction forms. results of antemortem microbiologic examination for routine bacterial and viral respiratory pathogens from the 21 individuals with probable sars were negative. however, on the basis of postmortem examination, 3 of these patients had evidence of coinfection with other organisms: 2 women had evidence of coinfection with aspergillus species, and 1 patient had evidence of cytomegalovirus infection. this study was reviewed and approved by the research ethics boards of the mount sinai hospital and the university health network, toronto, canada, and by the chief coroner's office of ontario, canada. a total of 212 discrete postmortem organ samples, including lung, liver, spleen, kidney, small bowel, large bowel, lymph nodes, heart, and skeletal muscle, were prospectively collected from the 21 patients who died of sars and underwent autopsies. two of these patients died 1100 days after disease onset, and all organ samples tested were determined by real-time reverse-transcriptase polymerase chain reaction (rt-pcr) to be negative for sars-cov. these patients have been excluded from all subsequent analyses. as controls, 228 postmortem organ samples from the same tissues were prospectively obtained from 51 subjects who died of causes other than sars. all samples collected at the time of autopsy were snap frozen in a mixture of absolute ethanol and dry ice and were subsequently stored at ϫ70њc until analyzed. all samples were coded and were independently processed and examined. specimen analysis and interpretation were performed blinded to other diagnostic investigations. for rt-pcr, organ tissue samples were homogenized, in rlt buffer (qiagen), by use of disposable tissue grinders (kendall precision), and rna was isolated by use of the rneasy mini kit (qiagen). the rt-pcr was performed using the commercially available realart hpa-coronavirus lc-rt assay (artus) on a lightcycler real-time pcr platform, as described elsewhere [10] . viral load was calculated from a standard curve based on 4 quantification standards. a standard preparation of sars-cov isolated from cell culture supernatants of veroe6 cells was used as a calibrator in each run. in addition, a second heterologous amplification system (i.e., an internal control) was included to ensure the quality of rna isolation and to identify pcr inhibition. the sensitivity and specificity of this standardized realtime rt-pcr assay for the detection of sars-cov in postmortem lung tissues were previously assessed at 100% [10] . specificity of amplicons was verified by nucleic acid sequencing. results. the 19 patients who died within 51 days after infection and underwent a postmortem examination had a mean age of 68 years (range, 43-99 years). ten of the patients were female. the mean duration of illness was 22.5 days (range, 5-51 days). sars-cov was found in 100% (19/19 patients, each with specimens collected from multiple sites, totaling 66 discrete samples) of lung samples, 73% (11/15) of bowel samples, 69% (9/13) of lymph node samples, 41% (7/17) of liver samples, 40% (7/18) of heart samples, 38% (6/16) of kidney samples, and 12% (2/17) of skeletal muscle samples. the viral loads for each organ are presented in tables 1 and 2. of interest, particularly high viral loads were observed in a recent lung-transplant recipient. all 206 postmortem samples from the 51 non-sars fatalities that occurred during the sars outbreak were negative for sars-cov. the corresponding sensitivity and specificity of the real-time rt-pcr assay were 100% (95% confidence interval [ci], 94.6%-100%) and 100% (95% ci, 98.2%-100%), respectively, for the detection of sars-cov in lung tissue within 51 days of disease onset. pathologic findings in sars-cov-infected lung specimens showed diffuse alveolar damage; however, histopathologic changes in bowel were minimal, despite the presence of sars-cov in 170% of these cases, often at high viral loads (data not shown). there were no changes on gross examination, and only minimal inflammatory changes were observed on microscopic examination. similarly, despite evidence of elevated antemortem levels of transaminases and the presence of sars-cov in the liver at autopsy in 41% of these cases, there were only minor inflammatory changes observed in the liver on microscopic examination. of note, 100% (7/7) of patients who had sars-cov detected in the liver had abnormal antemortem liverfunction test results, whereas abnormal results were found in 40% (4/10) who did not have sars-cov detected. discussion. there are limited data available detailing viral loads and extrapulmonary dissemination of sars-cov in humans. in this study, we used a standardized and validated realtime rt-pcr assay to detect and determine the viral load of sars-cov in multiple organs from a large number of individuals who died of sars during the toronto outbreak, compared with those who died of other causes. we demonstrate that, even 51 days after disease onset, sars-cov was consistently found in multiple lung lobes, suggesting that there is widespread dissemination of the virus throughout the lung at time of death. further, we observed extrapulmonary dissemination of the virus into all major organs, especially the bowel and lymph nodes. these data have implications for the clinical manifestation, disease course and outcome, and transmission of sars-cov. sars-cov viral dissemination has previously been examined in a simian model [11] . necropsy results indicated the widespread presence of sars-cov in lung but only sporadic presence of the virus in the duodenum, kidney, and spleen. although our findings that sars-cov is detected in postmortem these data, combined with evidence of viral shedding in stool and urine, viral replication in the gut, and reports of transient viremia and relatively low viral loads in the blood, suggest that viral overspill is perhaps a less likely explanation for the extrapulmonary dissemination of sars-cov that we observed in humans [12] . of interest, angiotensin-converting enzyme 2 (ace2), the putative functional receptor of sars-cov [13] , is expressed in many of the organs in which we observed sars-cov dissemination, including the gastrointestinal tract, heart, kidney, lung, lymph nodes, skeletal muscle, liver, and spleen [14] . gastrointestinal complaints-and watery diarrhea, in particular-are frequent symptoms of sars, reported at presentation by у20% of infected individuals [12] and developing in as many as 70% of individuals during the course of illness [15] . in this study, we demonstrated that the majority of patients had evidence of sars-cov in both large and small bowel, often at high viral loads. these data suggest that sars-cov displays tissue tropism for the bowel and provide a putative mechanism for the frequent occurrence of gastrointestinal symptoms in this population. this hypothesis is supported by observations that sars-cov is frequently and persistently identified in fecal specimens [1, 2, 11] and by recent electronic microscopic evidence indicating viral replication and recovery of sars-cov from postmortem small-bowel specimens [12] . collectively, these observations indicate that enteric involvement is common in sars and have implications for infection-control measures and the potential for fecal-oral transmission in community outbreaks. evidence of hepatic dysfunction is common in sars, and elevated serum alanine aminotransferase levels are observed before death in у40% of patients [6] . in this study, we observed a relationship between the presence of sars-cov in the liver and antemortem abnormal liver function tests, supporting a role for sars-cov in mediating hepatic dysfunction. furthermore, splenic atrophy and lymphadenopathy with tissue necrosis have also been reported in sars [2, 7, 8] . these findings can potentially be explained by our observations of elevated viral loads at these sites. ding et al. hypothesized that the degenerative changes observed in these organs are most likely due to disturbed cell metabolism caused by rapid viral replication [8] , but the authors lacked the viral-load evidence we present here to support these contentions. although organ damage in patients with sars may be the immunopathologic consequence of an exuberant host response [2] , evidence of elevated viral loads and of extrapulmonary viral dissemination suggests that regional viral replication could also potentially contribute to organ dysfunction and exacerbate the host response at these sites. although sars-cov has previously been detected in urine and stool [1, 2, 11] from patients with sars, to our knowledge there is only 1 report of sars-cov detection in kidney [3] . in addition, kuiken et al. found pcr evidence of sars-cov in kidney, duodenum, and stomach in 1 of 4 sars-cov-infected macaques, but no viral loads were given [11] . our findings of high viral loads in the gut and liver and moderate viral loads in the kidney are consistent with previous reports of elevated viral shedding in the stool and moderate to low shedding in urine [1, 2] . the current findings provide insight into multiorgan sars-cov dissemination and viral load at the time of death, on the basis of a prospective and systematic examination of a large number of fatal sars cases. sars-cov was consistently identified in the lungs of patients who died of sars and was not found in control individuals, supporting a direct role for sars-cov in contributing to fatal outcomes. sars-cov disseminates to other organs, which may explain, at least in part, the clinical manifestations and pattern of viral shedding observed in sars-cov-infected patients and may provide insight into the selection of appropriate clinical samples in the event of a future outbreak. identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence the severe acute respiratory syndrome outbreak of severe acute respiratory syndrome in hong kong special administrative region: case report the clinical pathology of severe acute respiratory syndrome (sars): a report from china sars coronavirus: a new challenge for prevention and therapy absolute association of coronavirus in lung tissue from fatal cases of severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study we thank the health care providers of the greater toronto region for their dedication and devotion to patient care during the severe acute respiratory syndrome (sars) outbreak. in addition, we thank nate kreiswirth, olga imas, george moussa, and the office of the chief coroner of ontario, for expert technical assistance with this study. we also thank hans-wilhelm doerr (university of frankfurt, frankfurt, germany) and matthias niedrig (robert koch-institute, berlin, germany) for generously providing the standard preparation of sars coronavirus cell culture isolate. key: cord-268490-e8jub01m authors: moscona, anne title: csi microbiology: emerging pathogens and a staged strategy for detection and discovery date: 2007-12-15 journal: j infect dis doi: 10.1086/524313 sha: doc_id: 268490 cord_uid: e8jub01m nan disease, both by enabling target selection for vaccine and drug development and by guiding patient care. in recent years, there has been substantial progress in applying molecular biologic advances to respiratory virus diagnosis [1] ; nonetheless, a major gap has been the lack of a costeffective, systematic way to identify the causes of respiratory diseases in populations. the article by renwick et al. [2] in this issue of the journal makes 2 unique contributions to this field: it demonstrates the need to consider and identify rhinoviruses as a cause of serious acute respiratory disease in children, and it establishes the masstag polymerase-chainreaction (pcr) multiplex platform as a practical tool for microbial surveillance. the masstag pcr method, developed by renwick et al. [2] and used in their study investigating the etiology of respiratory disease in hospitalized children, provides a paradigm for new detection strategies for early recognition and containment of a wide range of respiratory pathogens. this multiplex technology, in its first evaluation in 2005, was shown to be effective in identifying all the main respiratory viral pathogens, including respiratory syncytial virus, human parainfluenza viruses 1-3, metapneumovirus, influenza, and sars-cov as well as s. pneumoniae and h. influenzae [3] . in 2006, lamson et al. [4] , in the same group, went on to report the use of the method to investigate both undiagnosed influenza-like illness in new york state and the discovery of a novel genetic clade within the picornaviruses; "human rhinovirus new york" was the first new agent to be detected by use of masstag pcr. rhinoviruses have long been a relatively underappreciated cause of acute respiratory infection, a view that is beginning to change [5] . in their study in this issue of the journal, performed in collaboration with the robert koch institute (berlin, germany), renwick et al. [2] report clear evidence that links these viruses to severe respiratory disease: 75% of viruses detected among 97 nasopharyngeal aspirates from children hospitalized with acute respiratory infection-with no pathogen identified by routine methods-were rhinoviruses. independently, in publications following that by lamson et al. [4] , other investigators found similar evidence that associates severe respiratory disease with rhinovirus infection, implicating known serotypes of rhinovirus groups a and b as well as the same, novel viruses found in new york in pediatric lower-respiratory-tract infection [6] or asthma exacerbations [7] . with work now reported by 3 research groups employing different methods, the likely contribution of rhinoviruses to acute respiratory disease cannot be ignored and deserves further study. the diagnostic strength of highly multiplexed systems, such as the masstag pcr platform applied by renwick et al., in the lipkin group, builds on a series of advances by this group [2] . in 1987, the first application of subtractive cloning in microbiology led to the identification of a novel pathogen, borna disease virus, the prototype of a new family in the order mononegavirales [8, 9] . another new strategy-namely, domain-specific differential display pcr-ultimately led to the recognition of west nile virus as the cause of the outbreak of encephalitis in new york in 1999 [10, 11] . the recent development of pathogen microarrays, combined with a comprehensive panmicrobial sequence database, has added to the repertoire of methods for sensitive and broad differential diagnosis of infectious disease [12, 13] and has facilitated differential diagnosis of filovirus versus malaria in samples obtained during an outbreak of hemorrhagic fever. in early 2007, the same research group [14] pioneered the use of unbiased, high-throughput sequencing to assemble a comprehensive inventory of microflora from honeybee colonies affected by colony-collapse disorder [14] . the transition of cutting-edge pathogendiscovery technologies from research to clinical laboratories will not occur immediately. nonetheless, it is not premature to plan for this in the near future. key to implementation will be the development of a staged strategy for pathogen detection that enables low-cost and efficient differential diagnosis of infectious diseases [15] . one approach might be to begin with a multiplex pcr screen, such as the masstag pcrbased assays described here. if the initial screen fails, microarrays could be em-ployed. more labor-and resource-intensive unbiased, high-throughput sequencing would be reserved for the most difficult challenges. considerable emphasis has been placed on ensuring global access to vaccines and drugs for treatment of infectious diseases. less attention has been focused on the importance of understanding microbiological data in the context of public health. to this end, efforts such as those undertaken by the lipkin group, directed at training and equipping an international cast of investigators in state-of-theart methods for molecular diagnostics, are critical. it is no surprise that the list of investigators collaborating on papers from their laboratory often reads like a united nations of science. such worldwide capability in global infectiousdisease surveillance is important to local as well as international security. commitment to technology transfer and global collaboration is essential if we are to have the agility required to keep pace with emerging infectious diseases. pathogen surveillance and discovery can promote global interaction via collaborations on matters that know no national or political boundaries but simply reflect our common humanity. the cell biology of acute childhood respiratory disease: therapeutic implications a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany diagnostic system for rapid and sensitive differential detection of pathogens masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during rhinovirusassociated hospitalizations in young children characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity isolation and characterization of borna disease agent cdna clones genomic organization of borna disease virus identification of a kunjin/west nile-like flavivirus in brains of patients with new york encephalitis genetic analysis of west nile new york 1999 encephalitis virus panmicrobial oligonucleotide array for diagnosis of infectious diseases detection of respiratory viruses and subtype identification of influenza a viruses by greene-chipresp oligonucleotide microarray a metagenomic survey of microbes in honey bee colony collapse disorder pathogen surveillance and discovery key: cord-013049-7d436sqg authors: sobhanie, mahdee; matsuoka, yumiko; jegaskanda, sinthujan; fitzgerald, theresa; mallory, raburn; chen, zhongying; luke, catherine; treanor, john; subbarao, kanta title: evaluation of the safety and immunogenicity of a candidate pandemic live attenuated influenza vaccine (plaiv) against influenza a(h7n9) date: 2016-03-15 journal: j infect dis doi: 10.1093/infdis/jiv526 sha: doc_id: 13049 cord_uid: 7d436sqg background. we evaluated a candidate a/anhui/2013(h7n9) pandemic live attenuated influenza vaccine (plaiv) in healthy adults, and assessed the ability of 1 or 2 doses to induce immune memory. methods. healthy subjects in 2 age groups (18–49 years and 50–70 years) with undetectable hemagglutination-inhibiting (hai) antibody to h7n9 were enrolled. younger subjects received either 1 or 2 intranasal doses of 10(7.0) fluorescent focus units of a/anhui/1/2013 plaiv, while older subjects received a single dose. all subjects received a single 30-µg dose of unadjuvanted, antigenically matched a/shanghai2/2013(h7n9) pandemic inactivated influenza vaccine (piiv) 12 weeks after their first dose of plaiv. results. both vaccines were well tolerated. serum hai antibody responses were detected in 0 of 32 younger subjects and 1 of 17 older subjects after 1 dose of plaiv and in 2 of 16 younger subjects after a second dose. strong serum antibody responses were detected after a single subsequent dose of piiv that was broadly reactive against h7 influenza viruses. conclusions. an a(h7n9) plaiv candidate was safe in both age groups. priming with plaiv resulted in responses to subsequent piiv that exceeded those seen in naive subjects in previous reports. the a(h7n9) plaiv induces strong immune memory that can be demonstrated by exposure to subsequent antigenic challenge. clinical trials registration. nct01995695 and nct02274545. severe human disease due to influenza a(h7n9) virus is continuing to occur in china [1, 2] . in contrast to the experience with influenza a(h5n1) virus in humans, severe disease and hospitalization due to influenza a(h7n9) virus have predominantly affected older adults and those with chronic illnesses [3] . new cases have been recognized each winter, and influenza a (h7n9) virus is considered to pose a pandemic threat. vaccines for control of influenza viruses with pandemic potential are under development, and results of clinical trials have suggested that these vaccines will require the use of adjuvants and multiple doses to induce substantial serum antibody responses [4, 5] . an alternative approach is the development of pandemic live attenuated influenza vaccines ( plaivs). since seasonal laivs appear to be immunogenic and highly effective in children [6] [7] [8] , they would in theory be an excellent option for pandemic control. evaluation of a number of plaiv candidates have shown them to be well tolerated in healthy adults but infrequently associated with serum antibody responses [9] [10] [11] [12] . two studies have now demonstrated that plaiv recipients respond to subsequent doses of antigenically matched piiv with a rapid and vigorous antibody response that suggests that plaivs established immunologic memory [13, 14] . in a previous study involving h7 vaccines, recipients of 2 doses of an a/netherlands/ 219/03(h7n7) plaiv but not recipients of 1 dose of an antigenically distinct a/chicken/british columbia/cn-6/04(h7n3) plaiv had a vigorous serum antibody response to an unadjuvanted a(h7n7) piiv given 18 months later [14] . it was unclear in that study whether the absence of a response to the piiv by a (h7n3) plaiv recipients was because of antigenic mismatch or because of the number of doses of plaiv administered. in the current study, we evaluated a candidate a(h7n9) plaiv and directly compared the safety, viral shedding pattern, and immunogenicity in younger subjects who received either 1 or 2 doses of plaiv. because of the age distribution of individuals infected with influenza a(h7n9) virus, we also enrolled a second cohort of older subjects who received a single dose. all subjects received a single dose of antigenically matched unadjuvanted a(h7n9) piiv 12 weeks after plaiv receipt. we found that plaiv primed both older and younger subjects to respond to a single subsequent dose of piiv. these results suggest that a(h7n9) plaiv can effectively prime the immune system to respond to subsequent doses of unadjuvanted a(h7n9) piiv. the plaiv used in this study was generated by plasmid rescue in vero/primary chicken embryo kidney cell coculture as a 6-2 reassortant deriving the hemagglutinin (ha) and neuraminidase genes from human influenza a/anhui/1/2013(h7n9) virus and all other gene segments from the cold-adapted influenza a/ann arbor/6/60 master donor virus and was produced in embryonated hen's eggs. this vaccine was shown to provide protection against challenge with wild-type influenza a(h7n9) and a(h7n7) viruses in the ferret model [15] . sequence analysis of the neuraminidase gene predicted it to be sensitive to the antiviral drug oseltamivir. the inactivated vaccine used in this study was an unadjuvanted split-virion a(h7n9) vaccine derived from the antigenically identical influenza a/shanghai/2/2013(h7n9) virus (sanofi pasteur, swiftwater, pennsylvania), provided by the biomedical advanced research and development authority. the potency of the vaccine was determined to be 30 µg per 0.5-ml dose by reverse-phase high-pressure liquid chromatography [16] . evaluation of the a/anhui/1/2013 plaiv was performed using previously published methods [9, 12, 17] . the first stage enrolled subjects aged 18-49 years, and eligible subjects were assigned, based on their preference, to receive either a single dose of a (h7n9) plaiv or 2 doses separated by 28 days. after we determined that the a(h7n9) plaiv candidate was well tolerated in younger subjects, healthy older subjects aged 50-70 years were subsequently enrolled and received a single dose of a(h7n9) plaiv. for each dose of a(h7n9) plaiv, subjects were admitted to an isolation facility and observed for 2 days, and then received 10 7.0 fluorescent focus units of the vaccine virus in a volume of 0.5 ml by intranasal spray in open label fashion. physical examination and assessment of reactogenicity events were performed daily after inoculation, until discharge. among younger subjects, a nasal wash specimen was obtained daily for virus detection, while among older subjects, a nasal swab specimen was obtained daily. the presence of influenza virus was determined by inoculation of madin-darby canine kidney (mdck) cells at 33°c and by real-time reverse-transcription polymerase chain reaction (rt-pcr) analysis as previously described [18] . subjects were discharged from the facility on day 9 after inoculation if they had at least 2 consecutive real-time rt-pcr tests negative for vaccine virus. subjects returned approximately 12 weeks after their first dose of plaiv to receive a single booster dose of 30 µg of the unadjuvanted a(h7n9) piiv by intramuscular injection. the study was approved by the university of rochester institutional review board, and written informed consent was obtained from all participants. viral rna was extracted from nasal wash and swab samples, using the nuclisens easymag system (biomérieux). the super-script iii one-step rt-pcr system and platinum taq dna polymerase were used to reverse transcribe viral rna and amplify complementary dna (invitrogen). rt-pcr products were amplified in nested pcr reactions using the herculase ii fusion dna polymerase enzyme with the dntp combo kit (agilent). nested pcr-amplified products were run on 1% agarose gels, and gels containing a visible band denoting the expected fragment size were purified and sent to sequetech (mountain view, california) for sequencing. the sequences were then assembled and aligned to the reference vaccine a (h7n9) sequence. the 3 temperature-sensitive/attenuated loci of the polymerase basic 1 (pb1) gene of the cold-adapted influenza a/ann arbor/6/60 master donor virus [19] were also sequenced with a similar strategy to ensure that the viral rna originated from the a(h7n9) laiv. sera were tested by hemagglutination-inhibition (hai) and microneutralization (mn) assays against the vaccine virus and by mn assays against other wild-type h7 viruses. wild-type influenza a/anhui hai assays were performed on sera after treatment with receptor-destroying enzyme (denka seiken), using 0.75% or 1% horse erythrocytes [12] with 4 hemagglutination units of virus, beginning at a 1:4 dilution of serum. mn assays were performed on mdck cells as previously described [20] , except that assays using the plaiv viruses were performed at 33°c. for serum hai and mn antibody assays, subjects were defined as responders if they achieved a ≥4-fold increase in antibody titer, compared with the baseline value, at any time point after vaccination. sera in the younger cohort were also tested for ha-specific antibody by an enzyme-linked immunosorbent assay (elisa). ninety-six-well nunc maxisorb plates (thermal scientific) were coated with purified baculovirus-expressed h7 ha protein from influenza a/anhui/1/2013 virus (bei resources) at 0.25 µg/well and blocked with 5% nonfat dry milk. sera were tested at a starting dilution of 1:100, and binding was detected with alkaline phosphatase-conjugated isotype-specific goat anti-human immunoglobulin g (igg), immunoglobulin m (igm), or immunoglobulin a antibody (invitrogen, frederick, maryland). the end point titer was the highest dilution giving an optical density at least twice that of background. a 4-fold increase in titer over baseline was considered a response. sera collected before and 28 days after plaiv and piiv boost in younger subjects were also tested for antibody that could mediate antibody-dependent cellular cytotoxicity (adcc) using a modified flow-based assay as previously described [21, 22] . briefly, wells of a 96-well elisa plate (maxisorp, nunc) were coated with 600 ng/well of recombinant ha protein (sino biological) overnight at 4°c. wells were washed with 1 × pbs and incubated with diluted human sera (1:20-1:81 920) for 2 hours at 37°c. wells were washed with 1 × pbs and incubated with 100-500 000 nk-92 cells stably expressing human cd16 (conkwest) for 5 hours at 37°c in 10% co 2 . following incubation, cells were stained with cd107a apc-cy7 (clone h4a3; bd) and fixed with 10% paraformaldehyde. cells were analyzed via flow cytometry, and the end point titer was defined as the highest dilution of antibody inducing cd107a expression from natural killer (nk) cells at a level that was at least twice the background level in antigen-negative wells. mann-whitney rank sum tests were used to compare the duration of viral shedding between groups, and the fisher exact test was used to compare rates. because real-time rt-pcr positivity on the day after vaccination could represent input virus, infection was considered to be present on the basis of a positive result of the real-time rt-pcr assay on any day after day 1, isolation of vaccine virus in cell culture at any time after administration, or a 4-fold increase in serum hai antibody titer between the specimen obtained before vaccination and the specimen obtained 28 days after vaccination [12] . administration of 1 or 2 doses of a(h7n9) plaiv to serosusceptible young adults was well tolerated ( table 1 ). the frequency of systemic complaints in the 9 days following plaiv receipt was low, with mild headache being the most frequent complaint, and there were no differences in systemic reactogenicity between the first and second dose. nasal symptoms were more frequent, but there were no complaints of greater than mild severity. the frequency of complaints of runny nose and stuffy nose was less after the second dose than after the first dose, but the differences were not statistically significant. all complaints had resolved by the time of discharge from the isolation facility. because of the age distribution of disease due to influenza a (h7n9) virus in humans, we also evaluated a single dose of a (h7n9) plaiv in adults 50-70 years of age, an age group in which plaiv candidates have not been tested previously. the reactogenicity profile in this age group was very similar to that in younger adults, consisting primarily of mild nasal symptoms and headache. two of the young adult subjects in the single-dose plaiv group did not return for further visits, leaving 47 subjects across the 2 age cohorts who received the subsequent a(h7n9) piiv boost. the inactivated vaccine was also well tolerated, with the most common adverse event being mild tenderness at the site of injection (table 2) . local tenderness was slightly more common in the older age group. there were no severe complaints, and there was no difference in the reactogenicity of the piiv between those who received 1 dose and those who received 2 doses of plaiv. for each subject, the most-severe event is reported. recovery of the plaiv virus from the nasopharynx of study subjects was infrequent. (table 3 ). virus was primarily detected only by real-time rt-pcr, mostly on the day immediately following vaccine administration. virus was recovered in cell culture after 8 of 65 vaccine doses (12.3%), with roughly equal frequency in young adults after dose 1 and dose 2 and slightly higher frequency in older subjects. if vaccine virus infection is defined as detection of the virus in culture or by real-time rt-pcr at any time after day 1 or as development of a ≥4-fold increased hai antibody response, then approximately 20%-30% of subjects were infected after each dose of plaiv. there did not appear to be significant differences in the infectivity of the vaccine between the first and second dose in the 2-dose group. among those who received 2 doses, infection occurred in 3 of 16 (18%) after the first dose. among the 3 subjects with evidence of infection after dose 1, 1 subject (33%) was also infected after dose 2, while of the 13 subjects without evidence of infection after dose 1, 2 (15%) were infected after dose 2. detection of vaccine virus was more frequent and of longer duration in the older subjects. one older subject in particular had shed virus detected by real-time rt-pcr daily for 8 days following inoculation and was also culture positive on days 3-8 after inoculation. to facilitate discharge from the isolation facility, the subject was treated with 75 mg of oseltamivir twice daily beginning in the afternoon of day 8 and was real-time rt-pcr and culture negative on days 9, 10, and 11. sufficient signal for sequencing was only obtained from 1 sample from the younger group and during the very prolonged period shedding in the older subject. a sample from day 5 after inoculation of dose 2 in a younger subject showed 100% identity to the open reading frame of the n9 vaccine reference sequence. the 3 temperature-sensitive and attenuated loci in the pb1 gene were present, indicating that the sequenced viral rna originated from the administered vaccine. sequencing of samples from day 4 and from day 8 of shedding from the older subject with prolonged shedding also determined that the ha and neuraminidase genes on both days were identical to the original vaccine sequences and that the none of the differences between groups reached statistical significance. cold-adapted and temperature-sensitive loci on the pb1, pb2, and nucleoprotein genes were unchanged from the vaccine. two younger subjects, both in the 2-dose group, had a 4-fold increase in hai antibody (from <1:4 to 1:32) and mn antibody (from <1:10 to 1:20) titers between day 28 and day 56 after receipt of plaiv ( table 4 ). one of these subjects shed virus beyond day 1 after dose 1 but not dose 2, while the other subject did not shed virus beyond day 1 after either dose. in the older group, the subject who exhibited prolonged shedding of vaccine virus after inoculation also had an increase in both hai and mn antibody to a(h7n9) plaiv. no other subjects had measurable serum antibody hai or mn responses to administration of either dose of plaiv. abbreviations: hai, hemagglutination-inhibition assay; iga, immunoglobulin a-specific enzyme-linked immunosorbent assay; igg, immunoglobulin g-specific enzyme-linked immunosorbent assay; mn, microneutralization assay; nt, not tested. a defined as a ≥4-fold increase in titer between baseline and day 56, for plaiv, or between the time before piiv receipt to day 28 after piiv receipt. in contrast, 57% of younger subjects in the 1-dose plaiv group and 64% in the 2-dose group had ≥4-fold increased serum hai responses, and 81% and 93% had serum mn responses to a subsequent dose of unadjuvanted a(h7n9) piiv. similarly, 59% of older subjects had a hai response to a subsequent piiv boost after a single dose of plaiv, and 47% had a mn response. all of the subjects who responded achieved hai titers of >1:40 against the a/anhui/1/2013(h7n9) plaiv virus. the frequency of mn and hai responses was similar in younger and older subjects who received a single priming dose of plaiv and somewhat higher in those who received 2 doses of the antigenically matching a(h7n9) plaiv, but the differences were not statistically significant. responses detected by elisa to the h7 ha were primarily igg, and no igm responses were detected (data not shown). the kinetics of the antibody response among those subjects who had a ≥4-fold increased response are shown in figure 1 . increases in the antibody titer to a(h7n9) could be detected as early as 7 days after piiv in both younger and older subjects. antibody was still detectable in these subjects 180 days after receipt of the inactivated vaccine. there were no significant differences when the titers of antibody achieved by responders were compared between the 1-dose and 2-dose primed groups or between the 1-dose younger and older groups. sera obtained from the younger subjects after piiv receipt were also tested for neutralization activity against the wildtype influenza a/anhui we also used a modified nk cell activation assay to measure adcc responses to the h7 ha in the younger cohort. in subjects that received either a single dose or 2 doses of a(h7n9) plaiv, there was no detectable rise in adcc titer ( figure 3a and 3b) . however, all subjects had a significant rise in h7-specific adcc following piiv receipt, with geometric mean titers increasing from 93 to 2128 ( figure 3c ). further, there was a significant difference in the post-iiv adcc titer in subjects who had been primed with 1 versus 2 doses of plaiv ( figure 3d ). the adcc titer strongly correlated with the hai titer following piiv receipt, suggesting that a(h7n9)-specific hai antibodies may also have adcc activity (r = 0.7850; p < .001; data not shown). thus, the plaiv-iiv regimen induced robust levels of h7-specific adcc-mediating antibodies in healthy adults. consistent with the findings of other studies of pandemic formulations of laiv based on this master donor virus, the a(h7n9) plaiv was well tolerated in younger subjects, with a minority of subjects experiencing mild, self-limited nasal symptoms following vaccination. the safety of plaiv candidates has not been previously evaluated in older subjects, but because the primary age group affected by influenza a(h7n9) virus infection has been older, with a mean age of 63 years [3] , the results of the current study, which suggest that plaiv is well tolerated in this age group, are reassuring. the nasopharyngeal replication of the a(h7n9) plaiv was extremely limited in both younger and older subjects. the reasons for the highly restricted replication of the vaccine virus in most subjects are unclear, since previous studies have demonstrated that similar vaccine viruses replicate well in animal models [15] . however, some subjects demonstrated prolonged replication of the vaccine virus, which, in 1 subject, did not terminate until oseltamivir was administered. serum antibody responses to plaiv were only detected in 3 subjects. although the amount of virus detected after the second dose in younger subjects was somewhat reduced as compared to the amount detected after the first dose, there was no evidence that individuals with detectable virus after dose 1 were more or less likely to have detectable virus after dose 2. detection of virus shedding by pcr or culture did not predict antibody response to plaiv or boosting, although the single subject with prolonged viral replication manifested a detectable serum hai and mn response. emerging data suggest that, although cold-adapted plaiv candidates do not induce easily detectable primary immune responses, they prime individuals for vigorous serum antibody responses to subsequent doses of poorly immunogenic inactivated subvirion vaccines [13, 14] . these studies have evaluated subjects who received 2 doses of plaiv and were given inactivated vaccine ≥2 years later. therefore, in this study we directly compared the priming effect of a single dose of plaiv to that of 2 doses administered 28 days apart and whether boosting could be detected at approximately 12 weeks after priming, as suggested by studies evaluating inactivated vaccine boosting after dna vaccination [23, 24] . both 1 and 2 doses of plaiv primed subjects for a response to inactivated a/shanghai/2/2013(h7n9) vaccine. although we did not have a concurrent control group of naive subjects who received the inactivated vaccine, in a previous study of the same a(h7n9) piiv performed in naive subjects [4] , serum hai antibody responses were detected in only 2 of 99 subjects (2%) after a single dose of 45 µg of piiv, and mn responses were detected in 1 of 99 recipients (1%). even 2 doses of 45 µg of unadjuvanted a(h7n9) generated hai and mn responses in only 5% and 21% of subjects, respectively. the induction of antibodies that have nonneutralizing effector functions, such as adcc, may provide some enhanced protection from severe influenza virus infection. we have previously shown moderate to low h7-specific adcc in the general population [22] . in the current study, we found that approximately half of the younger subjects (16 of 30) had low, but detectable cross-reactive adcc toward recombinant h7 ha before they received the initial dose of plaiv. while the a (h7n9) plaiv did not induce substantial increases in adcc toward h7 ha, a single subsequent dose of a(h7n9) piiv induced significant levels of h7-specific adcc in all subjects. passive transfer studies in mice suggest that the protective ability of anti-ha stem antibodies is mediated by adcc [25, 26] . assessment of adcc responses to pandemic vaccines could be helpful in defining their protective potential. other differences between the 1-dose and 2-dose groups were not statistically significant. however, there was a trend toward more-frequent hai and mn responses to inactivated vaccine in those who received 2 priming doses of plaiv. the titers of antibody achieved by those who responded were not different in the 2 groups, suggesting that the major contribution of the second dose was to prime additional subjects who, for unknown reasons, were not primed by the first dose. in summary, with 3 different plaiv-piiv combinations (a [h5n1], a[h7n7], and a[h7n9]), we have demonstrated rapid, robust, high-quality, cross-reactive antibody responses after the boost with antigenically matched piiv. the observation of priming for subsequent responses to piiv in the absence of an antibody response to the priming immunization is similar in some ways to the response to a(h5n1) piiv in recipients of dna vaccine [23, 24] . although the specific immune mechanisms responsible remain to be elucidated, these observations expand the options potentially available for eliciting robust responses to avian h5 and h7 has through vaccination. clinical findings in 111 cases of influenza a (h7n9) virus infection case-control study of risk factors for human infection with influenza a(h7n9) virus in jiangsu province comparison of patients hospitalized with influenza a subtypes h7n9, h5n1, and 2009 pandemic h1n1 serological responses for an avian influenza a/h7n9 vaccine mixed at the point-of-use with mf59 adjuvant: a randomized clinical trial a cell culture-derived mf59-adjuvanted pandemic a/h7n9 vaccine is immunogenic in adults live attenuated versus inactivated influenza vaccine in infants and young children superior relative efficacy of live attenuated influenza vaccine compared with inactivated influenza vaccine in young children with recurrent respiratory tract infections comparison of the efficacy and safety of live attenuated cold-adapted influenza vaccine, trivalent, with trivalent inactivated influenza virus vaccine in children and adolescents with asthma evaluation of two live attenuated cold-adapted h5n1 influenza virus vaccines in healthy adults an open label phase i trial of a live attenuated h6n1 influenza virus vaccine in healthy adults an open-label, phase i trial of a live attenuated h2n2 influenza virus vaccine in healthy adults a live attenuated h7n3 influenza virus vaccine is well tolerated and immunogenic in a phase i trial in healthy adults a live attenuated influenza a (h5n1) vaccine induces long-term immunity in the absence of a primary antibody response live attenuated h7n7 influenza vaccine primes for a vigorous antibdy response to inactivated h7n7 influenza vaccine development of a high yield live attenuated h7n9 influenza vaccine that provides protection against homologous and heterologous h7 wild-type viruses in ferrets optimization and qualification of a quantitative reversed-phase hplc method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays a live attenuated h9n2 influenza vaccine is well tolerated and immunogenic in healthy adults design and performance of the cdc real-time reverse transcriptase pcr swine flu panel for detection of 2009 a (h1n1) pandemic influenza virus multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (flumist) derived from cold-adapted a/ann arbor/6/60 detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays cross-reactive influenzaq-specific antibody-dependent cellular cytotoxicity antibodies i the absence of neutralizing antibodies cross-reactive influenza-specific antibody-dependent cellular cytotoxicity in intravenous immunoglobulin as a potential therapeutic against emerging influenza viruses dna priming and influenza vaccine immunogenicity: two phase i open label randomised clinical trials prime-boost interval matters: a randomized phase i study to identify the minimum interval necessary to observe the h5 dna influenza vaccine priming effect broadly neutralizing hemagglutinin stalk-specific antibodies require fc[gamma]r interactions for protection against influenza virus in vivo a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins financial support. this work was supported by the laboratory of infectious diseases, national institute of allergy and infectious diseases potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-299835-92karhpl authors: ho, khek y.; singh, kamaljit s.; habib, abdulrazaq g.; ong, benjamin k.; lim, tow k.; ooi, eng e.; sil, bijon k.; ling, ai-ee; bai, xin l.; tambyah, paul a. title: mild illness associated with severe acute respiratory syndrome coronavirus infection: lessons from a prospective seroepidemiologic study of health-care workers in a teaching hospital in singapore date: 2004-02-17 journal: j infect dis doi: 10.1086/381558 sha: doc_id: 299835 cord_uid: 92karhpl background. severe acute respiratory syndrome (sars) is a newly recognized infectious disease that has recently emerged in east asia and north america. although the clinical features of acute infection have been well described, mildly symptomatic or asymptomatic infections have not been well characterized. objective. to assess the spectrum of illness in health-care workers (hcws). methods. a prospective seroepidemiologic cohort study was conducted on 372 hcws in a large teaching hospital in singapore who were both exposed and not exposed to patients with sars. participating hcws completed a questionnaire and provided paired serum samples, which were analyzed by 2 different laboratories blinded to clinical data, by use of an enzyme-linked immunosorbent assay based on a protocol developed by the centers for disease control and prevention and a dot-blot immunoassay, with confirmation by a viral neutralization assay. results. a total of 21 patients with sars were treated at our hospital. they were associated with transmission to 14 staff members, patients, and visitors in our hospital. of the 372 hcws participating in the present study, 8 were found to have positive antibodies to the sars coronavirus in both samples by use of both test methods, and 6 had pneumonia and had been hospitalized for either probable or suspected sars infection, whereas 2 had fever but did not have changes on chest radiographs. all seropositive hcws had been exposed either directly or indirectly to patients with sars. no asymptomatic, nonexposed staff members were found to be seropositive. there was a trend towards protection for hcws who, while fully protected, had had contact with patients with sars. conclusions. although the majority of cases of sars are associated with pneumonia, a small number of mildly symptomatic individuals do seroconvert. hcws who are exposed to patients with sars can be infected with sars, regardless of the intensity of exposure. this has implications for surveillance and infection control planning, in the event that sars returns next winter. severe acute respiratory syndrome (sars) is a newly recognized coronavirus infection that recently emerged in east asia, with subsequent global spread [1] [2] [3] . in singapore, cases of sars were diagnosed in early march were in the same rooms as patients with unrecognized sars [5] . the clinical features of typical sars have been well described in large clinical studies [6] [7] [8] . atypical presentations with no fever but with changes on chest radiographs have also been reported [9] . asymptomatic or mild infections with no respiratory symptoms and no changes on chest radiographs have, however, not been previously reported to have occurred in contacts of patients with sars, with the exception of 1 case report [10] . this has been documented in other emerging viral infections and has potential implications for the transmission and control of this emerging infection [11, 12] . the national university hospital (nuh), singapore, is a 900-bed teaching hospital that employs ∼3000 doctors, nurses, allied health professionals, and clerical staff members. between 18 march and 29 april 2003, a total of 21 patients with sars were treated in the hospital wards and emergency department before being transferred to the designated sars hospital. an escalated policy on the use of more-complete personal protective equipment (ppe) and isolation of suspected patients was instituted, beginning with the required wearing of n-95 masks, gowns, and gloves for isolation-ward personnel only. later, this was extended to personnel in all areas of the hospital, with regular audits of all hcws. we conducted a seroepidemiologic study of hcws in our hospital to assess the spectrum of illness seen in hcws infected with the sars virus, in particular to document whether asymptomatic or mild infections with no respiratory symptoms and no changes on chest radiographs occur in individuals with sars. after giving written, informed consent, unselected hcws were recruited on a voluntary basis from all areas of the hospital, beginning with high-risk areas and extending to low-risk areas, including outpatient clinics and offices. they completed a simple questionnaire describing their workplaces, contact with patients with sars, use of ppe, and symptoms experienced during the preceding 4 weeks. they also provided paired serum samples, which were collected initially at the peak of the outbreak and subsequently at a median interval of 31 days (range, 17-53 days) after initial collection. samples were anonymized to ensure that the confidentiality of hcws was preserved. the study was approved by the hospital institutional review board. all policies and procedures of the ministry of health, singapore, good clinical practice were followed in the conduct of this study. serum samples were stored at ϫ80њc and subsequently were sent to 2 different external laboratories for serologic testing. the laboratory staff were unaware of the clinical details of the patients. the first laboratory used an elisa based on a protocol and using antigens provided by tom ksiazek (centers for disease control and prevention, atlanta, ga) [1] . samples found to be positive for sars by elisa were confirmed by use of an indirect immunofluorescence assay. the second test was done at the national environmental agency, singapore, and used a dot-blot immunoassay using antigens derived from virus culture supernatant. positive samples, at a titer of у1:100, were then subjected to a virus neutralization assay, in serial 2-fold dilution, starting at 1:10-1:320 and using a microneutralization format described elsewhere for human enterovirus 71 [13] . a neutralizing antibody titer of у10 was considered to be positive for sars. serum samples from volunteer patients with sars and from nonexposed laboratory staff were included in all serologic assays as positive and negative controls, respectively. thus, all the serum samples were tested by use of 2 screening tests (i.e., elisa and dot-blot immunoassay), and the positive serum samples were confirmed by indirect immunofluorescence assay and virus neutralization assay, respectively. results of the serologic testing from each laboratory were not revealed to the other laboratory until after completion of the study. definitions. a seropositive individual was defined as having provided a serum sample that received a positive confirmatory result by both the indirect immunofluorescence assay and the virus neutralization assay. an exposed hcw was defined as having worked in an area where a patient later confirmed to have sars had been cared for. direct contact was defined by use of the world health organization (who) definition of having cared for, having lived with, or having had direct contact with respiratory secretions and/or body fluids of an individual with sars [14] . hcws who worked in the same ward but did not have direct responsibility for patients with sars or did not come into physical contact with respiratory secretions and/or body fluids of an individual with sars would thus be defined as exposed-only hcws. patients with sars were defined by use of who criteria [14] for probable cases, which included fever (temperature 138њc), respiratory symptoms, and radiographic evidence of pneumonia or respiratory distress. mildly symptomatic individuals were defined as those with fever significant enough to warrant evaluation at the staff clinic or emergency department but with no evidence of pneumonia on chest radiographs and prompt resolution of symptoms (within 48-72 h of symptomatic therapy). statistical analysis. differences between groups were assessed by the x 2 test, fisher's exact test, mann-whitney u test, kruskal-wallis test, or relative risk ratios with 95% confidence intervals, as appropriate. the results of these comparisons were reported as p values. all statistics were analyzed by use of the spss (version 11.5.1; spss) and stata (version 7.0; statacorp) software. the first patient with sars seen in our hospital was a cardiology resident from ttsh who entered our emergency department on 18 march 2003. since then, a total of 21 patients with sars, including 5 hcws from our hospital, have been seen in our wards and emergency department. all of these patients had positive antibodies to the sars coronavirus. six of the 7 tested also had sars coronavirus isolated from stool samples, blood samples, and/or respiratory secretions. these 21 patients stayed in nuh a mean ‫ע‬ sd of days from admission or 3.9 ‫ע‬ 4.8 onset of symptoms to transfer to ttsh. there were 14 known nosocomial transmissions to staff members, visitors, and other patients, all of whom were eventually transferred to ttsh for treatment; 11 of these were linked to a single atypical case [15] . initial policies on the use of ppe for hcws, instituted on 17 march 2003, confined the mandatory use of gloves, gowns, and n95 masks to isolation wards only but, by 28 march, were extended to include intensive-care units and the emergency department. on 9 april, after the identification of an atypical case of sars in an open general medical ward, full use of ppe was made mandatory for all staff in contact with patients. a total of 372 staff members participated, of whom paired serum samples were obtained from 303 (81.5%). serum was drawn at 17-53 day intervals, beginning on 22 april and continuing to 5 june 2003-that is, beginning 4 weeks after the first case of sars in nuh and ending 10 weeks after the last case was transferred to ttsh. overall, mean ‫ע‬ sd age was years, and 287 (77.2%) were women, 103 (27.7%) 34.2 ‫ע‬ 9.0 were physicians, 205 (55.1%) were nurses, and the rest were allied health professionals and clerical staff members. one hundred twelve (30.1%) worked in areas where patients with sars had been cared for (i.e., the exposed group), and the rest had no exposure at all to patients with sars (i.e., the nonexposed group). the characteristics of the hcws are listed in table 1. the exposed group was younger and included more nurses, probably because of a higher representation of emergency department and intensive-care unit staff members in this group. a large number of staff members reported a variety of symptoms during the study period, including fever ( . there was a n p 69 n p 22 significant difference in the frequency of fever between exposed (28.6%) and nonexposed (11.9%) hcws ( ). twenty-p p .0001 note. mildly symptomatic subjects, health-care workers who had fever significant enough to warrant evaluation at the staff clinic or emergency department but who had no evidence of pneumonia on chest radiographs and had prompt resolution of symptoms (within 48-72 h of symptomatic therapy). one hcws (5.6%) in our study cohort were hospitalized during this period, of whom 6 were classified by clinical criteria as probably infected with sars. twenty-one (5.6%) of the participants or their spouses had traveled to other sars-affected areas during the study period. serologic data on hcws (table 2) . samples from 8 hcws (2.2%) were found to be positive for sars-associated coronavirus by elisa, and these results were confirmed by indirect immunofluorescence assay using the cdc protocol, and samples from the same hcws were found to be positive by use of the dot-blot method, and these results were confirmed by viral neutralization assay. both samples tested for each hcw were found to be positive for all 8 hcws, and no increase in titer could be demonstrated on their paired samples, since, from all 8 subjects, samples were obtained 14 weeks after the onset of symptoms. all 6 hcws hospitalized at ttsh (including 5 who were first treated in our hospital) for probable sars infection were found to be seropositive, and 2 additional individuals who had fever but did not meet sars criteria (i.e., had symptoms !4 days in duration and had no changes on chest radiographs) were also seropositive. on the basis of data submitted anonymously for the study, as well as a review of hospital epidemiology and contact-tracing data, a profile of these 2 individuals could be constructed without compromising confidentiality: both worked in the emergency department at a time when patients with sars were being treated in the department, wore full ppe, and did not have direct personal contact with any patients with sars. one was admitted to an isolation room with fever, chills, and myalgias, which resolved completely within 3 days of symptomatic treatment; she had no changes on serial chest radiographs. the other was treated as an outpatient in the staff clinic; she presented with fever and upper respiratory symptoms. again, chest radiographs were normal, and symptoms resolved within 3 days. no secondary cases resulted from any of the infected hcws. no hcw who was completely asymptomatic was found by serologic testing to have sars-associated coronaviral infection. it is interesting to note that, even after removing these 8 seropositve hcws from the exposed and febrile groups in table 1, the difference between the exposed and nonexposed groups, in relation to being febrile, remains statistically significant (23.1% vs. 11.9%; ). p p .0095 relationship between serologic test results and exposure history. when analyzed by contact history, all of the seropositive hcws worked in areas where patients with sars had been cared for (table 3) . although only 4 of 8 had had direct contact by who definition, the remaining 4 worked in the same ward where the patients were located but did not have direct responsibility for these patients or come into physical contact with these patients' body fluids. although the numbers of subjects in our study are small, there was a trend towards protection for hcws who reported use of ppe 100% of the time or who, while fully protected, had contact with patients with sars. sars is a novel coronavirus that has recently emerged in southern china and has caused widespread disruption to health-care services and international trade, especially in east asia [16] . the vast majority of infections, with a few notable exceptions, have occurred in hospitals. as with all emerging infections, the clinical picture is only beginning to be described completely. the initial descriptions included atypical pneumonia that followed a prodrome with fever and myalgia [6] [7] [8] and that progressed almost universally to a severe respiratory illness, with a variety of changes on chest radiographs [17] . in approximately one-sixth of cases, this eventually progressed to acute respiratory distress syndrome and death [18] . later reports highlighted gastrointestinal symptoms as a major element of a large community outbreak of sars that was thought to be linked to environmental contamination [19] . atypical presentations have also been reported, but all of these eventually led to the typical pattern of progressive respiratory distress with frank changes on radiographs [9] . the strengths of the present study include the prospective nature of the collection of serum samples and acquisition of clinical data, an adequate sample size with representative subjects from all areas of a large teaching hospital, use of paired serum samples, and application of 2 previously validated serologic tests, which were performed independently. the present study is the first to document sars infection in hcws with normal chest radiographs. two of our seropositive hcws had fever for !1 week and responded to symptomatic therapy for upper respiratory-tract infections. their chest radiographs were repeatedly normal. thus, they did not meet the clinical criteria for probable sars infection. the possibility that our 2 individuals with mild illness were false positives is diminished by the fact that they were found to be positive on both assays, which were done by laboratory staff who were blinded to clinnote. any exposure, working in an area where a patient later confirmed to have sars had been cared for; direct contact, having cared for or having had direct contact with respiratory secretions and/or body fluids of an individual with sars; ci, confidence interval; exposure only, working in the same ward but not having cared for and not having come into physical contact with respiratory secretions and/or body fluids of an individual with sars; no exposure, not working in an area where a patient later confirmed to have sars had been cared for; ppe, personal protection equipment; rr, relative risk. ical data. that none of the nonexposed hcws had evidence of infection and the unpublished reports that blood donors in a variety of settings had no serologic evidence of sars support our hypothesis that these were indeed mild infections. given our experience with other viral respiratory tract infections in which a spectrum of infections is the rule, the detection of mildly symptomatic infections is not surprising. we also failed to detect seroconversion in totally asymptomatic individuals. this was not the experience of other investigators screening contacts of the nipah virus or avian influenza, but those were retrospective serologic studies done some time after the outbreak, and mild clinical symptoms might not have been detected [11, 12] . given the fact that the sars coronavirus seems to be a completely novel pathogen with minimal genetic relatedness to other coronaviruses of humans and animals [20] , it is perhaps not that surprising that it produced at least some clinical disease in all infected individuals with no preexisting cross-protective immunity. this is supported by our observation that, in our hospital, individuals who were exposed to patients with sars had a significantly higher prevalence of fever. the second important finding we observed is that individuals who did not have direct contact with patients with sars also both developed clinical sars and experienced seroconversion with milder illness. in such a setting, transmission is likely to occur on either environmental surfaces or the hands of other hcws. however, our study strikingly shows the complete absence of transmission to individuals working in the same building but in different areas from locations where patients with sars had been cared for. this also has implications, since it bears out our experience with the global epidemiology of sars, in which, who global travel alerts notwithstanding, almost all transmissions in hong kong, taiwan, toronto, and singapore and the majority in mainland china could be traced back to specific household or health-care settings. this should be taken into consideration in the event that sars is detected again next winter, before widespread economic disruption is created by travel alerts and advisories. neither of the 2 individuals with mild cases of sars was associated with any secondary transmission, despite not being isolated or quarantined for any significant period of time. although the majority of cases of sars in singapore did not lead to any secondary infections [5] , it is reassuring that mildly symptomatic cases are hopefully associated with lower virus loads [19] and less likely trigger epidemics. these cases might, however, allow for a low level of transmission of the virus, which might remain "below the radar" if they are not actively sought. this has been a concern with the reemergence of sars in toronto after a period during which transmission was believed to have been halted [21] . data on sars continue to emerge. we have shown that, as with most viral infections, there is a spectrum of illness associated with sars, from mild febrile illness to severe respiratory distress. with data emerging about an animal reservoir [22] for sars, it will be critical to detect periodic human infections, even if mild. although sars seems to have disappeared in the summer months, broad surveillance using more-sensitive assays will be critical in the event that sars reappears next winter. a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome update: outbreak of severe acute respiratory syndrome-worldwide severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts update: severe acute respiratory syndrome-singapore a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada clinical features and short-term outcomes of 144 patients with sars in the greater toronto area atypical presentations of sars mild severe acute respiratory syndrome a survey of nipah virus infection among various risk groups in singapore risk of influenza a (h5n1) among healthcare workers exposed to patients with influenza a (h5n1), hong kong seroepidemiology of human enterovirus 71 world health organization. global surveillance for severe acute respiratory syndrome (sars) preventing local transmission of sars: lessons from singapore coronavirus as a possible cause of severe acute respiratory syndrome severe acute respiratory syndrome: radiographic and ct findings acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection update: severe acute respiratory syndrome-toronto, canada isolation and characterization of viruses related to the sars coronavirus from animals in southern china we are grateful to all the health-care workers who participated in this study. we would also like to thank the medical students mo-yee chau, hui-yi chia, cherylin foo, and teck-wei tan for help with data entry. key: cord-269654-473kac75 authors: voo, teck chuan; clapham, hannah; tam, clarence c title: ethical implementation of immunity passports during the covid-19 pandemic date: 2020-06-24 journal: j infect dis doi: 10.1093/infdis/jiaa352 sha: doc_id: 269654 cord_uid: 473kac75 a number of countries are planning the use of “immunity passports” as a way to ease restrictive measures and allow infected and recovered people to return to work during the covid-19 pandemic. this paper brings together key scientific uncertainties regarding the use of serological tests to assure immune status and a public health ethics perspective to inform key considerations in the ethical implementation of immunity passport policies. ill-conceived policies have the potential to cause severe unintended harms that could result in greater inequity, the stigmatization of certain sectors of society, and heightened risks and unequal treatment of individuals due to erroneous test results. immunity passports could, however, be used to achieve collective benefits and benefits for specific populations besides facilitating economic recovery. we conclude that sector-based policies that prioritize access to testing based on societal need are likely to be fairer and logistically more feasible, while minimizing stigma and reducing incentives for fraud. clear guidelines need to be set out for which sectors of society should be prioritized for testing, and rigorous mechanisms should be in place to validate test results and identify cases of reinfection. the coronavirus disease 2019 (covid-19) pandemic has resulted in severe movement restrictions in many countries. these come at tremendous social cost, and there are ethical and economic imperatives to use proportionate strategies that minimize the duration of disruption. a key challenge is that social distancing measures can keep transmission in check but, without widely available vaccines, easing restrictions risks surges in transmission. this will occur until a sufficient fraction of the population has been infected and developed immunity, such that epidemics can no longer be sustained. "immunity passports" or certificates have been proposed to ease restrictions on infected and recovered individuals, allowing some people to return to work and kick-start economic recovery. these passports would rely on verifying an individual's serological status using tests to detect antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2). such tests are increasingly widely available, but key scientific questions remain. specifically, the correlation between presence of antibodies and immunological protection, and the duration of immunity to sars-cov-2, are unclear. additionally, the lag between infection and antibody development can affect test accuracy, as infected individuals may test negative if testing is done before antibodies develop. based on these scientific considerations, the world health organization (who) currently does not support the use of immunity passports but will review their guidance as the evidence evolves [1]. besides these scientific challenges, implementation of immunity passport policies raises important ethical questions [2] . here, we use a public health ethics framework to examine ethical implications of immunity passport policies. a central public health ethics principle is that of least restrictive intervention, which requires policy makers to choose measures that would least interfere with the valuable freedoms of individuals to achieve public health objectives [3] . if scientifically supported, allowing individuals who are immune and pose little risk of sars-cov-2 transmission to regain their liberties through immunity certification satisfies this principle [4] . while immunity passports could lead to unequal liberties and privileges between immune-certified and noncertified individuals, this is not inherently discriminatory if founded on evidence-based risk stratification [4, 5] . these arguments provide in-principle support for immunity passport policies. however, the acceptability of such policies depends on mitigating ethical concerns related to their implementation. here, we examine ethical concerns related to equity, stigma, and unintended harms, highlight some potential benefits of immunity passport policies, and make recommendations for their ethical implementation from a public health perspective. even if serological tests deployable en masse become available, supplies will initially be limited; prioritizing access to testing is a key consideration. crucially, equity principles do not imply equal access, but require needs assessment. globally, more vulnerable economies should be prioritized over more resilient ones. nationally, needs also vary substantially and should inform prioritization. workers in certain sectors are needed more urgently, including emergency services personnel, law enforcement, and teachers. others, including health care and social workers, have contact with more vulnerable populations. social distancing measures also impact certain groups more severely, including those with limited mobility who rely on services provided by others. financial barriers to testing should also be considered. globally, allowing market forces to dictate access will exacerbate inequities, allowing wealthier countries that can afford mass testing to recover sooner without assured access to testing for low-income countries. pooling of funds from international agencies and public-private partnerships should be considered to help finance testing in lowincome countries. at the individual level, if cost is a barrier to testing, those needing to return to work more urgently, such as day-wage earners, migrant laborers, low-income families, and those who cannot work while in isolation may be less able to pay for testing that could allow them to resume work. if the implementation of social distancing measures, and the mitigation of their impacts, is a state responsibility, then the cost of testing for immunity certification should be borne by the state, either directly or through employer-based financing schemes. classifying certain individuals as fit for work based on immunological status could result in stigma from inability to resume normal activities, and create resentment at the stratification of society according to "immunological fitness. " ironically, here stigma could be associated with people who have yet to be infected. in certain settings, such as schools, such stigma could be amplified, contributing to disparities in mental and physical health, and access to education. public health measures that result in stigma are not inherently unethical insofar as stigma is not an intended effect to achieve the public good [6] . nevertheless, policy makers are obligated to ensure that any stigma resulting from immunity passports is mitigated and short term. in this regard, immunity passport policies should be recognized and communicated as stop-gap measures within wider, multipronged approaches to help society transition out of restrictive measures, until effective vaccines become widely available. the ability to return to work sooner may provide perverse incentives to deliberately increase one's sars-cov-2 exposure. such behavior is highly undesirable, given the known risk of severe illness and the current strain on scarce intensive care resources in most countries. another concern is the potential for fraud through falsified blood samples or test results, which could enable susceptible individuals to resume normal activities, thus posing an increased infection risk to themselves and others. such behaviors run counter to a general duty not to harm others and the heavy emphasis in many countries on collective responsibility and cooperation with public health measures to protect others. similar perverse incentives have been reported from countries that require proof of a negative test of active sars-cov-2 infection from individuals travelling between regions; in some countries this has led to a black market for counterfeit certificates [7] . such fraud could be mitigated through digital verification systems. for example, blockchain applications are in development to link verified test results with individual identity [8] . such technologies, however, require public trust in mechanisms for privacy protection, and legal and regulatory mechanisms will be needed to limit access to and abuses of such data. aside from fraud, inappropriate easing of restrictions for some individuals could occur simply because of limitations on the predictive ability of serological tests. ideally tests should minimize false positives (which would allow susceptible individuals to return to work) and false negatives (which would deny recovered individuals the chance to return to work sooner). of particular interest here is the probability that a person who tests positive is actually immune (the positive predictive value). this depends on test accuracy, but also on population infection prevalence. initial seroprevalence and modelling studies from a number of highly affected countries indicate that around 5% of the general population has been infected during the first epidemic wave [9, 10] . at this level of population infection prevalence, a person testing positive by a test that is 95% sensitive and 95% specific has even odds of being a false positive (figure 1) . thus, if the proportion of the population infected is low, a substantial fraction of positive test results will be falsely positive and of little value in determining who should resume normal activities. in addition, viral antigenic drift could result in diminished immune protection to emergent virus strains among seropositive individuals, as happens with other coronaviruses and influenza viruses. conversely, some individuals may never yield a positive serology test, because of immune deficiencies or other factors inhibiting antibody responses. clear guidelines will be needed to determine when such individuals are allowed to resume normal activities, given that individuals' infection (and subsequent immunity) status can change over time. given current uncertainty in how measured immune responses correlate with the level and duration of protection, policies must also consider the duration of validity of immunity passports and what consequences there would be for passport holders if they subsequently became infected with sars-cov-2. waning immunity is a recognized feature of coronavirus infections, including sars and mers [11, 12] . serological responses might thus signify time-limited reduction in risk of reinfection (or severe disease) rather than life-long protection. commitment would therefore be needed for access to regular testing to monitor individual changes in serostatus over time. notwithstanding the above challenges, immunity passports have benefits beyond helping individuals to resume normal activities and facilitating economic recovery. firstly, policies such as remote working are unfeasible for workers in some sectors. allowing recovered individuals to resume work sooner could help optimize available state compensatory measures, by focusing support on those whose movements are still restricted due to susceptibility to infection, but who are unable to work. additionally, information on the serological status of workers in different sectors could inform prioritization of vaccine allocation, for example by identifying essential workers who are still susceptible to infection. appropriate care could also be reestablished for vulnerable patient groups impacted by no-visitor policies [13] , by allowing visits from family members with verified serological status. lastly, immunity passports could help reduce risk of international epidemic spread by serving as a permit for travel, akin to currently used vaccination certificates. this would require globally accepted test standards and compliance with the international health regulations (2005) [14, 15] , particularly with regards to seeking "any available specific guidance or advice from the who" (see article 43.2 on additional health measures [14] ). international health regulations allow state parties to implement additional measures as a precaution, but these should not "be more restrictive of international traffic and not more invasive or intrusive to persons than reasonably available alternatives that would achieve the appropriate level of health protection" (article 43.1 [14] ). potential strategies to implement immunity passport policies require a comprehensive assessment of benefits and harms, and what would least restrict individual liberties without significantly heightening the threat of covid-19. current scientific uncertainty on the extent and duration of antibody-mediated immunity to sars-cov-2 makes this challenging. some countries are likely to push ahead with an immunity passport program to accelerate economic recovery. however, ill-conceived policies have the potential to cause severe unintended harms that could result in greater inequity, the stigmatization of certain sectors of society, and heightened risks and unequal treatment of individuals due to erroneous test results. the risk of such harms could be reduced test characteristics 80% sensitivity, 90% specificity 90% sensitivity, 90% specificity 95% sensitivity, 95% specificity 95% sensitivity, 99% specificity figure 1 . positive predictive value (ppv) of a serological test (y-axis) at varying levels of population infection prevalence (x-axis), for tests with different sensitivity and specificity profiles. the ppv indicates the probability that an individual who tests positive truly has been infected with severe acute respiratory syndrome coronavirus 2 (sars-cov-2). for a test with 95% sensitivity and 95% specificity (dashed line), if population infection prevalence is 5%, there is a 50% chance that an individual who tests positive truly has been infected (and a 50% chance that the result is a false positive). through a centralized policy with clear guidelines on which sectors of society to prioritize for testing and rigorous mechanisms to validate test results and identify cases of reinfection. sector-based policies that prioritize access to testing based on societal need are likely to be fairer and logistically more feasible, while minimizing stigma and reducing incentives for fraud. the context of covid-19 swiss national covid-19 science task force. policy brief. ethical, legal, and social issues associated with "serological passports an ethics framework for public health the ethics of covid-19 immunity-based licenses privileges and immunity certification during the covid-19 pandemic the stigmatization dilemma in public health policy-the case of mrsa in denmark indonesia clamping down on fake medical certificates used to circumvent covid-19 travel curbs covid-19 'immunity passport' unites 60 firms on selfsovereign id project estudio ene-covid19: primera ronda estudio nacional de sero-epidemiología de la infección por sars-cov-2 en españa: informe preliminar estimating the burden of sars-cov-2 in france mers-cov antibody responses 1 year after symptom onset longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes dementia care during covid-19 international health regulations do not violate the international health regulations during the covid-19 outbreak potential conflicts of interest. c. c. t. has received funding from roche for research unrelated to this work. all other authors report no potential conflict. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-261472-qcu73sdu authors: yao, yong xiu; ren, junyuan; heinen, paul; zambon, maria; jones, ian m. title: cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein date: 2004-07-01 journal: j infect dis doi: 10.1086/421280 sha: doc_id: 261472 cord_uid: qcu73sdu severe acute respiratory syndrome (sars) coronavirus (scov) spike (s) protein is the major surface antigen of the virus and is responsible for receptor binding and the generation of neutralizing antibody. to investigate scov s protein, full-length and individual domains of s protein were expressed on the surface of insect cells and were characterized for cleavability and reactivity with serum samples obtained from patients during the convalescent phase of sars. s protein could be cleaved by exogenous trypsin but not by coexpressed furin, suggesting that the protein is not normally processed during infection. reactivity was evident by both flow cytometry and western blot assays, but the pattern of reactivity varied according to assay and sequence of the antigen. the antibody response to scov s protein involves antibodies to both linear and conformational epitopes, with linear epitopes associated with the carboxyl domain and conformational epitopes associated with the amino terminal domain. recombinant scov s protein appears to be a suitable antigen for the development of an efficient and sensitive diagnostic test for sars, but our data suggest that assay format and choice of s antigen are important considerations. and is responsible for virus attachment to receptors, entry by fusion, and the development of neutralizing antibody [9] . of interest, most of the residue changes identified within s protein lie in a region that, in other covs, causes significant alteration in tropism [10] , suggesting that drift toward a virus more capable of using a human cell-surface receptor could occur. the structure and function of scov s protein is, therefore, an integral part of studies of viral tropism and of the development of a receptor-blocking antibody response. during the convalescent phase of sars, antibody against the whole virus can be detected at day 10 and increases to a peak titer by day 28 [11] . however, the spectrum of the antibody response, its role in viral clearance, and its use as a correlate of protection has not been described. because scov s protein is such a pivotal viral protein, we have applied a high-throughput expression strategy to the production of recombinant s protein for biochemical and antigenic characterization. here, we describe high-level expression of 6 overlapping fragments of s protein on the surface of insect cells, a context that allows high-level production and maintains sensitive conformation [12, 13] . recombinant s protein showed strong serum reactivity in convalescent-phase serum samples by use of both flow cytometry and western blotting (wb), suggesting that it could be used in the development of sars-specific diagnostic techniques. reactivity was not equivalent between fluorescence-activated cell sorting (facs) and wb, however, and was not equal on all fragments, providing evidence of a variety of antibodies in the host response. cell culture. spodoptera frugipurda (sf9) cells were routinely cultured in sf900-ii medium (life sciences) at 28њc in suspension culture. for plaque assays, cells were allowed to settle onto polystyrene dishes for 1 h before virus inoculation. after virus adsorption, cells were sequentially overlaid with 1.6% lowmelting-temperature agarose and then with sf900-ii supplemented with 5% fetal calf serum and were cultured for 4 more days before staining with neutral red. vector construction. a cdna encoding the full-length s protein minus the signal peptide and transmembrane (tm) domain (aa 18-1193) of the hong kong isolate of scov [7] was supplied by andrew davidson and stuart siddell (university of bristol, bristol, uk) as a reverse-transcription polymerase chain reaction-amplified fragment and was first cloned into ptopo (invitrogen). several overlapping fragments of s protein, with end points chosen after bioinformatics analysis, were amplified from the original clone. all fragments generated were flanked by dissimilar restriction sites for the enzyme sfii. each fragment was cloned into a set of 3 vectors (18 variants in all), to provide his-tagged, maltose binding protein-tagged, and cell surface-displayed forms of s protein. only the cell surface-displayed cassette is described in detail here. the baculovirus display transfer vector pacvsv gtm [12] , modified to include sfii sites at the junction of the signal peptide and the vesicular stomatitis virus (vsv) g protein tm domain, was used to provide expression at the cell surface. expression in insect cells. for expression in insect cells, recombinant baculoviruses were constructed by use of a new, rapid recombination technique after cotransfection of each transfer vector with a linearized, modified form of bacmid dna capable of growth only after recombination [14] . coexpression of mouse furin or human calnexin was done by coinfection of viruses at mois 15 [15] . wb. protein samples to be analyzed were separated on precast 10% tris-hcl sds-polyacrylamide gels (bio-rad) and transferred onto immobilon-p transfer membranes (millipore). wb was performed with human convalescent-phase serum or rabbit serum to the vsv g protein tm domain (research diagnostics), followed by peroxidase-coupled secondary antibodies (sigma). the membrane was finally developed by use of bm chemiluminescence (roche). facs analysis (flow cytometry). cells were plated in a 6well plate ( cells/well) and overlaid with 2 ml of me6 1 ϫ 10 dium. after virus inoculation, cells were cultured for a further 2 days at 28њc, harvested, washed with pbs, fixed with cellfix (becton dickinson), and stained with serum and conjugate. data acquisition and analysis were performed by use of a fac-scan flow cytometer and cellquest software (both from becton dickinson). human serum. serum samples from patients with sars and human cov 229e were obtained from the respiratory disease reference laboratory of the health protection agency (colindale, uk). nine probable sars cases occurred in the united kingdom, but only 4 conformed to the clinical definition of sars in use at the time. serum samples from patients known to be positive for sars were derived from the patients with clinically confirmed cases, whereas serum samples used as probable but unconfirmed sars cases were derived from the remaining 5 patients. all patients recovered from sars. no human cov oc43 serum samples were available for the present study, so the reactivity of cov oc43 with sars s protein could not be assessed. similarly, only 2 serum samples from cov 229e-infected patients were available. serum samples were used in facs and wb analyses at 1:50 and 1:400 dilutions, respectively, unless otherwise stated. the time-course serum samples, available from 1 of the patients with confirmed sars, were assayed by use of elisa using lentil lectin-captured, fulllength s protein, and the 50% end-point titer was compared. the class of antibody bound was ascertained by use of classspecific, secondary anti-human antibodies (sigma). the scov s protein is ∼1200 residues and has 23 glycosylation triplets clustered toward the amino and carboxyl termini. because glycosylation and protein folding are often linked, we focused our expression studies on the use of a highyielding but eukaryotic-based expression system: baculovirusmediated expression in insect cells. we examined a number of biochemical features, to characterize the protein expression before use for antibody binding. the s protein signal peptide and tm domain were substituted for regions known to function well in this expression system-a signal peptide from the major baculovirus glycoprotein gp64 and a tm domain from the vsv g protein already present in pacvsv g tm [12] . this vector efficiently displays proteins on the surface of insect cells and is a method of expression that maintains the conformational integrity of even problematic proteins [13] . in addition to the full-length s protein, fragments representing the predominantly b-sheet and a-helical domains (residues 18-713 and 714-1193, respectively), a fragment from the n terminus (residues 18-410) predicted to form a distinct folded domain when analyzed by folding predication software [16] , and fragments representing residues 411-1193 and 411-713 were expressed in the same way ( figure 1a) . abundant expression of all s protein fragments was achieved by use of this strategy, as shown by wb with a vsv g tm domain antibody that showed 2 predominant bands for each construct representing nonglycosylated and glycosylated proteins, with apparent molecular weights consistent with those predicted (figure 1b). interaction with calnexin and s protein cleavage. the s proteins of all covs are heavily glycosylated, and improper glycan trimming can result in poor surface-expression levels through retention of the viral glycoprotein by the glycoprotein chaperone calnexin [17, 18] . to assess whether the calnexin pathway was relevant to expression of s protein, we coinfected recombinant full-length s protein with a virus-expressing calnexin and re-examined the level of glycosylated and nonglycosylated protein at 3 days after infection. compared with expression of s protein only, coexpression of s protein with calnexin led to an increase in the ratio of glycosylated to nonglycosylated product, calculated by use of gel scan to be ∼20% ( figure 2a) , showing that s protein interacts with calnexin during glycosylation. however, no overall increase in s protein yield was observed, despite improved glycoprotein processing. some, but not all, cov s proteins are cleaved around the center of the molecule to form the receptor-binding outer domain s1 and the inner-membrane fusion domain s2 [19, 20] . the full-length s protein was not cleaved in insect cells, with the fully glycosylated form having an apparent molecular weight of ∼180 kda ( figure 1b) . we assessed the potential for s protein to be cleaved by 2 proteases, the subtilisin-like furin (provided by coinfection, as described elsewhere [15] ) and by trypsin (added exogenously). furin is a classic viral glycoprotein maturation enzyme [21] . the consensus furin cleavage site does not occur in the s protein sequence, but several dibasic sites, which also can be recognized by the enzyme [15] , are present, allowing the possibility of furin cleavage. recombinant s protein produced in the presence of coexpressed furin showed no evidence of cleavage (figure 2b), suggesting that furin is not associated with scov envelope maturation. s protein on the surface of infected insect cells was also treated with trypsin under conditions found to cleave murine cov s [19] and was reanalyzed after the cleavage reaction, by wb using the vsv g tm domain and patients' antibody. treatment with trypsin caused the loss of the ∼180-kda glycosylated s protein band and concomitant appearance of 2 new bands, the most intense of which was at ∼70 kda and whose mobility was indistin-guishable from that shown by s-f3r1 (figures 1b and 2c). the f3/r3 junction, which was chosen wholly on the basis of predicted secondary structure, is at residue 713, and a single lys residue occurs at position 714. on the basis of these data, we suggest that trypsin can access the s protein under nondenaturing conditions, to cleave at lys 714 and produce a b-sheetrich s1 domain and a a-helix-rich s2 domain. interestingly, molecular modeling of the scov s protein has also suggested that the s1-s2 junction lies within the amino acid sequence 680-727 [22] . no s protein was found in the nonpellet fraction after the addition of trypsin, suggesting that the 2 s protein domains remain associated even after cleavage (data not shown). when probed with patients' serum samples, the presumed s2 domain at ∼70 kda was also highlighted, but there were also a number of smaller breakdown products. no distinct band at the predicted size of the s1 domain was visible, suggesting that it is degraded by trypsin or is not detected efficiently by the available human serum samples (see below). whether s protein is cleaved before or during scov cell entry remains undetermined. reactivity with human serum samples. the abundant, stable, and characterized surface expression of a suite of s protein-related fragments prompted us to use these reagents to investigate the antibody response to s protein in the serum samples from infected individuals. serum samples used were obtained from several uk patients (for patients' details, see materials and methods); in addition, serum samples representing a time course from disease onset were obtained from 1 reference patient. we assessed the reactivity of each serum sample with each fragment by use of flow cytometry and wb, to represent native and denatured sources of antigen, respectively. cytometry profiles showed the greatest reactivity of patients' serum samples with the protein s-f1r2 and s-f1r1, but all other fragments also reacted, with the exception of s-f2r3 (aa 411-713), which represents the middle section of the s coding region and failed to react significantly with most patients' serum samples, despite high levels of expression ( figure 1b) . the same general pattern of binding was apparent for all the patients' serum samples, although the exact degree of reactivity with each fragment varied somewhat (of the 2 serum samples shown in figure 3 , the data for serum sample 1 were the more typical pattern). when serum reactivity was assessed by use of wb, however, the pattern of binding was substantially different. reactivity was seen with the full-length protein s-f1r1, but strong binding was also seen with s-f2r1 and s-f3r1 ( figure 3, bottom) . one serum sample (2908, the most broadly reactive of all the samples) showed some reactivity with s-f1r2, s-f1r3, and s-f2r3 but was very weak, compared with fragments spanning the c-terminal half of the s protein, and was wholly absent in other serum samples (typified by the wb using serum sample 1 in figure 3, bottom) . quantitation of the serum response to s protein by densitometry (wb) and relative fluorescence (facs) gave the reactivity orders and f3r1 1 f2r1 1 f1r1 111 others , respectively (figure 4). f1r1 p f1r2 1 f1r3 p f2r1 p f3r1 thus, there was a clear shift in reactivity with the same serum samples, depending on the assay format. specificity of serum reactivity with s protein fragments. the possible use of insect cell-displayed s protein for diagnostic application was assessed by examining fragment reactivity with serum samples from patients infected with human cov 229e and also with serum from a patient with suspected but clinically unconfirmed sars (serum sample 3118). preliminary data have indicated that serum samples from some cov 229e-infected individuals can cross-react with purified scov nucleocapsid protein (p.h. and m.z., unpublished data), so the use of a more specific test for seroconversion, based on s protein, may be valuable. serum samples from 2 patients with confirmed cov 229e infection did not react with s-f1r2 by use of facs (figure 5), whereas the serum sample from a patient with suspected sars showed a weak but clear shift in fluorescence. wb could also distinguish the serum samples, although the discrimination between the samples was not as good as that achieved by use of facs (data not shown), suggesting that the most discriminatory test in uncertain cases should use optimized s protein fragments presented in a nondenaturing assay format. time course of antibody response. a set of serum samples representing a time course from 6 to 40 days after the onset of sars for 1 patient was obtained and used in an s protein-specific elisa, to determine the increase in s protein titer over time. the titer of s protein antibodies was significant from day 10, similar to the serum responses reported for whole infected cells [11] and isolated n protein [23] , and increased to the latest time point (40 days after onset) ( figure 6 ). serum samples were also used in wb assays to examine the changes of reactivity with each individual s protein fragment over time. the earliest wb-positive serum sample (10 days after onset) gave a pattern of binding similar to that of the other serum samples tested (figures 3 and 6). the 40-day time point provided a much stronger signal, but the pattern of binding was not altered ( figure 6 ). blots were also probed with isotype-specific conjugates, and the results were compared with blots probed with an igg-specific conjugate. we found evidence for the development of some igm response late in the recovery period, but it was weak, compared with igg (data not shown). only serum antibodies were available for the present study, and it remains possible that secreted antibodies are also present in respiratory secretions early during the infection. our data provide the first profiles of antibody binding to the scov s protein. efficient expression of the s protein as a specific source of antigen was achieved by use of a highly productive expression system, recombinant baculoviruses, combined with the use of signal and tm sequences proven to efficiently direct proteins to the surface of the expressing cell [12] . this format offered assays based on antibody binding to the cell surface, mimicking infected cells but with singularly high levels of expression of s protein, as well as denatured antigen prepared by cell lysis. characterization of the expressed products provided some evidence for potential interaction with a known glycoprotein chaperone (calnexin), although the stimulation in glycan processing was marginal (20% of the total), and for cleavage of s protein by trypsin-like, but not furin, proteases. cleavage of surface glycoproteins is a key factor in pathogenesis for some viruses [24, 25] , and it will be interesting to evaluate the role of s protein cleavage, if any, in scov infectivity in both animal and human hosts. in the present study, full-length s protein on the surface of insect cells did not bind a variety of species of red blood cells, suggesting that it does not hemagglutinate. s protein released by detergent lysis also failed to bind to any discrete proteins in far-western blot assay of vero cell membranes (data not shown). accordingly, our data do not address the nature of the receptor for scov, in particular the early suggestion that it may be cd13 [14] . recent data show angiotensin-converting enzyme 2 to be at least 1 functional scov receptor [26] . efficient sources of several recombinant s protein fragments allowed us to evaluate the predominant antibodies present in convalescent serum samples. interestingly, the patterns of s reactivity with available serum samples was similar within each assay format, suggesting that the immune response to s protein varies only quantitatively between individuals. this result would be in keeping with the apparently low immunological pressure suggested from the sequence variation observed in several isolates [7] . of the 2 assay formats we used, nondenatured s protein present on the cell surface provided the most sensitive detection of antibodies, with clear shifts in fluorescence for serum samples from patients with suspected but clinically unconfirmed sars. no shift was apparent with human cov 229e serum samples, suggesting that this format is highly specific. when reactivity of positive serum samples to individual s protein fragments was compared, we observed a strong differential binding, depending on the assay used. thus, although facs showed the highest reactivity with fragments, including the amino terminus of s protein, wb with the same serum samples showed preferential reactivity to the carboxyl terminal half of the molecule. differential antibody binding was not influenced by the high-mannose glycans present on insect-derived glycoproteins, because s protein prepared in mimic cells (invitrogen), which add complex glycans to the polypeptide [27] , showed the same pattern of reactivity with the serum samples used (data not shown). our data, which were obtained with only the few serum samples from patients with sars available in the united kingdom, clearly require confirmation with larger sets of serum samples but would be consistent with a level of conformational antibody present in the serum samples preferentially directed toward the globular n terminus of the protein and detected by use of facs but not by use of wb. this class of antibody includes those most useful for diagnosis (see above), suggesting that the most suitable assay format for a final diagnostic will be best configured with authentically folded protein, rather than, for example, peptides. our preliminary work with purified soluble full-length s protein suggests that sensitivity is maintained with the soluble protein, indicating that assay formats simpler than flow cytometry will be feasible. confirmation that the class of conformational antibody directed at the globular s1 domain includes neutralizing antibody, and whether it has any bearing on the outcome of infection, will be important if recombinant s protein is also to be considered as part of a possible vaccine for scov infection. identification of a novel coronavirus in patients with severe acute respiratory syndrome koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars coronavirus: a new challenge for prevention and therapy pathogenesis of chimeric mhv4/ mhv-a59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study non-polar distribution of green fluorescent protein on the surface of autographa californica nucleopolyhedrovirus using a heterologous membrane anchor baculovirus surface display of theileria parva p67 antigen preserves the conformation of sporozoite-neutralizing epitopes putative hapn receptor binding sites in sars-cov spike protein legitimate and illegitimate cleavage of human immunodeficiency virus glycoproteins by furin why are "natively unfolded" proteins unstructured under physiologic conditions? role of n-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control folding and oligomerization of influenza hemagglutinin in the er and the intermediate compartment conformational changes in the spike glycoprotein of murine coronavirus are induced at 37њc either by soluble murine ceacam1 receptors or by ph 8 the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex furin at the cutting edge: from protein traffic to embryogenesis and disease molecular modelling of s1 and s2 subunits of sars coronavirus spike glycoprotein antibody response of patients with severe acute respiratory syndrome (sars) to nucleocapsid antigen of sars-associated coronavirus furin: a mammalian subtilisin/kex2p-like endoprotease involved in processing of a wide variety of precursor proteins the pathogenesis of influenza in humans angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus engineering the protein n-glycosylation pathway in insect cells for production of biantennary, complex n-glycans we thank robin gopal (health protection agency, colindale, uk), for providing the original viral rna; andrew davison and stuart siddell (university of bristol, bristol, uk), for providing spike protein cdna; malik peiris (university of hong kong, hong kong), for distribution of source materials; christian drosten (bernard noch institute, hamburg, germany); and members of the virology group, for constructive criticism. key: cord-307918-8y89p11a authors: onyango, clayton o.; njeru, regina; kazungu, sidi; achilla, rachel; bulimo, wallace; welch, stephen r.; cane, patricia a.; gunson, rory n.; hammitt, laura l.; scott, j. anthony g.; berkley, james a.; nokes, d. james title: influenza surveillance among children with pneumonia admitted to a district hospital in coastal kenya, 2007–2010 date: 2012-12-15 journal: j infect dis doi: 10.1093/infdis/jis536 sha: doc_id: 307918 cord_uid: 8y89p11a background. influenza data gaps in sub-saharan africa include incidence, case fatality, seasonal patterns, and associations with prevalent disorders. methods. nasopharyngeal samples from children aged <12 years who were admitted to kilifi district hospital during 2007–2010 with severe or very severe pneumonia and resided in the local demographic surveillance system were screened for influenza a, b, and c viruses by molecular methods. outpatient children provided comparative data. results. of 2002 admissions, influenza a virus infection was diagnosed in 3.5% (71), influenza b virus infection, in 0.9% (19); and influenza c virus infection, in 0.8% (11 of 1404 tested). four patients with influenza died. among outpatients, 13 of 331 (3.9%) with acute respiratory infection and 1 of 196 without acute respiratory infection were influenza positive. the annual incidence of severe or very severe pneumonia, of influenza (any type), and of influenza a, was 1321, 60, and 43 cases per 100 000 <5 years of age, respectively. peak occurrence was in quarters 3–4 each year, and approximately 50% of cases involved infants: temporal association with bacteremia was absent. hypoxia was more frequent among pneumonia cases involving influenza (odds ratio, 1.78; 95% confidence interval, 1.04–1.96). influenza a virus subtypes were seasonal h3n2 (57%), seasonal h1n1 (12%), and 2009 pandemic h1n1 (7%). conclusions. the burden of influenza was small during 2007–2010 in this pediatric hospital in kenya. influenza a virus subtype h3n2 predominated, and 2009 pandemic influenza a virus subtype h1n1 had little impact. the coast of kenya, approximately 60 km north of mombasa. the district comprises a largely rural population of subsistence farmers and has an equatorial climate, with rain predominantly falling during april-july and october-december. kdh is the principal hospital facility for the population of the khdss. further details of the study area and respiratory disease surveillance at kdh and local clinics can be found in previous reports (2) (3) (4) 13) . nasopharyngeal wash or aspirate specimens were collected from eligible children aged 1 day to 12 years from january 2007 through december 2010 and were either stored in viral transport medium at −80°c (2007-2009) prior to molecular screening or screened prior to freezing in raw form. inpatients were eligible if they were admitted to the hospital with cough or difficulty breathing and either lower-chest-wall indrawing (defined as severe pneumonia) or 1 or more of the following: cyanosis, prostration, unconsciousness, or an oxygen saturation level <90% (a modification of the world health organization criteria for very severe pneumonia). children who were not residing in the khdss at admission, were admitted in extremis, were admitted for elective surgery, or received a diagnosis of neonatal tetanus were excluded. the following clinical and laboratory features obtained on admission or that relate to discharge outcome were compared between influenza-positive and influenza-negative children: duration of hospitalization >14 days, very severe pneumonia, wheezing, hypoxia (oxygen saturation level <90%, by fingertip pulse oximetry), circulatory shock (capillary refill time ≥3 seconds), severe anemia (hemoglobin level <5 g/dl), prematurity, congenital heart disease, positivity for hiv antibody (by 2 rapid tests), severe underweight (weight for age z score ≤3), slide positivity for plasmodium species, bacteremia, concurrent viral infection diagnosis, and death before discharge [2] . outpatient recruits were a convenience sample of children aged <13 years, enrolled for broad comparison with hospitaladmitted patients, who presented with either no signs of acute respiratory infection (non-ari) or signs of upper respiratory tract infection (urti) [2, 5] . individuals with urti had 1 or more of the following: cough, difficulty breathing, nasal discharge, runny or blocked nose, or sore throat. written informed consent was obtained from the parent or guardian of subjects. this study was approved by the kenyan national ethical review committee and the university of warwick biomedical research ethics subcommittee. diagnostic real-time reverse-transcription polymerase chain reaction (rt-pcr) rna was extracted from 140 µl of nasopharyngeal samples, using the qiagen viral rna miniprep kit (qiagen, united kingdom), or from 200 µl of nasopharyngeal samples, using the total nucleic acid extraction kit (roche applied science, germany) with a magna pure lc32 automated nucleic acid extractor, following the manufacturer's instructions. diagnostic screening for viral targets was performed using real-time rt-pcr. for specimens from kdh inpatients in 2007 and from outpatients in 2007-2008, reactions were tested by multiplex real-time rt-pcr, using fret hybridization probes as described by lassaunière et al [6] , with primers and probe targeting ns1 of influenza a virus and nucleoprotein of influenza b virus. all other samples were screened using the taqman qiagen quantifast multiplex method on the abi 7500 platform described by hammitt et al, targeting the matrix protein for influenza a and c viruses and targeting ns for influenza b virus [3] . concurrent targets in both rt-pcr assays included respiratory syncytial virus (rsv), adenovirus, rhinovirus, parainfluenza virus (piv) 1-3, human metapneumovirus, coronavirus (cov 229e, nl69, and oc43), and, differentially by assay, coronavirus hong kong [6] and piv4 [3] . an aliquot of each of the influenza a virus-positive specimens was shipped to the national influenza center (nic) in nairobi for subtyping. samples were subjected to rna extraction using the qiaamp viral rna isolation kit (qiagen). detection was performed using invitrogen superscript iii platinum one-step quantitative kit with primers and probes targeting seasonal influenza a virus h1n1 (a[h1n1]), a (h1n1)pdm09, and influenza a virus subtype h3n2 (a [h3n2]). statistical analysis was undertaken using stata, version 11.0 (statacorp, college station, tx) and microsoft office excel 2003 (microsoft, redmond, wa). the incidence of influenza among inpatients for age group i, i(i), per 100 000 population per year was estimated on the basis of the equation i(i) = [c (i)/n(i)p(i)].100,100, where c(i) is the average number of children per year admitted who were resident in the khdss in age group i, n(i) the midsurvey khdss population in age group i, and p(i) is the proportion of eligible children tested for influenza (ie, we assumed that children who were not tested would have had the same prevalence of influenza as those who were tested and scaled the incidence accordingly). for pneumonia incidence estimates, p(i) is set to 1. the kdhss population on 1 january 2009 was estimated to be 9451 individuals aged <1 year, 45 644 aged <5 years, and 108 708 aged <13 years. the population size and incidence estimation procedures have been described elsewhere [4] . the incidence estimation for the population proximal to the hospital was undertaken using cases involving children aged <5 years admitted from administrative sublocations within a 5km radius of the hospital and the corresponding midpoint population estimate from the khdss (12 339 as of 1 january 2009). the wilcoxon rank sum test was used to compare median ages; the χ 2 or fisher exact test was used to compare proportions, as appropriate; the score test ( procedure tabodds) was used to assess the trend in prevalence, by age; and the poisson probability distribution was used to assess whether observed cases of influenza in specified quarters of the year exceeded the expected number of cases. spearman rank correlation was used to test for a temporal association between monthly or quarterly numbers of influenza cases or influenza a virus infections and the number of cases of bacteremia or streptococcus pneumoniae infection. the analysis was undertaken with cases of influenza and bacteremia temporally in phase or between 1 and 4 months time step out of phase. the association between positivity for any influenza type and laboratory or clinical features on admission was assessed using logistic regression, adjusted for age group, to obtain odds ratios (ors) and 95% confidence intervals (95% cis). over the 4-year period, there were 2429 admissions to kdh involving individuals who were eligible for the study (57% were boys; median age, 9 months [interquartile range {iqr}, 3-22 months]). a total of 503 (21%) had very severe pneumonia (50% were boys; median age, 10 months [iqr, 2-34 months]; 55% were infants), and the in-hospital case-fatality rate was 6.5%. of the eligible inpatients, 2002 (82%) were tested for influenza (57% were boys; median age, 9 months [iqr, 3-21 months]; 58% were infants), and this percentage did not differ among those aged <1 year, 1-4 years, and ≥5 years (p = .333); 387 (19%) had very severe pneumonia (52% were boys; median age, 10 months [iqr, 2-34 months]; 53% were infants). stratified by severity, 84% of eligible inpatients with severe pneumonia were tested, compared with 77% of eligible inpatients with very severe pneumonia (p = .001). the case-fatality rate among inpatients who were untested was significantly higher than among those who were tested ( ; 37% were infants). compared with the median age of inpatients, the median ages of outpatients with non-ari (p = .004) or urti (p ≤ .0001) were higher. the prevalence of influenza virus of any type was 4.9% (99 of 2002 cases) among inpatients; 4.7% (76 of 1615) had severe pneumonia, and 5.9% (23 of 387) had very severe pneumonia (p = .299). among outpatients, the prevalence of influenza virus of any type was 3.9% (13 of 331) among those with urti and 0.5% (1 of 196) among those with non-ari. data stratified by virus type are presented in table 1 . influenza a virus was the most prevalent type among outpatients with pneumonia (3.5%) and outpatients with urti (3.3%). these proportions were unaltered by restricting the analysis to children <5 years of age. among outpatients classified as having the seasonal patterns of influenza a and b virus infection are shown in figure 1 . over the 4-year period, influenza a and b virus infection showed a significantly higher occurrence among inpatients during quarters 3 and 4, relative to the average for all quarters (22.5 cases expected vs 37.5 cases observed; p = .003). there was very little influenza activity in 2010. the majority of influenza b virus infections occurred in the fourth quarter of 2009. most influenza cases occurred after the main period of rainfall (april-july) and before peak temperatures (first quarter; figure 1 ). cases of bacteremia, and specifically s. pneumoniae infection, by quarter, are shown in figure 1 . no statistically significant correlation between influenza cases (or influenza a virus infections) and occurrence of bacteremia (or s. pneumoniae infection) was identified, either concurrently or delayed (p > .05). of figure 1a) , with around 50% of cases in infants (46% had influenza a virus infection, 47% had influenza b virus infection, and 66% had influenza c virus infection; p = .632). the proportion of inpatients with pneumonia who were found to be influenza positive showed a trend for increase with increasing age (supplementary figure 1b) . in the case of influenza a virus infection, the trend was significant: 1.7% of children aged 0-2 months had influenza a virus infection, compared with >5% of children aged ≥24 months (p = .005). analysis of the association between a diagnosis of infection with any influenza virus and components of a set of severity features yielded an increased odds of hypoxia among children with influenza, compared with those without influenza (ageadjusted or, 1.78; 95% ci, 1.04-1.96). no other severity feature was significantly associated with influenza (supplementary table 1 ). there were 4 deaths (age range, 7-32 months) among the 99 inpatients with influenza; one was infected with influenza a virus, 2 were infected with influenza b virus, and 1 was infected with influenza c virus. three were positive for hiv antibody, severely malnourished (weight-for-age z score, ≤4), and had a discharge diagnosis including immunosuppression, and 1 inpatient (who was infected with influenza a virus) had chronic heart disease. our study identified all 3 influenza viruses in circulation in this rural coastal kenya location among patients hospitalized with severe or very severe pneumonia and among outpatients with urti. however, the prevalence of infection with any influenza virus was low among inpatients with severe or very severe pneumonia (4.9%) and among outpatients with urti (3.9%). influenza a virus predominated, with identification in 3.5% of inpatients and 3.3% of outpatients with urti. correspondingly, relative to the incidence of severe or very severe pneumonia among hospitalized children aged <5 years (1323 cases per 100 000 per year), the incidences of influenza (64 cases per 100 000 per year) and influenza a virus infection (46 cases per 100 000 per year) were low. there was near absence of influenza in the convenience sample of children without signs of respiratory illness. our inpatient data are consistent with the results of a recent review of data on seasonal influenza from 15 published studies in sub-saharan africa [1] , which reported a median prevalence of 6.0% among hospitalized pediatric patients, with a range of 0%-16%. in the same review, the median prevalence of influenza among outpatients with ari was higher than we found, at 10% (range, 1%-25%; 11 studies), but comparisons should be cautioned because of a number of methodological differences. exploration of a range of severity features and concurrent illnesses revealed hypoxia to be more commonly associated with influenza among hospitalized children. calculation of the incidence of influenza-associated severe disease on the basis of hospital admission data is likely to underestimate the true burden in the community, as a result of the relationship between healthcare access and distance from the hospital. this is supported in the analysis, where it was shown that the incidence of influenza-associated admissions among children with severe or very severe pneumonia was about 70% greater in the population proximal to the hospital. we have previously shown a similar distance decay for severe rotavirus diarrhea [7] and severe rsv-associated pneumonia [4] and pneumonia and meningitis [8] . furthermore, a previous study of rsv among infants and young children in the hdss revealed that roughly 4 in 5 children identified with severe pneumonia in the outpatient setting were not admitted to the local hospital [9] . notwithstanding this underestimation, it is clear that the incidence of influenza-associated hospital admissions is significantly lower than that associated with either rhinovirus or rsv infection. while the etiology of rhinovirus as the causative agent of lower respiratory tract disease may be in question [10] , this is not the case for rsv, which is known to be a major cause of infant and childhood lower bronchiolitis and pneumonia in sub-saharan africa and globally [11] . while rsv is invariably among the most prevalent viruses in children admitted with lower respiratory tract illness, it is not always dominant over influenza [1] . in kenya, contemporary data on the relative prevalence of respiratory viruses among pneumonia-related admissions to the hospital are largely absent. further data are clearly needed in kenya to gauge the relative burden of disease due to respiratory viruses and thereby help support future health policy planning. in terms of seasonality, there was increased occurrence in the third and fourth quarters of each year, most notably for influenza a virus infection, except in 2010. these periods are characteristically times of lower rainfall levels (referred to locally as "second rains"), intermediate temperatures, and relative lower humidity. during the study period, a(h1n1)pdm09 entered kenya [12] , and cases of a(h1n1)pdm09 infection were identified in kdh from late 2010. it is possible that the introduction of a(h1n1)pdm09 disrupted the normal pattern of a(h3n2) activity in 2010; only a(h1n1)pdm09 was observed in the latter quarters of 2010. continued surveillance will reveal whether a (h1n1)pdm09 has any long-term effect on the circulation patterns of other influenza subtypes. however, in general, the contribution of a(h1n1)pdm09 to the burden of hospitalization-associated pneumonia was minimal in this setting. the possibility exists that the burden of influenza was underestimated because of associated, but delayed, invasive bacterial disease. however, we identified no evidence for an increased number of admissions in which bacteria (or s. pneumoniae, in particular) were detected in blood cultures during the quarter following peak occurrences of influenza. during the a(h1n1)pdm09 infection pandemic, antiviral therapy (oseltamivir) was prescribed to children admitted to kdh with severe acute respiratory illness on a presumptive basis (ie, prior to laboratory confirmation of influenza.) this would not have altered the pattern of observation of influenza described in this study, because nasopharyngeal specimens were collected prior to treatment with the antiviral. within the surrounding community, a(h1n1)pdm09 vaccination was undertaken in 2010 but was limited to target groups, including healthcare workers, pregnant women, and patients with chronic disease, totaling 2203 subjects (kenya ministry of health, personal communication). this number and the age group of subjects receiving vaccine would not have altered the pattern of a(h1n1) infection occurrence described in this study. half of the influenza cases occurred in infants, and the proportion of cases rapidly declined with age into older age groups. however, we noted that within the age group that we studied, the prevalence of influenza increased with age, suggesting that relative to other causes of cases of severe or very severe pneumonia associated with admission, those caused by influenza decline less rapidly with age. a limitation of the present study is the failure to recruit and test samples from approximately 20% of eligible children. the reasons for this have been previously described [2, 4] and were primarily due to parental refusal and, to a lesser extent, to discharge or death before sampling. failure to recruit and test was more common for critically ill children and for those who died while in the hospital. this almost certainly led to an underestimation of severity associated with influenza. a further limitation of the study is that results are based only on nasopharyngeal samples, and we now have definitive evidence that an oropharyngeal swab specimen provides added diagnostic value for detecting influenza in our setting (22% increased detection; 95% ci, 9-42), compared with nasopharyngeal specimens alone [3] . the low prevalence of influenza viruses in this study limits the power of the analysis to identify associations between virus presence and specific clinical features or coinfections. our study involved sampling of 2000 children; a definitive investigation of clinical associations will require a considerably larger sample size or a location with a markedly higher incidence of influenza a virus infection. we report very low prevalence of influenza in outpatient children without signs of acute respiratory infection, suggesting that influenza virus is rarely the cause of asymptomatic infection, and the data also suggest influenza is the cause of only 4% of urti cases. further interpretation of these data with regard to the association between influenza and severe disease is unwarranted since outpatient sampling was not contemporaneous throughout the period of surveillance of hospitalized patients, and collection was not frequency matched by age and location within the khdss. we therefore await the complete results of a larger and better-designed case-control study, which is ongoing. in conclusion, although the incidence of influenza was underestimated in this study, it is clear that influenza contributes only a small proportion of the total burden of hospitalizationassociated severe and very severe pneumonia among children in this rural coastal kenya setting. influenza a virus is the dominant influenza virus causing pediatric severe and very severe pneumonia. a seasonal signature for influenza was evident, but no temporal association was identified with invasive bacterial disease. although a(h1n1)pdm09 infection was observed, its contribution to disease was not substantial. hypoxia was more frequently identified among patients with influenza, and immunosuppression, severe malnutrition, or chronic heart disease were identified in all of the 4 influenza-associated deaths. given the low influenza prevalence, larger studies are required to investigate associations between influenza and disease severity or prevalent conditions, such as malaria, hiv infection, or malnutrition. additional comparative studies on viral diagnoses in severe pneumonia hospital admissions are warranted elsewhere in kenya. such data may be informative to the kenya ministry of health in their assessment of the role for influenza antivirals and vaccination in kenya. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org/). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. seasonal influenza epidemiology in sub-saharan africa: a systematic review viral etiology of severe pneumonia among kenyan infants and children added value of an oropharyngeal swab in detection of viruses in children hospitalized with lower respiratory tract infection incidence and severity of respiratory syncytial virus pneumonia in rural kenyan children identified through hospital surveillance increased detection of respiratory viruses in paediatric outpatients with acute respiratory illness by real-time polymerase chain reaction using nasopharyngeal flocked swabs a novel multiplex real-time rt-pcr assay with fret hybridization probes for the detection and quantitation of 13 respiratory viruses incidence and clinical characteristics of group a rotavirus infections among children admitted to hospital in kilifi sensitivity of hospital-based surveillance for severe disease: a geographic information system analysis of access to care in kilifi district respiratory syncytial virus infection and disease in infants and young children observed from birth in kilifi district viral respiratory infections in hospitalized and community control children in alaska global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis introduction and transmission of 2009 pandemic influenza a (h1n1) virus-kenya specimen collection for the diagnosis of pediatric pneumonia influenza surveillance of kenyan children with pneumonia acknowledgments. we thank the clinical and laboratory staff, for their hard work in collection and processing of the specimens; mwanajuma ngama, for the coordination of patient recruitment and sample collecpotential conflicts of interest. key: cord-003171-z22ekgtv authors: babu, tara m; perera, ranawaka a p m; wu, joseph t; fitzgerald, theresa; nolan, carolyn; cowling, benjamin j; krauss, scott; treanor, john j; peiris, malik title: population serologic immunity to human and avian h2n2 viruses in the united states and hong kong for pandemic risk assessment date: 2018-10-01 journal: j infect dis doi: 10.1093/infdis/jiy291 sha: doc_id: 3171 cord_uid: z22ekgtv background: influenza a pandemics cause significant mortality and morbidity. h2n2 viruses have caused a prior pandemic, and are circulating in avian reservoirs. the age-related frequency of current population immunity to h2 viruses was evaluated. methods: hemagglutinin inhibition (hai) assays against historical human and recent avian influenza a(h2n2) viruses were performed across age groups in rochester, new york, and hong kong, china. the impact of existing cross-reactive hai immunity on the effective reproduction number was modeled. results: one hundred fifty individual sera from rochester and 295 from hong kong were included. eighty-five percent of patients born in rochester and hong kong before 1968 had hai titers ≥1:40 against a/singapore/1/57, and >50% had titers ≥1:40 against a/berkeley/1/68. the frequency of titers ≥1:40 to avian h2n2 a/mallard/england/727/06 and a/mallard/netherlands/14/07 in subjects born before 1957 was 62% and 24%, respectively. there were no h2 hai titers >1:40 in individuals born after 1968. these levels of seroprevalence reduce the initial reproduction number of a/singapore/1/1957 or a/berkeley/1/68 by 15%–20%. a basic reproduction number (r(0)) of the emerging transmissible virus <1.2 predicts a preventable pandemic. conclusions: population immunity to h2 viruses is insufficient to block epidemic spread of h2 virus. an h2n2 pandemic would have lower impact in those born before 1968. influenza pandemics may occur when influenza a viruses of animal origin with a novel hemagglutinin (ha, or h) with or without neuraminidase (na, or n) subtypes to which the human population has little or no immunity infect humans and transmit efficiently from person to person. there were 3 influenza pandemics in the 20th century. in 1918, an influenza a virus of the h1n1 subtype emerged and caused widespread disease; subsequently, h1n1 viruses caused seasonal epidemics until 1957. in 1957 a new influenza a virus of the h2n2 subtype, sometimes referred to as the "asian" influenza, emerged to cause a second pandemic, and subsequently h2n2 viruses replaced h1n1 viruses as the cause of seasonal influenza from 1957 to 1968. in 1968 a third pandemic was caused by an h3n2 virus, which replaced h2n2 viruses and continues to circulate in humans to the present day. influenza a pandemics are associated with significant mortality, morbidity, and financial burden. for example, the pandemic of 1918-1919 resulted in at least 50 million influenza-related deaths [1] , while the pandemics of 1957 (h2n2) and 1968 (h3n2) combined had estimated economic losses around us $32 billion (estimated in 1995 dollars) [2] . although not designated a pandemic, the reemergence of h1n1 in 1977 also shared some characteristics with the other 3 pandemics. the 2009 h1n1 pandemic was caused by a virus subtype that was then circulating in humans and was unexpected because it was generally assumed that that population immunity would prevent emergence of a pandemic virus of a subtype currently endemic in humans. there continues to be concern regarding the potential for new influenza a viruses to be transmitted to humans, with documented severe zoonotic disease caused by influenza a h5 and h7 infections. however, because h2n2 virus has already caused a pandemic, and h2 subtype viruses are currently circulating in wild and domestic birds [3] , reemergence of an h2n2 virus is one of the most likely scenarios for a new pandemic. anti-ha antibody, anti-na antibody, and cell-mediated immunity have all been correlated with protective immunity in both experimental animals and in humans [4] [5] [6] [7] . anti-ha antibody in peripheral blood sera, as assessed by the hemagglutination inhibition (hai) assay, has a strong correlation with protection against influenza infection and disease. although complicated by significant interlaboratory variation, an hai titer of 1:40 is generally accepted as a marker of reduced susceptibility [4] . therefore, analysis of the population level of hai antibody can be used to estimate the population susceptibility to infection [8] . for example, the seroprevalence of hai antibody to ph1n1 in individuals older than 65 years correlated with a significantly decreased influenza-associated mortality for these individuals during 2009 [9] . it has been suggested that exposure to antigenically related h1n1 influenza virus 50-60 years earlier provided older adults with some degree of immunity against the h1n1pdm09 virus [10] . similarly, individuals who were exposed to h2n2 viruses during the period from 1957 to 1968 may have persistent antibody to these viruses and be relatively protected from an emerging h2n2 virus. however, more than two-thirds of the world population in 2016 was born after 1968 [11] , suggesting that there may be substantial susceptibility to these viruses. because pandemics spread across the world within weeks after emergence [12] , much faster than the process of developing and rolling out a vaccine to the newly emerged pandemic virus, which takes >6 months [12] , attention has recently focused on developing systematic risk assessment algorithms for animal viruses of possible pandemic threat so that preemptive preparations including the development of vaccine seed strains can be initiated in advance. examples of these include the influenza risk assessment tool and the tool for influenza pandemic risk assessment [13] . an integral aspect of this risk assessment process is assessment of population immunity to the relevant virus. in this study, we evaluated population immunity using hai assays against human and avian h2n2 influenza strains in different age groups in the united states and hong kong. we then estimated the impact of existing cross-reactive hai immunity on reducing the effective reproduction number (r) of a potentially pandemic h2 subtype virus and characterized the minimum basic reproduction number (r 0 ) that such a virus must possess to cause a pandemic. samples in rochester were collected between 18 january 2010 and 14 march 2014 from nonimmunosuppressed individuals who were healthy or had stable medical conditions and were from 6 months to 80 years of age. in the united states, influenza activity typically peaks in january or february. according to the centers for disease control and prevention, in 2010-2011 influenza activity peaked in early february and in 2011-2012 it remained low through february and did not peak until mid-march. in 2012-2013, 2013-2014, and 2014-2015, influenza activity peaked in late december with some variability [14] . sera from children and adults in hong kong were collected as part of a previous serological study between 24 august 24 and 19 december 2011, prior to the winter influenza season, which typically commences around february-march in hong kong [15] . the preceding influenza season peaked in february-march 2011 and the dominant virus subtype was pandemic h1n1. two hundred ninety-five serum samples from this study were selected for testing in age strata. selection of viruses for hai testing was based on 3 phylogenetic lineages of the h2 influenza virus subtypes: human and avian eurasian lineages. viruses were selected from each lineage to represent temporal and geographic diversity. a/singapore/1/57(h2n2) and a/berkeley/1/68(h2n2) represented the human lineage, whereas a/mallard/ england/727/06(h2n2) and recent h2n2 virus isolate a/ mallard/netherland/14/07(h2n2) were of the eurasian avian lineage [16] . the a/mallard/england/727/06 virus was generated by plasmid-based reverse genetics with ha and na of a/ mallard/england/727/06 and other internal virus genes of a/ puerto rico/8/34 origin. antigenic relatedness of the selected test viruses was determined by reciprocal hai assays shown in supplementary table 1 . viruses with pandemic potential were handled in a us department of agriculture-approved animal biosafety level 3 (absl3)-enhanced facility. β-propiolactone (bpl)-inactivated virus using standard procedures for use as antigen in the hai test was prepared. the bpl-treated virus preparation was inoculated into 10-day-old hen's eggs following standard virus culture procedures to confirm complete inactivation of the virus. the antigen was then removed from the absl3-enhanced laboratory for hai analysis. in rochester and hong kong, hai studies were performed in biosafety level 2 conditions. serology hai tests were performed using turkey red blood cells in rochester, and chicken red blood cells in hong kong, otherwise the 2 laboratories used the same procedure for the test. sera were pretreated with receptor-destroying enzyme (denka seiken co ltd) and tested at a starting dilution of 1:10. hai was performed using "v" bottom microtiter plates as previously described [17] . the positive control sera used were ferret hyperimmune sera against a/kruitt/63, a/mallard/netherlands/31/2006, a/swine/ missouri/2124514/2006, a/mallard/netherlands/14/2007, and a/bakker/68 viruses, and goat hyperimmune sera against a/ japan/305/57 and a/singapore/1/57. negative controls consisted of antigen alone wells and the reagent control contained phosphate-buffered saline with red blood cells. statistical significance was analyzed using graphpad prism software (graphpad, san diego, california) using 1-way analysis of variance, followed by bonferroni post hoc analysis. p values <.05 were considered statistically significant. the reproduction number of each virus in each population was calculated as follows. we partitioned the population into n = 8 age groups (0-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, ≥71) and m = 4 hai titer levels (<1:20, 1:20, 1:40, ≥1:80). let p i be the proportion of population in age group and s ij be the proportion of age group i with the jth hai titer. the age distribution p i was based on the most recent census data from the united states (2012) and hong kong (2011). to estimate s s for each age group, we used bayesian inference with dirichlet y i was the number of individuals in age group i in our serologic study and x ij was the number of subjects in age group i with the jth hai titer level [18] . we assumed noninformative priors, that is, all priors were dirichlet distributions with parameters a j = 1 for j m = ¼ 1, , , and hence the joint posterior distributions of s s we assumed that an hai titer of <1:20, 1:20, 1:40, and ≥1:80 reduced susceptibility by 0%, 25%, 50%, and 75%. as such, the proportion of the population that were immune was ij j , where z j was the reduction in susceptibility conferred by the jth hai titer level (ie, , and z 4 0 75 = . ). the basic reproduction number r 0 was the largest eigenvalue of the matrix q ij { } , where q ij was the average number of secondary cases in age group i generated by an infector in age group j when everyone in the population was susceptible [19] . we constructed the matrix q ij { } using the united kingdom social contact matrix [20] because analogous data are not available from the united states and hong kong. because the immune proportion of age group i was table 1 . the demographics of the study population matched the demographics of the source population (data not shown). the results of hai testing of the sera from rochester and hong kong gave very similar results, despite the 2 populations and 2 different laboratories. the geometric mean titers (gmts) of antibody in the 2 populations against the test viruses by decade of birth are shown in table 2 . as expected, there were substantial levels of anti h2 hai antibody in the sera of persons old enough to have been infected with h2n2 viruses between 1957 and 1968, and essentially no detectable antibody in persons born after 1968. among persons born before 1957, the gmt of antibody against the early human a/singapore/18/57 was significantly higher compared with titers against the later human a/berkeley/1/68 virus, whereas in persons born from 1961 to 1970 there was a trend toward relatively higher titers against the a/berkeley/1/68 virus. titers against the avian h2n2 viruses were lower. there were no significant differences in the gmt against a/singapore/1/57, a/berkeley/1/68, or a/ mallard/england/727/2006 in sera tested in hong kong and rochester, but sera tested in rochester had significantly higher titers against a/mallard/netherlands/14/07 than the sera tested in hong kong. the prevalence of titers ≥1:40 against the test viruses is shown for sera from rochester and hong kong in persons born before the 1957 h2n2 pandemic, during the years that h2n2 circulated (1957-1968), or after 1968 is shown in figure 1 . ninetyeight percent of individuals from rochester and hong kong born before 1957 had titers ≥1:40 against the a/singapore/1/57 virus, whereas >63% of subjects born between 1957 and 1968 had titers ≥1:40. in contrast, none of those born after 1968 had titers >1:40 to a/singapore/1/1957. generally, persons born prior to 1957 had a lower prevalence of titers ≥1:40 to a/ berkley/1/1968, while the prevalence of titers ≥40 in persons born during circulation of these viruses was similar against a/ (7) 38 (13) 1951-1960 20 (13) 39 (13) 1961-1970 18 (12) 37 (13) 1971-1980 11 (7) 38 (13) 1981-1990 32 (21) 40 (14) 1991-2000 29 (19) 40 (14) 2001 or later 23 (15) 39 (13) data are presented as no. (%). singapore/57 and a/berkley/68. the prevalence of titers ≥1:40 against the 2 avian h2n2 viruses tested were significantly lower in these groups. no person born after 1968 had titer >1:40 against any of the h2 viruses. after combining the results from both populations, the cumulative distribution of antibody titers against the 4 test viruses in these 3 age groups is shown in figure 2 . eightyfive percent of individuals born in both the united states and hong kong before 1968 had hai titers ≥1:40 against a/ singapore/1/57 ( figure 2 ). more than 50% of subjects had hai titers ≥1:40 to a/berkeley/1/68 if born before 1968. the frequency of titers to avian h2n2 viruses was 62%, and 24% of subjects born before 1957 had titers of ≥1:40 to a/mallard/ england/727/06 and a/mallard/netherlands/14/07, respectively, with such titers seen in 21% and 8% of individuals born from 1957 to 1968. successful pandemic emergence and spread of a virus depends on the proportion of the population that is immune and the initial r of the potentially pandemic strain. the median r for the 1957 a/h2n2 pandemic was 1.65 (interquartile range [iqr], 1.53-1.70) [21] . to assess the impact of these age-dependent population immunity profiles on the pandemic potential of each of these viruses if they were to emerge in the human population, we computed the impact of population immunity in reducing r (figure 3 ). the current population immunity in the united states and hong kong would reduce the initial r of a/singapore/1/1957 by around 15% (12%-18%) and that of a/ berkeley/1/1968 by around 12% (10%-17%). as such, a pandemic of a/singapore/1/1957 and a/berkeley/1/1968 would be prevented if initial r of the emerging virus was below 1.18 (1.14-1.22) and 1.14 (1.11-1.20), respectively. the threshold r below which a pandemic with the avian subtype h2 viruses a/mallard/england/727/2006 and a/mallard/netherlands/14/2007 would be prevented was slightly lower. the comparability of data from 2 geographically separated areas of the world, rochester and hong kong, argues for the representativeness and generalizability of such studies that aim to assess population immunity to viruses of pandemic concern. our study suggests that those individuals born prior to or during the period of h2n2 virus circulation were more likely to have higher hai titers against the human h2n2 viruses than those born after 1968 when h2 infection in humans had been displaced by the h3n2 virus. in our study, we also confirmed evidence of cross-reactive hai antibodies to unrelated avian h2 viruses, albeit at lower prevalence and titer. the prevalence of such cross-reactive antibodies was higher in those born prior 1957 and the gmt of these cross-reactive antibodies was higher in those born prior to 1960 than in the birth cohort of 1961-1970. this is possibly because those who were infected in the early pandemic waves of the h2n2 virus in 1957-1958 were reinfected some years later by antigenically drifted h2n2 viruses, possibly broadening cross-reactive immunity. as reported by others, we also observed low hai titers <1:40 spanning across the age groups and this could be due to nonspecific data are presented as geometric mean titer (95% confidence interval). a samples in hong kong and rochester were tested at a starting dilution of <1:10 and negative tests are given an imputed value of 5. inhibition by the sera. however, we checked the sera for nonspecific agglutinins where about 7% of the samples contained nonspecific inhibitors. since the assays were run on sera which were confirmed not to contain nonspecific inhibitors, these low titers may be due to a certain type of antigen exposure that needs further investigation. sera from those born after 1963 had higher hai titers to a/berkeley/1/68 than to a/singapore/1/57 whereas the converse was true in those born before 1963. the older group of individuals who were first infected by a/singapore/1/1957like viruses likely had a boost of these titers when they were subsequently infected by later drift variants (ie, a/ berkley/1/1968-like viruses), the phenomenon known as "original antigenic sin" [22] . the broader cross-reactivity may also occur due to targeting different antigenic sites, as a recent study points out that antibody response against h2 is mainly to the receptor binding domain resulting in a greater degree of cross-reactivity whereas for h1 or h3 viruses it is the hypervariable regions of ha resulting in a lesser cross-reactivity [23] . soon after the h2n2 pandemic of 1957, studies done on sera collected prior to this pandemic were carried out and it was reported that people born prior to 1887 had detectable hai antibody to h2 viruses. it was therefore suggested that the historical pandemic that was believed to have occurred in 1889-1890 was likely caused by an h2 subtype virus [24] . this prior exposure has also shown to elicit cross-reactive antibody for h2 strains that are currently circulating in animals, potential candidate pandemic stains [25] . sera tested in rochester yielded higher titers against a/mallard/ netherlands/14/07 than the sera tested in hong kong. the differences between laboratories are not unexpected given the known laboratory-to-laboratory variation in the hai test, and what remains remarkable is the closeness of results. one technical difference between the 2 laboratories was the source of erythrocytes for the hai test; rochester used turkey erythrocytes whereas hong kong used chicken erythrocytes. it is not known if this may contribute to this difference in hai test results. an alternative reason for the discrepancy in titers between countries may be attributed to variation in vaccination rates. vaccine uptake in hong kong is lower than in the united states. potentially, repeated vaccination may increase cross-reactive antibody to this avian h2 strain. assuming that the h2 seroprevalence in rochester and hong kong reflects global population seroprevalence, we modeled the impact of such immunity on possible emergence of an h2n2 pandemic. this model was based on the candidate pandemic h2 strain originating from escape of the human adapted h2n2 virus or from the avian gene pool that acquires transmissibility in humans. we estimate that the current levels of population immunity would reduce r of a/singapore /1/1957a recent risk assessment for viruses currently known to be circulating in wild birds has been carried out and the risk of these viruses acquiring transmissibility in humans was assessed to be low; most isolates replicated in human bronchial epithelial cells and ferrets. several did transmit between ferrets by direct contact, but all has retained a preference for avian-like α2,3-linked sialic acid receptors [26] . these viruses still remain primarily in wild birds and have not been established in mammalian species including swine. our study provides the systematic assessment of the impact of human population immunity that goes toward such an overall assessment of pandemic risk. a limitation of the study is that other potential contributors to population immunity, such as cross-reactive na-inhibiting antibody, stalk-specific antibody, cell-mediated immunity, and heterosubtype reactive hai or neutralizing antibody [4] [5] [6] [7] 27] , were not assessed. furthermore, in many parts of the world (especially in developing countries), the proportion of individuals born in 1968 or later might be larger than that in rochester and hong kong. the level of immunity against a(h2n2) in these populations would be lower than that estimated here and the pandemic potential of a(h2n2) in these populations would thus be higher than estimated here. in summary, we find that levels of population immunity to h2 subtype viruses are not substantial enough to block epidemic spread of an h2 virus that had acquired efficient transmissibility between humans. furthermore, the existing levels of population immunity to h2 viruses will continue to decline with new birth cohorts being added to the human population. given that human-adapted h2n2 viruses that arose subsequent to the 1957 pandemic are present in many laboratories worldwide, these findings support the need to have preparedness for h2 viruses as a credible pandemic threat. the approach used in the case study of h2 viruses is more broadly applicable in defining the impact of population immunity to viruses to which there is some level of cross-reactive hai antibodiesfor example, swine h1 and h3 viruses and avian h9n2 viruses [27] . reliable methods for assessing population immunity are a key to reliable risk assessment of viruses for pandemic threat. it was the lack of such risk assessment of population immunity to the h1n2 triple reassortant swine viruses that led to these viruses not being recognized as potentially pandemic viruses prior to 2009; indeed, it was the triple reassortment swine ha that was the key protective antigen for the 2009 h1n1 pandemic virus. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. figure 3 . estimations of overall population-level immunity against h2 viruses and the potential impact of population immunity on reproduction number, using 3 potential titer cutoffs for 100% immunity. bars updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic origin of the pandemic 1957 h2 influenza a virus and the persistence of its possible progenitors in the avian reservoir correlates of protection to influenza virus: where do we go from here? antibody to influenza virus neuraminidase: an independent correlate of protection preexisting influenza-specific cd4+ t cells correlate with disease protection against influenza challenge in humans cellular immune correlates of protection against symptomatic pandemic influenza seroprevalence to influenza a(h1n1) 2009 virus-where are we? complex patterns of human antisera reactivity to novel 2009 h1n1 and historical h1n1 influenza strains cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus vaccinate for the next h2n2 pandemic now the infection attack rate and severity of 2009 pandemic h1n1 influenza in hong kong pandemic preparedness and the influenza risk assessment tool (irat) relative incidence and individual-level severity of seasonal influenza a h3n2 compared with 2009 pandemic h1n1 adaptation of pandemic h2n2 influenza a viruses in humans world health organization. manual for the laboratory diagnosis and virological surveillance of influenza the bugs book: a practical introduction to bayesian analysis inferring influenza infection attack rate from seroprevalence data social contacts and mixing patterns relevant to the spread of infectious diseases estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature original antigenic sin responses to influenza viruses host versus flu: antibodies win a round? pre-epidemic antibody against 1957, strain of asiatic influenza in serum of older people living in the netherlands studies on the content of antibodies for equine influenza viruses in human sera risk assessment of h2n2 influenza viruses from the avian reservoir safety and antigenicity of whole virus and subunit influenza a/hong kong/1073/99 (h9n2) vaccine in healthy adults: phase i randomised trial key: cord-266573-vfl08i2p authors: largent, emily a; lynch, holly fernandez title: paying participants in covid-19 trials date: 2020-05-29 journal: j infect dis doi: 10.1093/infdis/jiaa284 sha: doc_id: 266573 cord_uid: vfl08i2p trials are in development and underway to examine potential interventions for treatment and prophylaxis of coronavirus disease 2019 (covid-19). how should we think about offering payment to participants in these trials? payment for research participation is ethically contentious even under ideal circumstances. here, we review 3 functions of research payment—reimbursement, compensation, and incentive—and identify heightened and novel ethical concerns in the context of a global pandemic. we argue that covid-19 trial participants should usually be offered reimbursement for research-related expenses, and compensation for their time and effort, as for other types of research under usual circumstances. given increased risk of undue influence against pandemic background conditions, incentive payment should be avoided unless essential to recruitment and retention in important trials whose social value outweighs this risk. where essential, however, incentives can be ethically permissible, so long as reasonable efforts are made to minimize the possibility of undue influence. with much of the globe rushing to respond to coronavirus disease 2019 (covid19) , clinical trials to evaluate safe and effective options for treatment and prevention are critical. the trials are diverse, examining new and repurposed drugs and vaccines at various stages of development, and involving a variety of designs with a range of opportunity for direct benefit. a wide variety and number of research participants are needed to enroll in these trials, reflecting a spectrum of experience with covid-19-including healthy individuals, individuals exposed to the virus, individuals experiencing different levels of disease severity, and recovered individuals-as well a diversity of age, sex, race and ethnicity, socioeconomic status, medical comorbidities, and more. we have already seen that many individuals are eager to try nearly anything that has exhibited some promise against this disease, which may facilitate trial recruitment [1] . but what role will payment play in encouraging trial participation-and what role should it play? paying research participants is ethically contentious under ideal circumstances and pandemic circumstances are far from ideal. concerns about offering payment are therefore likely to be heightened in this context [2] . existing frameworks for evaluating the ethics of paying research participants offer a strong foundation for assessing the acceptability of payment in covid-19 trials. nonetheless, consideration must be given to the unique medical, financial, and institutional circumstances wrought by the pandemic. we argue that participants in covid-19 trials should be offered reimbursement for research-related expenses and compensated for their time and effort, just as they should be for any other type of trial. given the pandemic's devastating economic effects, as well as the fact that risks may be higher or more uncertain in covid-19 trials than in nonpandemic research, there is an increased likelihood of undue influence stemming from incentive payments. still, it may be permissible to offer incentives when necessary to recruit and retain participants for important trials offering the possibility of social value sufficient to outweigh these concerns, so long as steps are taken to minimize the risk that participants will be unduly influenced. paying research participants serves several discrete functions [3] . first, participants may be reimbursed for out-of-pocket expenses incurred as a result of research participation, such as copayments or travel costs. second, payment can compensate participants for the fair value of their time and effort expended in research participation. third, incentive payments go beyond what is necessary to either reimburse or compensate, with the intention of improving recruitment or retention. offers of payment that fall into any of these categories can be ethically acceptable; however, each function raises distinct ethical considerations influenced by the pandemic. offering reimbursement for reasonable, research-related expenses to restore participants to their financial baseline should be viewed as the ethical default in covid-19 trials, as in research more broadly. this treats participants fairly by preventing them from having to pay to contribute to socially valuable research that may not, or is not expected to, benefit them directly. where direct benefit is possible, covering expenses can also promote distributive justice by making those potential benefits more widely accessible without regard to participants' financial need or wealth [4] . considering that a high percentage of the population is likely to be infected with severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the current absence of curative therapies or a vaccine, and the widespread financial hardship and exacerbation of economic disparities caused by the pandemic, covid-19 trial participation must not be limited only to those who can afford it. making participation widely accessible is especially important in light of the disproportionate impact this virus is having on minority communities [5, 6] . while there are of course many barriers to trial participation, financial barriers are among the most easily modified [7] . although reimbursement should be the rule, there will be exceptional circumstances when it can be ethically acceptable to proceed without it. in the context of covid-19, for example, investigators and institutions may be initiating trials without traditional sources of public research funding or with only limited support, such as the provision of product, by commercial companies. in these instances, there may not be adequate funding to reimburse participants. lack of funding should not preclude important trials from proceeding, so long as participants are made aware of the financial implications of enrollment and reasonable efforts are made to minimize financial burden. if a limited budget is available for reimbursement, it may be acceptable to reimburse only select participants-such as those with the greatest financial need-or to cover expenses up to a prespecified limit without necessarily reimbursing them all [8] . compensation also should be viewed as the ethical default because it helps to minimize the chance participants will be exploited by receiving benefits that are disproportionately low compared to the burdens they undertake and the value they contribute to research. while the prospect of direct benefit is relevant to avoiding exploitation, research benefits are not always present and are never certain. thus, it often makes sense to compensate participants for their work via a wage-payment model, using a fair local wage for similarly burdensome nonresearch endeavors as a benchmark [9, 10] . this is not intended to make participants better off as compared to their financial baseline or even to fully compensate for participants' opportunity costs but rather to acknowledge the value of their time and effort. compensation can also help distinguish research activities, with their distinct goals and risks, from clinical care, signaling that participants are contributing to science and that individual benefit may not result from their research participation [11] . this is likely to be particularly important for covid-19 patients, because the dearth of compelling treatment options means that clinical care will often incorporate experimental methods and research. nevertheless, it may be ethically acceptable to proceed without compensation when reasonable budget constraints preclude it, as above. when compensation is offered, 2 ethical concerns can arise, which sometimes point in opposite directions. first, if the amount is unfairly low, it will not adequately address the possibility of exploitation; this concern can be addressed by offering more compensation. yet, relatively higher compensation may be linked to a second concern, which arises when circumstances extrinsic to a trial transform payments intended as compensation into de facto incentives. this is especially likely in the context of covid-19. compensation is intended to render research participation as financially attractive as other, nonresearch opportunities that demand a similar amount of time, burden, and skill. however, the availability and type of those nonresearch opportunities can be impacted by a variety of factors. even for those who have remained healthy, the covid-19 pandemic is causing a human tragedy with significant economic impact, including widespread layoffs, small business closures, hiring freezes, and dramatic market fluctuations [12] . these challenges have exacerbated longstanding economic inequalities and laid bare the fragility and inadequacy of the social safety net in many countries, including the united states. these economic and social challenges are only likely to increase as the pandemic continues. against this backdrop, many prospective covid-19 research participants will no longer have meaningful alternatives for paid work, which may contribute to a perception-and perhaps reality-that paid research participation is their best opportunity to make money. this perception may be heightened if compensation is increased to the extent necessary to avoid exploitation, even if the total amount of money remains relatively modest. these difficult background circumstances do not make offers of payment to covid-19 research participants impermissible. it would be perverse to withhold fair compensation simply because participants are facing economic hardship [13] . rather, in light of pandemic circumstances-similar features of which may be replicated in other contexts, including research conducted in low-and middle-income countries or with participants whose nonresearch options are limited even in the absence of a pandemic-offers of compensation may raise ethical concerns akin to incentives [14] . thus, institutional review boards (irbs) should consider whether it is sometimes appropriate to treat them as such. rather than aiming to satisfy obligations of fairness to participants or to set research on a par with other payment-generating activities, incentives aim to push trial enrollment higher on an individual's choice set-or incidentally have that effect based on available alternatives. as such, incentives are not ethically obligatory. however, to the extent incentives can contribute to recruitment and retention, and thus to efficient trial completion with adequate statistical power, they can be ethically important [15] . incentives may also advance distributive justice by increasing willingness to enroll, thereby spreading the burdens of research participation over a larger swath of the population and avoiding concentrating those burdens exclusively on individuals who are the least well off financially by making research more broadly attractive. yet, incentives can be ethically fraught. some people worry that incentives may coerce research participants. the prevailing view is that coercion entails a threat to violate an individual's rights or not fulfill an obligation to her unless she complies with some request, in a circumstance in which she has no reasonable alternative but to comply [16] . by this definition, genuine offers of payment are not coercive because they are not threats [17] . importantly, feeling that there is no reasonable alternative to make a similar amount of money-a feeling likely to arise for at least some prospective covid-19 trial participants-is not the same as being coerced. a more salient ethical concern is that incentives may unduly influence participants, although this is more complex than just making a decision motivated by a desire for financial benefit. undue influence occurs when an excessive reward leads the recipient to make a choice that is unreasonably against her selfdefined values, interests, or responsibilities [16, 18] . a decision would be objectively unreasonable if it reflected a level of risk an irb would not or should not approve for participants in the target population. for example, it would be objectively unreasonable for persons with a known hypersensitivity to hydroxychloroquine to participate in a trial assessing that drug's effect on the progression of covid-19 [19] . approval of a trial protocol by a well-functioning irb should generally eliminate concerns that incentives will lead to objectively unreasonable decisions. in contrast, research participation may be subjectively unreasonable if it is discordant with an individual's particular values and interests or if the risks (should they materialize) would be particularly burdensome for the individual to bear [20] . because irbs are tasked with making population-level judgments, they cannot be expected to assess the subjective reasonableness of trial participation for every individual. thus, irb review cannot eliminate the possibility that that incentives will lead to subjectively unreasonable decisions-and, therefore, to undue influence. for instance, a jehovah's witness considering participation in a trial of intravenous immunoglobulin-a blood plasma product-for covid-19 might have consciencebased concerns. similarly, someone who is highly risk-averse may prefer to avoid the uncertainties associated with research participation. either of these individuals could be encouraged by offers of incentive payment to set aside those concerns. while this may seem worrisome, in practice, it is quite hard to distinguish between cases in which an offer of payment has unproblematically tipped the balance in favor of an otherwise undesirable but not unreasonable choice and those in which it has problematically tipped the balance in favor of an unreasonable or irrational choice. in many cases, a decision to enroll in research will reflect a participant's reasoned judgment that, in these circumstances, participation is aligned with her overall interests, even if certain considerations might have weighed in the other direction. decisions are often multifactorial, with various pros and cons; the fact that all cons have not been resolved does not necessarily render a decision subjectively unreasonable. acknowledging this challenge, the best irbs can do is to minimize the possibility of undue influence for trial participants on the whole by making it unlikely for research participation to constitute an objectively unreasonable choice for members of the target study population. they should also make sure the consent process alerts participants to factors that might make participation subjectively unreasonable [20] . although careful irb review generally constitutes a critical bulwark against undue influence, there are additional considerations when evaluating the potential for undue influence in covid-19 trials. first, given that there is still much to be learned about sars-cov-2 and so little is known about the various interventions under study in this context, we may justifiably be concerned that it will be difficult for irbs to make risk-benefit determinations confidently. therefore, participation even in irb-approved studies may not always be truly objectively reasonable for the target study population, or there may be disagreement about what is objectively reasonable. moreover, the challenge of evaluating this research may be exacerbated by the heightened burdens currently facing irbs. the sheer volume of covid-19 research proposals being put forward [21] and the dire need for clinical advancement mean that members are being asked to review a tremendous number of protocols as quickly as possible, often while meeting remotely to promote social distancing, all of which may influence the quality and nature of review. second, the global scale of the pandemic and associated morbidity and mortality may render even quite risky research objectively reasonable on the basis of high social value. for example, we have already seen vaccine trials proceed without the usual animal trials, and there is increasing discussion of using challenge trial design to speed vaccine development, a design far riskier for sars-cov-2 than for viruses used in other recent challenge trials given the lack of a proven cure [22] [23] [24] [25] . it is widely accepted that trials can be justified on the basis of benefits to society; in the absence of direct benefits, it may be desirable to offer larger incentive payments similar to hazard pay offered to emergency workers or others performing dangerous work-as noted above, this can help make participation attractive across a wider socioeconomic swath [26] . yet, larger incentives might also increase the likelihood of undue influence by making it more likely that people will make subjectively unreasonable decisions [20] . a third concern is that incentives, because they go beyond amounts available through participating in other activities, may cause individuals "to lie, deceive, or conceal information that, if known, would disqualify them as participants in a research project" [27] . such obfuscation can have 2 effects [20, 28] . first, it may expose individuals to research-related risks that exclusion criteria were designed to shield them from. second, it may jeopardize the scientific integrity of the research. this can be of particular concern when it is not possible to rely on objective, independently verifiable measures to confirm trial eligibility [29] . in response to these challenges, one might consider the most conservative course of blocking incentives for covid-19 trials. however, this approach could inhibit recruitment, retention, or both, impeding the conduct of critically important research, in turn creating a greater possibility that participants will be exposed to research risks without realizing the social benefits that initially justified them. there are 2 potential errors here: (1) inhibiting ethically acceptable trials out of an abundance of caution, or (2) risking undue influence and deception by incentivizing participation in covid-19 trials [30] . given the importance of research in response to the pandemic, as well as limited, albeit encouraging, empirical data suggesting that higher offers of payment increase participants' perceived risk as well as time spent reviewing research-related risks [11, [31] [32] [33] , our view is that incentive payments can be ethically permissible, despite the residual risk of undue influence and deception. rather than blocking incentives for covid-19 trials due to the concerns raised above, the better approach is to differentiate those circumstances in which incentive payments are truly essential to boost recruitment and retention for important research. if they are not, as may be the case for covid-19 research that offers participants other benefits or that can rely on altruism born of social solidarity, it is best to avoid incentive payments. this is also the more efficient approach; why spend resources on incentives that are unneeded? however, incentives sometimes will be critical. for example, covid-19 trials that offer no or low potential for direct benefit and impose substantial burdens and risks, such as early phase trials focused on dosing and safety, may otherwise fail to adequately enroll participants. of course, the fact that participants may have qualms about participating that need to be overcome by offering incentives might suggest that concerns about undue influence are highest in these circumstances. yet these are also the covid-19 trials for which irb attention is likely to be most intense and focused, potentially reducing concerns about objectively unreasonable decisions to enroll. risks of subjectively unreasonable decisions remain, but should be viewed in a similar context to other research risks, meaning that they can be justified when both minimized and reasonable in relation a study's potential for benefit. thus, incentives should be limited to covid-19 trials with adequate time and research personnel to facilitate robust informed consent processes that can help prospective participants carefully consider the risks, burdens, and discomforts of participation, as well as those that can adopt objective measures of eligibility and adherence. they should also be limited to those trials with sufficient importance to the battle against covid-19 that their potential benefits can overcome residual concerns about undue influence. relatedly, care should be taken to avoid incentive payments being used to draw participants into lower priority trials [34] . irbs are tasked with minimizing-not eliminating-the possibility of undue influence; we should accept that minimization may look different for covid-19 trials compared to other research. offering payment for trial participation intended to combat a pandemic that is coupled with economic distress raises unique considerations. we argue that reimbursement and compensation should be offered in covid-19 trials as a matter of fairness, as is true for other types of clinical research. yet the economic stressors of the pandemic may cause compensation to be experienced as an incentive, raising concerns about undue influence, while the usual protections against undue influence may also be weakened by pandemic circumstances. rather than abandoning the utility of incentives, we recommend that they be limited to those covid-19 trials that truly need them, that will permit undue influence to be minimized, and whose social value and importance can outweigh residual risks of undue influence. this suggests that financial incentives will be most appropriate for covid-19 trials without the prospect of direct benefit, although in the face of such a massive global threat, altruism and a call to duty may render incentives even less critical. hospital orders for old malaria drugs have spiked amid coronavirus pandemic conventional wisdom versus actual outcomes: challenges in the conduct of an ebola vaccine trial in liberia during the international public health emergency fernandez lynch h. a framework for ethical payment to research participants what makes clinical research ethical covid-19 and african americans evidence mounts on the disproportionate effect of covid-19 on ethnic minorities addressing financial barriers to enrollment in clinical trials differential payment to research participants in the same study: an ethical analysis what's the price of a research subject? approaches to payment for research participation human research subjects as human research workers informative inducement: study payment as a signal of risk kff health tracking poll -early economic vulnerability and payment for research participation precarity, clinical labour and graduation from ebola clinical research in west africa the continuing unethical conduct of underpowered clinical trials paying research participants: regulatory uncertainty, conceptual confusion, and a path forward payment for research participation: a coercive offer undue inducement: nonsense on stilts? hydroxychloroquine in outpatient adults with covid-19 paying research participants: the outsized influence of "undue influence a real-time dashboard of clinical trials for covid-19 [published online ahead of print 24 researchers rush to test coronavirus vaccine in people without knowing how well it works in animals human challenge studies to accelerate coronavirus vaccine licensure ethical considerations for zika virus human challenge trials ethics of controlled human infection to address covid-19 compensation for cures: why we should pay a premium for participation in 'challenge studies on paying money to research subjects: 'due' and 'undue' inducements association between financial incentives and participant deception about study eligibility exploring ethical concerns about human challenge studies: a qualitative study of controlled human malaria infection study participants' motivations and attitudes making the case for completion bonuses in clinical trials empirical assessment of whether moderate payments are undue or unjust inducements for participation in clinical trials perceptions of financial payment for research participation among african-american drug users in hiv studies the influence of risk and monetary payment on the research participation decision making process when clinical trials compete: prioritising study recruitment key: cord-003567-h8uq5z8b authors: crank, michelle c; mascola, john r; graham, barney s title: preparing for the next influenza pandemic: the development of a universal influenza vaccine date: 2019-04-15 journal: j infect dis doi: 10.1093/infdis/jiz043 sha: doc_id: 3567 cord_uid: h8uq5z8b nan structural biology, protein engineering, and antigen delivery amenable to platform manufacturing approaches. molecularand atomic-level information about the immune-viral interface combined with new capacities for surveillance and rapid response to pandemics are shaping a new conceptual framework for vaccine development. as a result of these advances, highlevel, broad, and durable immunity against the large universe of influenza viruses may now be within reach [20] . this issue of the journal of infectious diseases was motivated by the confluence of the 1918 influenza pandemic centenary and the new opportunities afforded by technological advances and breakthroughs along with the improved understanding of influenza biology. we have gathered information, opinions, and ideas from thought leaders in immunology, virology, epidemiology, and vaccinology to address the challenge of developing a universal influenza vaccine and articulate some of the knowledge and technical gaps that remain. in 1918, there were approximately 1.8 billion persons living on earth; today, there are more than 7 billion [21] . if a pandemic similar to that of 1918 occurred today, how would the devastation compare? because of rapid international travel, the spread of a new virus would be faster than it was in 1918, when spread was largely related to the movement of troops at the end of world war i. today, population density is higher and cities are larger, providing a more favorable environment for a rapidly spreading virus. although we now have more sophisticated medical care, the availability of hospital beds and life support equipment would probably not be sufficient to manage an outbreak equivalent in magnitude to that of 1918. if there were sentinel events as in 1918 and in 2009-a small spring epidemic preceding the fall pandemic-current vaccine manufacturing approaches would not be sufficiently fast or scalable for worldwide distribution to preempt pandemic spread. therefore, development of a universal influenza vaccine that can reliably protect against drifted seasonal strains and pandemic strains without biannual reformulation is imperative. ideally, this vaccine would not need to be given every year; however, even if annual vaccination was required but antigenic components needed updating only every 5-10 years, it would still be a significant advance over the current system. s108 • jid 2019:219 (suppl 1) • crank et al there are some clear pathways to explore and knowledge gaps to fill in the immediate future using currently available technology, as described in the accompanying commentaries, outlined here: 1. by harnessing high-throughput sequencing and computational biology, more sophisticated algorithms based on sequence analysis, glycan patterns, and other features that may anticipate high transmissibility can be developed for predicting the next dominant strain [4] . the prudent study of gain-of-function mutations would allow scientists to learn more about what molecular signatures to look for. 2. improving strain selection for seasonal vaccines would increase the likelihood of an antigenic match between the vaccine and dominant circulating strains and thereby improve the utility of current vaccine technology [2] . the current vaccines could be further improved by better standardization of the neuraminidase content, adjustment of antigen doses, addition of improved adjuvants, and production in cell substrates that minimize the likelihood of viral adaptations and changes in protein sequences [2] . 3. precisely defining the b-cell repertoire and epitope-specific phenotypes involved in the response to influenza infection and vaccination would provide insight into the problem of "original antigenic sin" described by thomas francis in 1960 and the related phenomenon of immunodominance [22] . prior influenza immunity and poorly understood antigenicity patterns make it difficult to reshape and broaden the antibody response using current vaccines [7] . defining all the ways antibody can bind and neutralize influenza structurally and establishing a new nomenclature for describing antigenic sites across both influenza a groups as well as influenza b would reduce confusion and improve communication between scientists [5] . in addition, learning which features of vaccine-induced local or systemic immune responses result in sustained serum antibody responses may inform vaccine formulation and delivery approaches. 4. understanding more precisely the b-cell and antibody responses would allow the application of protein engineering for antigen design and display using molecular targets and antibody lineage end points to guide iterative design modifications [14] . 5. the role of cd4 + t cells in determining the efficacy of a b-cell response is an area of active investigation; however, more work in this area may be required to solve the problem of durability and maintenance of antibody responses [6] . 6. the direct role of cd4 + or cd8 + t-cell effector functions and whether those cells require localization in mucosal tissue or lymph nodes to effectively protect against respiratory viral pathogens are poorly understood. optimizing vaccine formulation and delivery route and modality is dependent on acquiring this type of knowledge [6] . 7. defining the importance of including specific antigenic targets, such as the head or stem domains of hemagglutinin, neuraminidase, or the m2 ectodomain in universal vaccines, and determining whether they are more effective when used in combination or alone could be accomplished through both vaccine protection and natural history studies that provide a better understanding of protective immunity [9] [10] [11] [12] . 8. understanding the mechanistic correlates of immunity generated by immunization with live attenuated vaccines may reveal the importance of secretory immunoglobulin a and intraepithelial t cells that require induction of immunity to occur at the mucosal surface [13] . 9. defining both the virological and host immune response patterns associated with transmissibility would allow better modeling of population dynamics and factors that could best interrupt transmission cycles [8] . this could be particularly important for identifying distinct vaccination strategies for different target populations, including in societal settings in which transmission dynamics and target populations vary [15] . 10. using human challenge studies and improving animal models of influenza infection and transmission may help answer some of these questions [3] . however, the utility of animal models hinges on selecting those that are most relevant to human pathogenesis and immunity. improving the characterization of and expanding the reagents for these models would not only benefit influenza vaccine development but would also provide answers to immunological questions relevant to other respiratory virus infections and emerging infectious diseases in general. recent estimates place the cost of influenza pandemics at upward of $500 billion per year [23] . in this context, an investment of at least $1 billion per year in the biomedical research effort to achieve a solution for protection against pandemic influenza seems justified. in addition, efforts to develop a universal influenza vaccine, even before reaching this goal, will likely lead to improved seasonal vaccines, with the potential to reduce morbidity rates and save tens of thousands of lives each year. solving this problem is potentially achievable with today's technology, and with an organized and sustained focus on interventions for influenza and other potential emerging infectious threats, more advanced approaches could be rapidly developed. finally, gaps remain in our understanding of influenza biology and immunity and methods to produce highly effective vaccines. to close those gaps, it will be important to align the interests of all the stakeholders preparing for the next pandemic. priorities of public health officials in lower-, middle-, and high-income countries, academic researchers, regulatory bodies, major funders, and pharmaceutical companies must be understood and collectively addressed to face the logistical and scientific challenges ahead [24] . thanks to scientific and technological breakthroughs of the past decade, vaccinology is experiencing a revolution. may we find the resolve, political will, and new business plans to take full advantage of these new opportunities and prepare ourselves before the next pandemic arrives. updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic influenza vaccines: good, but we can do better making universal influenza vaccines: lessons from the 1918 pandemic can we predict the next influenza pandemics? antibody determinants of influenza immunity the way forward: potentiating protective immunity to novel and pandemic influenza through engagement of memory cd4 t cells immunodominance and antigenic variation of influenza virus hemagglutinin: implications for design of universal vaccine immunogens dynamic perspectives on the search for a universal influenza vaccine universal influenza vaccine approaches employing full-length or head-only ha proteins universal influenza virus vaccines that target the conserved hemagglutinin stalk and conserved sites in the head domain the role of m2e in the development of universal influenza vaccines neuraminidase, the forgotten surface antigen, emerges as an influenza vaccine target for broadened protection how live attenuated vaccines can inform the development of broadly cross-protective influenza vaccines new vaccine design and delivery technologies influenza immunization in low-and middle-income countries: preparing for next-generation influenza vaccines novel vaccine technologies: essential components of an adequate response to emerging viral diseases emerging viral diseases from a vaccinology perspective: preparing for the next pandemic advances in antiviral vaccine development a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases historical estimates of world population on the doctrine of original antigenic sin pandemic risk: how large are the expected losses? vaccine development in the twenty-first century: changing paradigms for elusive viruses we thank our colleagues at the national key: cord-309786-8zyf9e3k authors: karron, ruth a; luongo, cindy; mateo, jocelyn san; wanionek, kimberli; collins, peter l; buchholz, ursula j title: safety and immunogenicity of the respiratory syncytial virus vaccine rsv/δns2/δ1313/i1314l in rsv-seronegative children date: 2019-10-12 journal: j infect dis doi: 10.1093/infdis/jiz408 sha: doc_id: 309786 cord_uid: 8zyf9e3k background: respiratory syncytial virus (rsv) is the leading global cause of severe pediatric acute respiratory tract illness, and a vaccine is needed. rsv/δns2/δ1313/i1314l contains 2 attenuating elements: (1) deletion of the interferon antagonist ns2 gene and (2) deletion of codon 1313 of the rsv polymerase gene and the stabilizing missense mutation i1314l. this live vaccine candidate was temperature-sensitive, genetically stable, replication restricted, and immunogenic in nonhuman primates. methods: a single intranasal dose of rsv/δns2/δ1313/i1314l was evaluated in a double-blind, placebo-controlled trial (vaccine-placebo ratio, 2:1) at 10(6) plaque-forming units (pfu) in 15 rsv-seropositive children and at 10(5) and 10(6) pfu in 21 and 30 rsv-seronegative children, respectively. results: in rsv-seronegative children, the 10(5) pfu dose was overattenuated, but the 10(6) pfu dose was well tolerated, infectious (rsv/δns2/δ1313/i1314l replication detected in 90% of vaccinees), and immunogenic (geometric mean serum rsv plaque-reduction neutralizing antibody titer, 1:64). after the rsv season, 9 of 20 vaccinees had increases in the rsv titer that were significantly greater than those in 8 of 10 placebo recipients (1:955 vs 1:69, respectively), indicating that the vaccine primed for anamnestic responses after natural rsv exposure. conclusion: rational design yielded a genetically stable candidate rsv vaccine that is attenuated yet immunogenic in rsv-seronegative children, warranting further evaluation. clinical trials registration: nct01893554. respiratory syncytial virus (rsv) is the most important cause of severe acute lower respiratory illness (lri) in infants and children worldwide [1, 2] , and the relative importance of rsv has increased as the burden of bacterial pneumonia has declined with vaccine implementation [3] . according to global estimates, rsv caused approximately 33 million cases of lri and approximately 118 000 deaths in children <5 years of age in 2015 [2] . more than 80% of all rsv-associated lris (rsv-lris) and more than half of the rsv-associated deaths in low-and middle-income countries were estimated to occur in infants ≥6 months old, highlighting the importance of developing rsv vaccines for active immunization of infants and children [4] . live attenuated intranasal rsv vaccines are attractive for pediatric immunization because they mimic mild natural infections and induce durable cellular, humoral, local and systemic immunity. furthermore, candidate live attenuated rsv vaccines [5] [6] [7] [8] [9] [10] [11] [12] have not caused the vaccine-associated enhanced rsv disease that was observed in children who received formalin-inactivated rsv [13] and that also seemed to be associated with administration of rsv subunit vaccines in experimental animals [14] [15] [16] . progress in the elucidation of rsv gene function [17] and the use of reverse genetics systems [18] led to the development of rationally designed attenuated rsv strains, including strains attenuated through deletion of the ns2 gene. ns2 is a virally encoded type i and iii interferon antagonist that interferes with interferon induction and signaling [19] [20] [21] . deletion of the ns2 gene diminished rsv replication in chimpanzees [22] . the increased interferon response to infection may enhance the adaptive immune response, as has been demonstrated for bovine rsv with ns1 or ns2 deletion in calves [23] . ns2 also functions as a pathogenicity factor, promoting epithelial cell shedding in vitro and in the hamster model, potentially contributing to small airway obstruction [24] . thus, deletion of ns2 may be beneficial for vaccine safety. recently, a candidate vaccine was developed that contains the ns2 deletion and the attenuating deletion of codon 1313 in the polymerase (l) gene, which also confers mild temperature sensitivity (shutoff temperature of 38ºc-39ºc [24] ;). this deletion was genetically and phenotypically stabilized by substitution of leucine (l) for isoleucine (i) at codon 1314. the resultant virus, rsv/δns2/ δ1313/i1314l, was attenuated and immunogenic in nonhuman primates [25] . based on this preclinical profile, we conducted a stepwise phase i evaluation of rsv/δns2/δ1313/i1314l in rsv-seropositive and rsv-seronegative children. in rsvseronegative children, rsv/δns2/δ1313/i1314l was restricted in replication, well tolerated, immunogenic, and primed for potent antibody responses after natural exposure to wild-type (wt) rsv. rsv/δns2/δ1313/i1314l was derived from a recombinant version of wt rsv strain a2 (rd46 [22] ; genbank accession no. kt992094) with the further modification of a 112 nucleotide phenotypically silent deletion in the sh noncoding sequence that stabilizes the complementary dna (cdna) during propagation in bacteria [26] . rsv/δns2/δ1313/i1314l contains 2 independent attenuating elements: (1) a 523 nucleotide deletion of the ns2 gene and (2) a codon deletion in the l gene (δ1313; deletion of s1313) plus the adjacent missense mutation i1314l that prevents the compensatory deattenuating mutation i1314t [25] . the virus was generated from cdna on world health organization vero cells by reverse genetics [18] , and clinical trial material (ctm) was prepared at charles river laboratories. sequence analysis confirmed that the seed virus and ctm were free of detectable adventitious mutations. the ctm had a mean infectivity titer of 10 7.3 plaque-forming units (pfu)/ml. ctm was stored at −70°c and diluted to dose on site using leibovitz l15 medium. l15 medium was used as placebo. this phase 1 trial was conducted at the center for immunization research (cir), johns hopkins bloomberg school of public health, between june 2013 and april 2018 (clinicaltrials.gov nct01893554). a single dose of rsv/δns2/δ1313/i1314l was evaluated sequentially in randomized, double-blind, placebocontrolled studies in rsv-seropositive children aged 12-59 months at a dose of 10 6 pfu and in rsv-seronegative children at a dose of 10 5 or 10 6 pfu ( figure 1 ). children were randomized 2:1 to receive vaccine or placebo, administered as nose drops (0.5 ml; approximately 0.25 ml per nostril). randomization, blinding, and unblinding were performed as described elsewhere [10] . children were enrolled between april 1 and october 31 each year, outside the rsv season. clinical assessments were performed and nasal wash (nw) samples obtained as described elsewhere (rsv-seropositive children, study days 0, 3-7 and 10; rsv-seronegative children, study days 0, 3, 5, 7, 10, 12, 14, 17, 19, 21, 28 ± 1 day at each time point) [10] . adverse events were collected through day 28 for rsv-seropositive and rsv-seronegative children; serious adverse events and lris were collected through day 56 for rsv-seronegative children, with additional physical examinations performed and nw samples obtained in the event of lri. fever, upper respiratory illness (uri; including rhinorrhea, pharyngitis, and hoarseness), cough, lri, and otitis media were defined as described elsewhere [27] . when illnesses occurred, nw samples were tested for other viruses and mycoplasma by means of real-time reversetranscription polymerase chain reaction (rt-pcr) (respiratory pathogens 21 kit; fast track diagnostics, luxembourg). rsv-seronegative participants were monitored for medically attended acute respiratory illness (maari), which included medically attended acute lri (maalri), during the first rsv season after inoculation [6] . after the first rsv season, parents of children in the 10 6 pfu dose cohort were asked to participate in an optional second season of rsv surveillance. during rsv surveillance (1 november through 31 march), families were contacted weekly to determine whether maari had occurred [6, 10] . for each illness, a clinical assessment was performed and a nw sample was obtained for adventitious agent testing. rsv-positive specimens were typed as rsv a or b [10] . illnesses are as described in the text. uri was defined as rhinorrhea, pharyngitis, or hoarseness, and lri as wheezing, rhonchi, rales, or diagnosis with pneumonia or laryngotracheobronchitis (croup). other symptoms included rash, conjunctivitis, nasal congestion, diarrhea, and vomiting. c infection with vaccine virus was defined as the detection of vaccine virus by means of culture and/or rt-qpcr and/or a ≥4-fold rise in rsv serum neutralizing antibody titer and/or serum anti-rsv f antibody titer. d shedding of vaccine virus as detected by means of culture and/or real-time rt-qpcr. the limit of detection of vaccine virus was 0.5 log 10 pfu/ml for culture and 1.7 log 10 copies/ml for rt-qpcr. e the lower limit of detection for the rsv immunoplaque assay was 0.5 log 10 pfu/ml. f the lower limit of detection for rt-qpcr was 1.7 log 10 copies/ml. g a >4 fold-rise in serum rsv neutralizing antibody was detected in a placebo recipient in the 10 5 pfu dose cohort, probably indicating infection with wild-type rsv between study days 28 and 56. vaccine virus in nw fluid was quantified by immunoplaque assay using a mix of 3 monoclonal antibodies (mabs) to rsv f (mabs 1129, 1243, and 1269 [28] ) and by quantitative rt-pcr (rt-qpcr) [10] . to verify the presence and genetic stability of the attenuating elements at time of peak vaccine shedding, viral rna was obtained from a single passage of nw fluid on vero cells. the presence of the ns2 gene deletion was verified by agarose gel electrophoresis, confirming an 855 base pair rt-pcr amplicon spanning the deletion. the presence of the 1313 deletion and the i1314l mutation was confirmed by sequence analysis of a 758 base pair pcr fragment of the l gene [12] . serum samples were obtained before inoculation and approximately 1 month after inoculation of rsv-seropositive participants and 2 months after inoculation of rsv-seronegative participants. to measure serum antibody responses after exposure to wt rsv, serum samples were obtained from rsv-seronegative participants in october of the calendar year in which the child was enrolled and in april of the following calendar year; for participants enrolled in september or october, the postvaccination serum samples also served as pre-rsv season serum samples. of 30 rsv-seronegative children enrolled in the 10 6 pfu dose cohort, 22 participated in second-year rsv surveillance, with serum samples obtained before and after the second surveillance season. serum samples were tested for rsv neutralizing antibodies using a complement-enhanced 60% rsv plaque-reduction neutralization assay [29] , and for immunoglobulin g (igg) antibodies to the rsv f glycoprotein using an enzyme-linked immunosorbent assay [10] . the plaque reduction neutralization titer (prnt) and rsv f igg titer are expressed as reciprocal log 2 values. antibody responses were defined as ≥4-fold increases in titer in paired specimens. infection with vaccine was defined as detection of vaccine virus by culture or rt-qpcr and/or a ≥4-fold rise in rsv prnt or in rsv f igg. the mean peak titer of vaccine virus shedding (in log 10 pfu/ml) was calculated for infected vaccinees only. prnt and rsv f igg titers were transformed to log 2 values for calculation of means, and the student t test was used to compare means between groups. rates of illness and antibody responses were compared using the 2-tailed fisher exact test. rsv/δns2/δ1313/i1314l was sequentially evaluated in 15 rsv-seropositive children (10 vaccinees and 5 placebo recipients), 22 rsv-seronegative infants and children at 10 5 pfu (15 vaccinees and 7 placebo recipients), and 30 rsvseronegative infants and children at 10 6 pfu (20 vaccinees and 10 placebo recipients; figure 1 and table 1 ) between april 2013 and april 2018. none were lost to follow-up or excluded from analysis. the mean age was 30.1 months (range, 13-51 months) for rsv-seropositive participants; 11.6 months (6-22 months) for rsv-seronegative participants in the 10 5 pfu dose cohort, and 12.8 months (6-23 months) for rsv-seronegative participants in the 10 6 pfu dose cohort. of the 67 participants, 49% were female, 69% white, 8% black, 3% asian, and 20% described as of mixed racial heritage; 8% were hispanic, and 92% were non-hispanic. in rsv-seropositive participants, uri was observed in 2 and cough was observed in 1 of 10 vaccinees during the 28-day postimmunization reporting period ( the acute phase in 11 of 15 vaccinees (73%) versus 4 of 7 placebo recipients (57%) for the 10 5 pfu dose cohort and in 11 of 20 vaccinees (55%) versus 8 of 10 placebo recipients (80%) for the 10 6 pfu dose cohort, including 2 lris in placebo recipients (with onset on days 33 and 45 after placebo administration) ( table 1 and figure 2 ]. although uri (rhinorrhea) occurred more frequently in vaccinees (in 73% vs 57% of placebo recipients in the 10 5 pfu cohort, and in 50% vs 40%, respectively, in the 10 6 pfu cohort) (figure 2 and table 1 ), these differences were not statistically significant, and the incidence of rhinorrhea did not increase with increased vaccine dose. one vaccinee and 1 placebo recipient had otitis media. other viruses were detected in 10 of 22 and 7 of 12 symptomatic rsv-seronegative vaccine and placebo recipients, respectively, including rhinovirus, enterovirus, adenovirus, coronavirus, bocavirus, and parainfluenza virus (piv) type 3. the nw samples obtained within 3 days after illness onset from the 22 symptomatic vaccinees revealed other respiratory viruses only (5 children), other respiratory viruses and vaccine virus (5 children), vaccine virus alone (5 children), or neither vaccine virus nor other respiratory viruses (7 children). interestingly, during the genetic stability testing of the vaccine isolates from nw sample, the presence of wt rsv a was detected, together with vaccine, on day 10 after immunization in a child who had received 10 6 pfu of vaccine. this vaccinee developed grade 1 rhinorrhea 4 days after detection of both viruses in the nw. however, there were many instances in which a potential causative agent was not detected. for example, only 7 of 12 symptomatic rsv-seronegative placebo recipients had other viruses detected by rt-pcr; of the 5 in whom none were detected, 2 had lri (1 episode of croup and 1 episode of croup and stridor). at the 10 5 pfu dose, vaccine virus was detected in nw samples by culture on a single day in 1 of 15 vaccinees (titer, 10 1.4 pfu) and by rt-qpcr in 11 of 15 vaccinees (mean peak copy number, 10 3.0 ; table 1 and figure 3a and 3b). infectivity and replication of this vaccine was significantly improved with an increased dose: at 10 6 pfu, vaccine virus was detected in 16 of 20 vaccinees by culture (p < 0.0001; fisher exact test) and in 18 of 20 by rt-qpcr (table 1 and figure 3a and 3b), and most vaccinees shed vaccine virus over several days ( figure 3c and 3d). for most vaccinees, the peak of vaccine shedding was detectable by culture and by rt-qpcr between days 5 and 10 ( figure 3c and 3d; triangles) . at both the 10 5 pfu and 10 6 pfu doses, the magnitude of vaccine virus replication was highly restricted (mean peak titer by culture, 10 0.6 and 10 1.8 pfu; mean peak copy number by rt-qpcr, 10 3.0 and 10 3.5 among those who were infected) ( table 1 and figure 3a and 3b), indicative of the substantial attenuation of this vaccine. rt-pcr and partial sequence analysis of nw isolates obtained at the peak of vaccine shedding from 18 of 20 rsv-seronegative vaccinees who received 10 6 pfu confirmed the presence of the ns2 deletion and the ∆1313 and i1314l mutations. none of the rsv-seropositive vaccinees had a ≥4-fold rise in rsv f serum igg titer or prnt ( table 2) table 2 and figure 4 ). for recipients of 10 5 pfu, the mean postvaccination prnt was 5.2 log 2 , or 1:37 (table 2) ; for recipients of 10 6 pfu, it was 6.0 log 2 , or 1:64 (table 2 and figure 4 ). there was no apparent correlation between the magnitude of viral shedding as measured by culture or rt-qpcr and neutralizing or rsv f igg antibody responses (data not shown). one placebo recipient in the 10 5 pfu dose cohort had a rise in rsv prnt at day 56 (4.8 log 2 , or 1:28); this child was presumed to have been infected with wt rsv between days 28 and 56 (table 2) . all 52 rsv-seronegative children participated in rsv surveillance during the first season after inoculation, and 22 of 30 (16 vaccinees and 6 placebo recipients) in the 10 6 pfu dose cohort participated during the second rsv season. all-cause maari was frequent, occurring in 52% of children ( (table 2) . unexpectedly, 1 vaccinee in the 10 5 pfu (not shown) and 1 in the 10 6 pfu dose cohort ( figure 5a , right) experienced laboratory-confirmed rsv-maari without an increase in prnt. in each case, several other viruses were detected, and these may have reduced rsv replication or immunogenicity. abbreviations: igg, immunoglobulin g; nd, not done; pfu, plaque-forming units; post, after inoculation; post-ss, after surveillance season; pre, before inoculation; pre-ss, before surveillance season; rsv, respiratory syncytial virus; sd, standard deviation. a antibody data are expressed as reciprocal mean log 2 titers. postinoculation antibody titers were measured at day 28 in rsv-seropositive children and at day 56 in rsv-seronegative children. for rsv-seronegative children, serum samples were also collected and assayed before and after the surveillance season; this was not done for rsv-seropositive children. rsv plaque reduction neutralizing titers (prnt) were determined by means of complement-enhanced 60% plaque reduction neutralization assay; serum igg titers to rsv f were determined by means of enzyme-linked immunosorbent assay (elisa). titers below the limit of detection were assigned values of 2.3 log 2 (prnt) and 4.6 log 2 (elisa). b a >4 fold-rise in serum rsv neutralizing antibody was detected in a placebo recipient in the 10 5 pfu dose cohort, probably indicating infection with wild-type rsv between study days 28 and 56. in the 10 6 pfu cohort, rsv-maari occurred in 4 of 20 vaccinees (3 rsv a and 1 rsv b) (figures 4a and 5a , left and right; triangles) and in 3 of 10 placebo recipients (2 rsv a and 1 rsv b) (figures 4b and 5b; triangles) . however, in 3 of 4 cases of putative rsv-maari in vaccinees and 1 of 3 in the placebo group, additional viruses were isolated, so causality is unclear. the fourth rsv-maari in a vaccinee was associated with rsv b infection. the placebo recipient with a viral coinfection experienced maalri (grade 3 croup), with both rsv a and piv type 2 detected. four fold or greater increases in rsv prnt after the first rsv surveillance season were detected in 8 of 20 vaccinees ( table 2 and figures 4a and 5a) , including 3 of 4 with rsv-maari ( figure 5a, left; triangles) , and in 8 of 10 placebo recipients ( table 2 and figures 4b and 5b ; triangles), including 3 with rsv-maari. the mean postsurveillance prnt in these 8 placebo recipients (6.1 log 2 ) ( figure 4b ; circled) was comparable to the mean postvaccination prnt in vaccinees in the 10 6 pfu dose cohort (6.0 log) ( figure 4a and table 2 ), suggesting that the rsv neutralizing antibody response to the vaccine was comparable to that induced by primary wt rsv infection. there were no differences in the magnitude of the prnt in children with medically attended or inapparent rsv infections (figure 4 ; dots compared to triangles). in all, 9 rsv-seronegative vaccinees who received 10 6 pfu had evidence of wt rsv infection during surveillance, as determined by rise in prnt and/or viral detection. of note, the postsurveillance prnt in these 9 vaccinees was significantly greater than in the 8 placebo recipients (9.9 log 2 vs 6.1 log 2 , or 1:955 vs 1:69; p < 0.0001) ( figure 4a and 4b) indicating that a single intranasal dose of rsv/δns2/δ1313/i1314l primed for potent anamnestic responses to wt rsv infection. all-cause maari was again frequent (13 episodes in 9 of 22 children; 41%), including 1 mild episode of rsv-associated maalri in a 28-month-old vaccinee with congestion, cough, and posttussive emesis beginning 473 days after vaccination. at physical examination, she was afebrile and not in respiratory distress, but crackles were heard on auscultation. rsv a was detected as a single pathogen. increases in rsv prnt of ≥4-fold were detected in 6 of 16 vaccinees and 2 of 6 placebo recipients ( figure 5a , middle, and figure 5b) . no vaccinee had a ≥4-fold increase in prnt during both seasons, but 2 placebo recipients did ( figure 5b ). rsv/δns2/δ1313/i1314l was created by reverse genetics, using knowledge of rsv gene function and known attenuating mutations engineered for genetic stability. the vaccine was attenuated by deletion of the interferon antagonist ns2 gene and the insertion and stabilization of an attenuating ts mutation, δ1313/i1314l [25] . sequence analysis of shed vaccine virus confirmed the stability of these mutations. previously, rsv vaccine candidates containing the ns2 deletion and other nonstabilized ts mutations were evaluated in phase 1 studies; these vaccines were either underattenuated or overattenuated [7] . however, 10 6 pfu of rsv/δns2/δ1313/i1314l infected all rsv-seronegative children and induced a primary antibody response in 90%. we did not observe lri after immunization, and rates of fever, cough, and otitis media were comparable in rsvseronegative vaccinees and placebo recipients. rhinorrhea occurred more often in seronegative vaccinees than in placebo recipients, although the differences were not statistically significant when the 10 5 pfu and 10 6 pfu dose cohorts were considered singly or together. rates of illness did not increase with the higher dose. other respiratory viruses were detected frequently in vaccinees and placebo recipients, consistent with previous studies [6, 30] , but we also encountered a substantial number of respiratory events without concurrent detection of any pathogen. this high incidence of background respiratory illness, typical for this age group, and the inability to detect potential causative agents in some symptomatic children, indicate that larger studies will be needed to determine whether administration of rsv/δns2/δ1313/i1314l is associated with an increased risk of mild respiratory illness. should administration of rsv/δns2/δ1313/i1314l be associated with transient rhinorrhea, this would probably be acceptable if efficacy against rsv-associated lri was demonstrated. as in previous studies [6-8, 10-12, 27] , we conducted surveillance for rsv-associated maari in rsv-seronegative participants, with clinical assessment performed for each medically attended illness and nw samples obtained for viral identification. in the current study, we also conducted surveillance for the first time during a second rsv season in a subset of children to assess the durability of the immune response. as previously demonstrated for all live attenuated rsv vaccines evaluated to date, there was no evidence of enhanced rsv disease in any vaccine recipient. of the children with presumed rsv-maari, 50% of vaccines and 33% of placebo recipients had ≥1 additional respiratory virus isolated, so attribution remains unclear. we note that the rates of rsv-maari in this study and in our previous studies [10] [11] [12] were low compared with some population-based epidemiologic studies, probably because we purposefully selected children without medical risk factors for serious rsv disease for enrollment in these phase 1 studies. comparison of rsv prnt before and after each surveillance season indicated that rsv/δns2/δ1313/i1314l primed for substantial anamnestic serum antibody responses; for example, during the first surveillance season, titers among vaccinees with prnt responses were approximately 14-fold higher than titers among placebo recipients with prnt responses (1:955 vs 1:69, respectively). the antibody responses in the vaccinees were memory responses, whereas those in the placebo recipients generally were primary responses. these remarkably high memory responses are a consistent and important feature of recently evaluated live attenuated rsv vaccines [10] [11] [12] . memory responses were also observed during the second season, indicating that priming is durable. during the second rsv surveillance season, mild rsvassociated lri was observed in 1 vaccinee. although an isolated event, this information suggests that it may be desirable to offer a booster dose of rsv/δns2/δ1313/i1314l to protect during a second rsv season. alternatively, the boost could consist of a piv-vectored bivalent rsv/piv vaccine [31] ; in animal studies, the replication and immunogenicity of piv-vectored rsv vaccines was unaffected by rsv-specific immunity from a prior immunization. these possibilities could be evaluated in future vaccine trials. when administered to rsv-seronegative children, a 10 6 pfu dose of rsv/δns2/δ1313/i1314l was highly infectious yet restricted in replication, induced serum prnt responses comparable to those observed after primary wt rsv infection, primed for substantial anamnestic serum antibody responses after natural exposure to wt rsv, and was genetically stable. these desirable vaccine characteristics have also been noted for live attenuated rsv vaccine candidates bearing the m2-2 deletion ( [10] and unpublished data). larger studies to directly compare the tolerability and immunogenicity of candidate vaccines bearing ns2 and m2-2 deletions will be needed; 1 such study 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and additively increase the quantity and quality of respiratory syncytial virus (rsv)-neutralizing antibodies induced by an rsv fusion protein expressed by a parainfluenza virus vector key: cord-299149-lc2dxvxz authors: chen, i-cheng mark; loh, jin phang; chuah, cheryl x p; gao, qiu han christine; sun, yinxiaohe; ng, sock hoon; koh, wee-hong victor; goh, ee hui; zhao, xiahong; tambyah, paul anantharajah; cook, alex r; chng, jeremiah; pang, junxiong; tan, boon-huan; lee, vernon j title: evidence for cross-protection against subsequent febrile respiratory illness episodes from prior infections by different viruses among singapore military recruits 2009–2014 date: 2019-06-15 journal: j infect dis doi: 10.1093/infdis/jiz046 sha: doc_id: 299149 cord_uid: lc2dxvxz background: few studies have evaluated the relative cross-protection conferred by infection with different groups of viruses through studies of sequential infections in humans. we investigated the presence of short-lived relative cross-protection conferred by specific prior viral infections against subsequent febrile respiratory illness (fri). methods: men enlisted in basic military training between december 2009 and december 2014 were recruited, with the first fri as the study entry point. resplex ii assays and real-time polymerase chain reaction assays were used to detect viral pathogens in nasal wash samples, and survival analyses were performed to determine whether infection with particular viruses conferred short-lived relative cross-protection against fri. results: prior infection with adenovirus (hazard ratio [hr], 0.24; 95% confidence interval [ci], .14–.44) or influenza virus (hr, 0.52; 95% ci, .38–.73) conferred relative protection against subsequent fri episode. results were statistically significant even after adjustment for the interval between enlistment and fri (p < .001). adenovirus-positive participants with fri episodes tended to be protected against subsequent infection with adenovirus, coronavirus, enterovirus/rhinovirus, and influenza virus (p = .062–.093), while men with influenza virus–positive fri episodes tended be protected against subsequent infection with adenovirus (p = .044) and influenza virus (p = .081). conclusion: prior adenovirus or influenza virus infection conferred cross-protection against subsequent fri episodes relative to prior infection due to other circulating viruses. viral interference describes the phenomenon whereby a prior viral infection potentially exerts some effect on subsequent infection with other viruses [1] . edward jenner first reported this when herpetic infections were observed to cross-protect against the subsequent development of vaccinia lesions [2] . then, in the 1950s, isaacs and lindenmann discovered the role of interferons in viral interference [3] . since then, cross-protection has been observed for animal viruses [2] , with possible mechanisms elaborated on in multiple animal models [4] [5] [6] . in human populations, time-series analyses have demonstrated how epidemics involving a particular virus influence the temporality of epidemics involving other families of respiratory viruses [7] [8] [9] [10] . several case-control and cross-sectional studies also show that co-detection of respiratory viruses is less frequently observed than if the infections caused by different respiratory viruses occur independently of each other [11] [12] [13] . however, evidence from studies of sequential infections in humans would provide more-robust evidence that viral infections can reduce the risk of subsequent infection with a different group of viruses and clarify whether such cross-protective effects differ between different types of viruses. as previously reported, the rollout of routine trivalent seasonal influenza vaccination in the singapore armed forces was accompanied by a dramatic decline in the incidence of laboratory-confirmed influenza virus infections but a much less noticeable decrease in the overall incidence of febrile respiratory illness (fri) episodes [14] . a subsequent increase in the incidence of adenovirus infections appeared, at least from time-series data, to account for some degree of replacement for the influenza virus infections averted through vaccination [15] . in this article, we set out to clarify the relationship between specific prior viral infections and subsequent fri episodes in young singaporean men undergoing basic military training (bmt). we reanalyzed the singapore armed forces fri surveillance program data by using survival analyses to assess whether there was any evidence, at the individual level, that infection with particular viruses was conferring short-lived relative cross-protection against subsequent fri episodes, including those caused by other virus groups. since may 2009, the singapore armed forces has operated a sentinel fri surveillance program at a major recruit-training center. recruits who develop fri would report to the primary healthcare clinic for assessment, where they would receive treatment and would typically be given home leave for 1-2 days. during regular consultation hours, recruits meeting our inclusion criteria (ie, a temperature ≥37.5°c plus either cough or sore throat) were asked to participate in the study. following receipt of written informed consent from the patient, we administered a questionnaire and collected nasal wash samples from both sides of the nose. we excluded repeat consultations if the patient was determined not to have recovered from the earlier illness episode. our study covered recruits undergoing bmt between 15 december 2009 and 31 december 2014, during which inactivated influenza vaccine was routinely administered to recruits who did not have any contraindications. this was initially a monovalent influenza vaccine (miv) containing only 2009 pandemic influenza a(h1n1) virus (a[h1n1] pdm09), for recruits who enlisted between 11 december 2009 and 4 october 2010. the miv was superseded by a trivalent influenza vaccine (tiv) that included a(h1n1)pdm09, for recruits who enlisted between 8 november 2010 and 10 december 2014). the study was reviewed and approved by the singapore military's joint medical committee for research by and the national university of singapore's ethics review committee (national university of singapore institutional review board reference 09-255). samples were sent in viral transport medium (copan diagnostics, murrieta, ca) for etiological testing within 24 hours of collection. during the 5-year study period, the resplex ii multiplex polymerase chain reaction assay was used from december 2009 through 29 june 2012 to detect viral pathogens, after which it was replaced by in-house viral multiplex polymerase chain reaction assays (supplementary materials) [16] . bmt is tailored to the physical, medical, and vocational needs of a recruit. the majority enlist into the main intake types, with standard durations of around 9, 17, and 19 weeks, with a small proportion having a mix of shorter courses (termed "others"). dropout rates are low, and our study clinic served most of the healthcare needs for recruits at the training center. we were hence able to reanalyze the individual fri episodes as a cohort study by linking consultation episodes through coded subject identifiers. the first fri episode (fri-1) for a given participant served as the point of entry into the study (t 0 ). individual participants then accumulated follow-up time ( figure 1a) , with subsequent fri episodes being the event of interest. those with no further episodes accumulated follow-up time until their exit date from bmt, while participants with ≥2 episodes contributed additional follow-up intervals after each subsequent episode. other than subsequent consultation for any fri episodes, additional definitions for events of interest were episodes positive for any of the respiratory viruses tested and specific groups of etiological agents. we grouped the agents by using broad categories that accommodated changes in testing protocol during the study period while reflecting virus taxonomy: adenoviruses (advs)-species b, e, and others for which the type was undetermined; coronaviruses (covs)-cov-229e, cov-nl63, cov-hku1, cov-oc43, and other covs; enteroviruses and rhinoviruses (ervs); human metapneumovirus (hmpv); influenza virus (fluv)-influenza a(h3n2) virus, 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09), influenza a(h1n1) virus, influenza a virus-positive samples for which the subtype was undetermined, and influenza b virus; human parainfluenza virus (hpiv)-types 1-4; and respiratory syncytial virus (rsv)-rsv-a and rsv-b. the virus categories described above were also our exposures of interest, to investigate whether an fri episode caused by one virus group conferred relatively greater protection against a subsequent fri episodes than fri episodes testing negative for that virus group. in individuals with ≥1 subsequent fri episode, exposure to prior infections was regarded as cumulative. for instance, for scenario 3 in figure 1a , with 2 subsequent fri episodes, hmpv exposure occurred only during interval c, cov exposure occurred during intervals b and c, and adv occurred during all 3 intervals. potential confounders included the year of the surveillance program, participant's age and ethnicity, type of bmt intake, history of smoking and asthma (including childhood asthma), and receipt of influenza vaccine before the fri episode. assessment of whether the participant received miv or tiv was based on self-reported vaccination history up to 1 year prior to the first fri episode (since antibody titers and vaccine effectiveness could wane thereafter [17] [18] [19] ), supplemented with singapore armed forces records for vaccines received after enlistment. since fri episodes are concentrated differentially during a bmt course, we adjusted for this effect using the fri incidence rate during successive 2-weekly phases after the time of enlistment for that intake type ( figure 1b and supplementary materials). observation time was split into subintervals if the observation period straddled different bmt phases and was analyzed in a multilevel modeling framework ( figure 1c ). the framework included random effects terms for the bmt companies that participants belonged to, within which were nested the intervals (delineated by consecutive fri episodes), within which, in turn, were nested the subintervals straddling bmt phases (levels 4 to 1 for model 2). models 1 and 3 only had 3 levels. model 1 omitted the subintervals used to adjust for fri incidence. model 3, which stratified subintervals by whether they ended <4 weeks or ≥4 weeks after the initial fri episode, used only the first fri episode for each participant (episode bmt phase, wk figure 1 . a, scenarios for febrile respiratory illness (fri; defined as consultations at the primary healthcare clinic in which a temperature ≥37.5°c was detected, plus either cough or sore throat) episodes and intervals in time-to-event analyses. in scenario 1 (no subsequent fri episode), a participant accumulates follow-up time from the first fri episode (fri-1) to the date of exit from basic military training (bmt). in scenario 2, follow-up time accrues between the first (fri-1) and second (fri-2) episodes (interval a, level was therefore redundant). models 2 and 3 adjusted for fri incidence rates by time from enlistment and, hence, excluded the bmt intake type termed "other" (for which data used to estimate incidence rates were unavailable). we present simple tabulations and visualizations of the data at the participant and episode levels, using χ 2 and fisher exact tests where appropriate. we then investigated how fri caused by specific virus families affected the subsequent risk of fri episodes relative to those who were unexposed, through kaplan-meier plots and log-rank tests, as well as with multilevel survival analyses (with survival time modeled using a weibull distribution), which adjusted for potential confounders. we also investigated whether exposure to fluv and adv affected the subsequent risk of all fri episodes and the risk of fri by specific pathogens, using kaplan-meier plots and log-rank tests. however, multivariable analysis was not performed because of sample size limitations when restricting the event of interest to specific pathogens. all analyses used stata 15 (stata; college station, tx), with 2-tailed p values of < .05 considered statistically significant. the study included 6138 fri episodes among 5677 participants (enrolled over approximately 5 years; a total of 3487 fri episodes (56.8%) were positive for ≥1 virus included in the panel (table 2) fri episodes with an identified virus were less likely than those without an identified virus to be followed by a subsequent fri episode (6.2% vs 9.2%, respectively; p < .001), and subsequent fri episodes with viruses from the same group were rare (<2% across all virus groups). figure 2d ); the former pattern was observed after and the latter before routine tiv administration (data not shown). among fri episodes with an identified virus, the proportion that were not followed by a fri episode decreased more gradually than that among fri episodes without an identified virus ( figure 3a) . relative protection against a subsequent fri episode was also observed for adv-positive versus advnegative episodes ( figure 3b ) and likewise for fluv infections ( figure 3c ); all 3 results were statistically significant at p < .001. however, no significant relationships were observed for cov, erv, hmpv, hpiv, and rsv (supplementary figure 1) . model 1, which accounted for the hierarchical data structure and other covariates, gave similar results (table 3 ). relative to episodes without an identified virus, only adv (hazard ratio [hr], 0.24; 95% ci, .14-.44) and fluv (hr, 0.52; 95% ci, .38-.73) infections conferred significant protection against subsequent fri episodes. adv and fluv infections remained significantly protective, after adjustment for fri incidence by time from enlistment (model 2) and after addition of a variable that stratified subintervals on whether they ended <4 weeks before or ≥4 weeks after the initial fri episode (model 3). interestingly, the hazard of a subsequent fri episode was significantly lower in subintervals ending <4 weeks after the initial episode (vs those ending ≥4 weeks after the initial episode; hr, 0.43; 95% ci, .30-.61). moreover, there was evidence for interaction with fluv (p interaction = .085); stratified hrs for fluv infection in subintervals ending <4 weeks and ≥4 weeks after the initial fri episode were 0.23 (95% ci, .08-.63; p = .005) and 0.58 (95% ci, .38-.87, p = .009), respectively. supplementary table 1 corroborates these findings through an alternative approach using crude fri incidence rates based on the underlying bmt population at risk. following adv and fluv infection, incidence rates for subsequent fri episodes were consistently lower than the average for all recruits in the 19-, 17-, and 9-week bmt intake types. moreover, compared with the full course duration, analyses censored 8 and 6 weeks after enlistment showed stronger protective effects from prior fluv infection. likewise, incidence rate ratios stratified by bmt intake type revealed increased protection by prior fluv infection with shorter course durations (table s2) . adv-positive episodes tended to protect against subsequent infection with adv (p = .090) and also 3 other virus groups all models were adjusted for study year, type of bmt intake, age, ethnicity, smoking history, history of asthma, and influenza vaccination. c not adjusted for fri incidence rate by time from enlistment or for timing of interval relative to the prior consultation episode. all episodes are included as exposures and events of interest. all bmt types are included. d adjusted for fri incidence rate by time from enlistment, expressed as fri episodes per 1000 enlistee days (as defined and illustrated in methods and figure 1b ), but not for timing of interval relative to the prior consultation episode. all episodes are included as exposures and events of interest. only the 19-week, 17-week, and 9-week bmt types are included. e adjusted for the fri incidence rate by the time since enlistment, expressed as the number of fri episodes per 1000 enlistee-days (as defined and illustrated in methods and figure 1b) , and for whether the interval assessed was <4 weeks or ≥4 weeks after the prior episode. the initial episode is the exposure of interest, and the second episode is the event of interest. only the 19-week, 17-week, and 9-week bmt types are included. figure 3a ). cross-protection between fluv episodes was not apparent in the full data set (supplementary figure 3b) . however, before routine administration of tiv (ie, on or before 4 october 2010), fluv-positive episodes tended to protect against subsequent fluv infection in enlistees who had not recently received tiv (p = .081; supplementary figure 3c ). none of the other virus groups conferred significant group-specific protection (supplementary table 3 ). our study provides evidence on how having a fri episode due to respiratory viruses from certain categories reduced the risk of a subsequent fri episode during basic military training relative to having a fri episode that was not due to those respiratory viruses. among the viruses, protective effects were strongest following adv and fluv episodes. adv infections conferred protection against subsequent infection with adv and crossprotection to other groups of commonly circulating viruses in bmt (cov, erv and fluv), while fluv infection conferred significant protection against subsequent adv infection. the observation of influenza's protection against subsequent episodes of influenza (mostly due to other types/subtypes in our data, in the period prior to routine tiv administration) is also consistent with reports by other human and animal model studies [6, 20, 21] . previous time-series analyses have demonstrated viral interference after accounting for seasonal factors, suggesting that epidemics of influenza virus and rhinovirus infection tend to shift the timing of epidemics for other viruses [8] . others have used case-control-type analyses of codetection data to demonstrate that rhinovirus infection, as an exposure, was inversely associated with the probability of observing adenovirus [12] and influenza virus infections, with the latter 2 viral infections framed as the outcomes of interest [12, 13] . however, in a study of military recruits in which adenovirus was the dominant fri agent, wang et al used a case-control analysis to demonstrate the reverse phenomenon-that adenovirus infection as an exposure reduced the odds of rhinovirus infection as the outcome of interest [11] . however, they noted how another interpretation of their results is that rhinovirus infection reduced the risk of subsequent adenovirus infection. the opposing framing of outcomes and exposures in these codetection studies highlights issues in interpreting results from such cross-sectional and casecontrol designs, in which the sequence of infections cannot be ascertained. what distinguishes this work is our use of a cohort study design by which we can identify the temporality and sequence of initial and subsequent fri episodes, which the previous studies could not do. observations positive for a specific virus group are essentially being compared against those negative for all viruses we tested for. therefore, this analyses does not assess whether, for example, infections by the erv group confers any protection relative to individuals who did not recently have any viral infection. supplementary tables 1 and 2 suggest that, relative to enlisted recruits for the main bmt intake types, incidence rates for subsequent fri episodes were higher over the full course duration and for 19-week bmt intake types, respectively. this applied to all virus groups except adv and fluv groups and was possibly due to confounding by common risk factors for repeated fris at the individual level [15] . however, in the shortest, 9-week bmt type and when censoring the data at shorter periods after enlistment, incidence rates were reduced for most virus groups relative to the average rates for recruits following enlistment. for fri episodes as a whole, incidence rate ratios were 0.49 (95% ci, .29-.77; p < .001) in the data censored 4 weeks after enlistment (supplementary table 1 ). these observations therefore suggest that viral causes of fri other than adv and fluv may also confer short, time-limited protection. they also corroborate findings from table 3 on how a reduced hazard of a subsequent fri episode occurs in the first 4 weeks following the initial fri episode, relative to latter periods, and how fluv is more strongly protective during the former period. the difference between the viruses may thus be one of degree, with most viruses conferring some weak, timelimited protection, with the stronger effects from adenovirus and influenza virus becoming apparent when aggregating data over the full course duration. one mechanism consistent with such short-lived cross-protection is the triggering of interferonstimulated genes and cytokines, causing nearby cells in the respiratory tract to enter an antiviral state [22, 23] . alternatively, transient cross-protection could result from infection-induced behavioral changes, such as improved hand hygiene following an fri and a short duration of reduced exposure to new pathogens while being furloughed on home leave. however, this on its own is likely inadequate to explain the extent of cross-protection observed with adenovirus and influenza virus infections relative to other fri episodes. other proposed mechanisms, such as competition for the same receptor-binding sites and intracellular host machinery for replication [24] , and an unfavorable physiological state of the host, such as high body temperatures upon an initial infection [11] , are more applicable to protection against coinfections but not, as demonstrated here, against sequential infection. our findings have several implications. they provide an explanation for what has previously been suspected on the basis of time-series analyses, such as how influenza epidemics potentially delay rsv epidemics [8, 25] . they also add to the body of evidence motivating the search for the causative mechanisms for cross-protection among viral infections while providing additional clues. given the relative ranking of virus families in conferring cross-protection, we suggest focusing the search for mechanisms on common factors induced by both adenovirus and influenza virus. notably, our previous studies suggest that infections with these 2 infections may be more severe, because they are more likely to present with febrile illness [26] and higher temperatures (≥38°c) than infections due to other common circulating virus families [16] . this again supports a cytokine-mediated antiviral effect. finding a means to induce such cross-protection without severe side effects could potentially lead to interventions that may be particularly useful in the context of exposure to dangerous viruses for which there is no specific vaccine or antiviral agent [27] . however, our study has some limitations. our study population largely comprises healthy young adults, and key circulating viruses in this group differs somewhat from those observed in the general population in singapore [28] . also, we excluded respiratory illness episodes that did not meet our inclusion criteria of having a temperature ≥37.5°c. we also acknowledge that our capture of data on fri episodes is likely incomplete, as consultations outside office hours and over the weekend (when trainees are on home leave) were missed by our surveillance program. however, we argue that restricting our study to febrile cases and the incomplete capture of data are biases toward the null and thus do not negate our main findings on the relative cross-protection against subsequent fri episodes conferred by adenovirus and influenza virus infections. in summary, our study demonstrated broad-based viral interference in a population of military recruits. infections from the adenovirus and influenza virus families conferred significant cross-protective effects against subsequent fri episodes relative to other circulating viruses. the duration of these crossprotective effects extend beyond those previously demonstrated in studies of viral coinfections. our study points the way for research into the underlying mechanisms for broad-based antiviral activity in the human host, which deserves greater study. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the 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and clinical characteristics rate of decline of antibody titers to pandemic influenza a (h1n1-2009) by hemagglutination inhibition and virus microneutralization assays in a cohort of seroconverting adults in singapore duration of influenza vaccine effectiveness: a systematic review, meta-analysis, and meta-regression of test-negative design case-control studies do antibody responses to the influenza vaccine persist year-round in the elderly? a systematic review and meta-analysis epidemiology and relative severity of influenza subtypes in singapore in the postpandemic period from viral interference: the case of influenza viruses toll-like receptors respiratory virus induction of alpha-, beta-and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells coinfections of the respiratory tract: viral competition for resources interference between outbreaks of respiratory syncytial virus and influenza virus infection epidemiologic analysis of respiratory viral infections among singapore military servicemen in 2016 oromucosal administration of interferon to humans detection of viral respiratory pathogens in mild and severe acute respiratory infections in singapore we thank our colleagues whose work on the project, along with our own, made this article possible; and benjamin tay, for assisting with extraction of additional data during manuscript revision.financial support. this work was supported by the ministry of education (tier 1 grant r-608-000-132-112 to c. x. p. c.) and by the centre for infectious disease epidemiology and research, saw swee hock school of public health (funded by the singapore ministry of defence). key: cord-279311-msh9wvsh authors: wang, fan; nie, jiayan; wang, haizhou; zhao, qiu; xiong, yong; deng, liping; song, shihui; ma, zhiyong; mo, pingzheng; zhang, yongxi title: characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia date: 2020-03-30 journal: j infect dis doi: 10.1093/infdis/jiaa150 sha: doc_id: 279311 cord_uid: msh9wvsh background: in december 2019, novel coronavirus (sars-cov-2) pneumonia (covid-19) was reported in wuhan and has since rapidly spread throughout china. we aimed to clarify the characteristics and clinical significance of peripheral lymphocyte subset alteration in covid-19. methods: the levels of peripheral lymphocyte subsets were measured by flow cytometry in 60 hospitalized covid-19 patients before and after treatment, and their association with clinical characteristics and treatment efficacy was analyzed. results: total lymphocytes, cd4(+) t cells, cd8(+) t cells, b cells, and natural killer (nk) cells decreased in covid-19 patients, and severe cases had a lower level than mild cases. the subsets showed a significant association with inflammatory status in covid-19, especially cd8(+) t cells and cd4(+)/cd8(+) ratio. after treatment, 37 patients (67%) showed clinical response, with an increase in cd8(+) t cells and b cells. no significant change in any subset was detected in nonresponsive cases. in multivariate analysis, posttreatment decrease in cd8(+) t cells and b cells and increase in cd4(+)/cd8(+) ratio were indicated as independent predictors of poor efficacy. conclusions: peripheral lymphocyte subset alteration was associated with clinical characteristics and treatment efficacy of covid-19. cd8(+) t cells tended to be an independent predictor for covid-19 severity and treatment efficacy. in december 2019, several cases of pneumonia of unknown origins were reported in wuhan, hubei province, china [1, 2] . rapidly, the disease spread throughout china. by genome-wide sequencing of samples of bronchoalveolar lavage fluid, the pathogen was confirmed to be a distinct clade of the β-coronavirus associated with human severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) [3] . the novel virus was officially named sars-cov-2, with the disease termed covid-19 [4] . lymphocytes and the subsets of cd4 + t cells, cd8 + t cells, b cells, and natural killer (nk) cells play an important role in the maintenance of immune system function. after virus infection, alteration in total lymphocyte numbers and the subsets varies with different virus types, indicating a potential association between lymphocyte subset alteration and viral pathogenic mechanisms [5] . recent studies indicated a clear decrease in peripheral lymphocytes in covid-19 patients but any alteration in the subsets was still unknown [6, 7] . in this study, we aimed to clarify the characteristics and clinical significance of peripheral lymphocyte subset alteration in covid-19, which might help elucidate the pathogenesis and develop novel biomarkers and therapeutic strategies for covid-19. we enrolled 60 patients with covid-19, which was confirmed by detecting sars-cov-2 rna in throat swab samples using a sars-cov-2 nucleic acid detection kit according to the manufacturer's protocol (shanghai biogerm medical biotechnology). all the patients were initially admitted to zhongnan hospital of wuhan university from 7 january to 14 february 2020. in addition, we had previously tested 245 healthy blood donors to establish interlaboratory reference ranges for various parameters. these reference values were used to provide data for the healthy controls in this study. the study was approved by the ethics committee of zhongnan hospital of wuhan university (no. 2020011). written informed consent was obtained from patients. the following information on each patient was extracted from electronic medical records: age, sex, medical history, symptoms, severity assessment on admission, laboratory findings, chest computed tomography (ct) or radiograph findings, treatment, and efficacy. on admission, severe illness was defined according to the following criteria: (1) breathing rate ≥30 times/min; (2) pulse oximeter oxygen saturation (spo 2 ) ≤93% at rest; and (3) ratio of partial pressure of arterial oxygen (pao 2 ) to fraction of inspired oxygen (fio 2 ) ≤300 mmhg. after 1 week of treatment, clinical response was defined according to the following criteria: (1) symptom alleviation (eg, fever, cough, chest distress, and shortness of breath); and (2) improvement in radiological abnormalities on chest ct or radiograph. cases not meeting these criteria were classified as nonresponsive. samples of edta anticoagulated peripheral blood (2 ml) were collected from patients with covid-19 before initial treatment and a second sample was collected after 1 week of treatment. all samples were tested within 6 hours of being obtained. briefly, cd3 + /cd4 + /cd8 + t-cell, cd19 + b-cell, and cd16 + cd56 + nk-cell counts (cells/μl) were measured by multiple-color flow cytometry with human monoclonal anti-cd3-fluorescein isothiocyanate (fitc), anti-cd4-phycoerythrin (pe), anti-cd8-allophycocyanin (apc), anti-cd19-pe, anti-cd16-apc, and anti-cd56-pe antibodies (bd multitest) according to the manufacturer's instructions. the cells were analyzed on a bd facs canto ii flow cytometry system (bd biosciences). categorical data were described as percentages and continuous data as median with interquartile range (iqr). a nonparametric comparative test for continuous data was used to compare variables between groups. multivariate analysis was used to identify independent predictors of lymphocyte subsets for the treatment efficacy in covid-19. receiver operating characteristic (roc) curve analysis was conducted to evaluate the ability of lymphocyte subsets in predicting treatment efficacy. bootstrap test was used to compare 2 correlated roc curves. all statistical analyses were performed using spss statistics version 21.0 software. p < .05 was considered statistically significant. sixty covid-19 patients were included in the study ( table 1) . the median age was 60 years (iqr, 38-66), and 22 patients (37%) were male. hypertension (15%) and diabetes (10%) were the most common comorbidities. fever (70%), cough (48%), and shortness of breath (32%) were the most common symptoms. according to ct or radiograph findings, 40 patients (67%) showed bilateral pneumonia. in blood tests, leukocytes, neutrophils, and platelets were below the normal range in 17 (32%), 11 (21%), and 11 (21%) patients, and above the normal range in 6 (11%), 7 (13%), and 1 (2%) patients, respectively. lymphocytes were below the normal range in 38 patients (72%). we initially analyzed the levels of lymphocyte subsets by flow cytometry in whole blood. compared to healthy controls, covid-19 patients had a significantly lower total lymphocytes (p < .0001), cd4 + t cells (p < .0001), cd8 + t cells (p < .0001), b cells (p = .0003), and nk cells (p < .0001) ( figure 1 ). no significant difference was observed in cd4 + /cd8 + ratio (p = .603). nineteen patients (32%) were categorized as experiencing serious illness on admission. compared to patients with mild illness, severe cases had significantly lower total lymphocytes (p = .0007), cd4 + t cells (p = .024), cd8 + t cells (p = .005), and b cells (p = .018) ( figure 2 ). no significant difference was observed in cd4 + /cd8 + ratio (p = .392) and nk cells (p = .177). inflammatory indicators erythrocyte sedimentation rate (esr), c-reactive protein (crp), and interleukin-6 (il-6) were abnormal in 36 (71%), 34 (72%), and 30 (70%) patients on admission. total lymphocytes and cd4 + t cells were negatively correlated with esr (p = .037 and p = .011, respectively). cd8 + t cells were negatively correlated with esr (p < .0001), crp (p = .001), and il-6 (p = .005) ( figure 3 ). cd4 + /cd8 + ratio was positively correlated with esr (p = .035), crp (p = .002), and il-6 (p = .003). b cells showed no significant correlation with esr (p = .778), crp (p = .945), or il-6 (p = .661). nk cells were negatively correlated with il-6 (p = .049). after hospitalization, 28 patients (47%) were treated with oxygen inhalation, 27 (45%) with intravenous corticosteroid, 41 (68%) with antiviral treatment, and 23 (38%) with immune enhancer. the most common antiviral treatment was arbidol administration (37%) and interferon inhalation (32%), and 50% of patients received more than 1 antiviral regimen. in immune enhancing treatment, 32% and 10% of patients received thymalfasin and immunoglobulin, respectively. after 1 week of treatment, 37 patients (67%) reached clinical response, and 18 (33%) had not reached clinical response. in responsive patients, total lymphocytes (p < .0001), cd8 + t cells (p < .0001), and b cells (p = .026) increased significantly after treatment, and no significant change was detected in cd4 + t cells, cd4 + /cd8 + ratio, and nk cells (p > .05) (figure 4 ). in nonresponsive patients, no significant change was detected in any lymphocyte subsets (p > .05). comparatively, corticosteroid treatment increased total lymphocytes significantly, while antiviral treatment increased total lymphocytes, cd4 + t cells, cd8 + t cells, and b cells significantly (data not shown). immune enhancer had no obvious improvement in any subsets (data not shown). in multivariate analysis, posttreatment decrease in cd8 + t cells (p = .011) and b cells (p = .010) and increase in cd4 + / cd8 + ratio (p = .032) indicated a poor efficacy when considering the factors of age, sex, disease severity on admission, oxygen inhalation, antiviral treatment, and use of corticosteroid and immune enhancer ( table 2) . roc curve analysis was conducted to evaluate the role of posttreatment alterations in peripheral lymphocyte subsets in predicting treatment efficacy ( figure 5 ). the area under the roc curve (auc) was 0.738 (95% confidence interval [ci], .586-.890) for cd8 + t-cell decrease, 0.605 (95% ci, .441-.769) for cd4 + /cd8 + ratio increase, 0.600 (95% ci, .434-.765) for b-cell decrease, and 0.781 (95% ci, .638-.923) for the integrated indicator. bootstrap testing indicated a higher predictive accuracy of the integrated indicator than the alteration in cd8 + t cells, b cells, cd4 + /cd8 + ratio, or total lymphocytes individually (p < .05). since december 2019, covid-19 has been reported in wuhan and has rapidly spread throughout china. as with mers-cov and sars-cov, sars-cov-2 is a member of the coronavirus family and belongs to the β-coronaviruses [8] . infection by these coronavirus can cause sustained responses of cytokines and chemokines (namely a cytokine storm), leading to a high incidence of immune disorders and mortality [9] . lymphocytes and their subsets play an important role in the maintenance of immune system function. as with immune diseases and other infectious disease, virus infection can also lead to dysregulation in the levels of lymphocyte subsets [10, 11] . cellular surface molecules to cd3 + , cd4 + , cd8 + , cd16 + , cd19 + , and cd56 + mark the lymphocyte t-helper cells (cd3 + cd4 + ) and cytotoxic t cells (cd3 + cd8 + ), b cells (cd19 + ), and nk cells (cd16 + cd56 + ). these cells are involved in the humoral and cytotoxic immunity against viral infection. thus, it is important to clarify the characteristics of lymphocyte subsets in covid-19, which could provide novel insights to explore the immune mechanism. in our study, lymphopenia was common in the patients with covid-19 (72%), indicating an impairment of the immune system during the course of sars-cov-2 infection. in addition, decreases in cd4 + t cells, cd8 + t cells, b cells, and nk cells were also observed in the covid-19 patients. these alterations were also found in patients with pneumonia caused by mers-cov and sars-cov [12] . in the study by cui et al on sars, the incidence of lymphopenia was 84%, cd4 + t cells decreased in 100% of patients, cd8 + t cells decreased in 87%, b cells decreased in 76%, and nk cells decrease in 55% [13] . in the study by assiri et al on mers, lymphopenia occurred in 34% of patients [14] . lymphopenia might be caused by virus attachment or indirectly by immune injuries from inflammatory mediators. moreover, exudation of circulating lymphocytes into inflammatory lung tissues might also lead to lymphopenia. among covid-19 patients, severe cases had a lower level of total lymphocytes, cd4 + t cells, cd8 + t cells, and b cells than mild cases, which was similar to the alteration in sars [15, 16] . cd8 + t-cell levels were negatively correlated with inflammatory indicators esr, crp, and il-6, while the cd4 + /cd8 + ratio was positively correlated. total lymphocyte and cd4 + t-cell levels were negatively associated with esr, and nk cells were negatively correlated with il-6. these findings indicate a more obvious change in cd8 + t cells than in other lymphocyte subsets after sars-cov-2 infection. thus, lymphocytes and their subsets, especially cd8 + t cells, might be a potential predictor for disease severity and clinical efficacy in covid-19. after 1 week of treatment, in responsive cases there was a significant increase in total lymphocytes, cd8 + t cells, and b cells, but in nonresponsive cases there was no significant change in any lymphocyte subsets. however, these findings might be confounded by therapeutic factors. first, the lympholytic effects of corticosteroid could reduce the lymphocytes directly [17] . for the patients with corticosteroid treatment, the recovery of lymphocyte population might be weakened by the lympholytic effects of corticosteroid. thus, we conducted a multivariate analysis to evaluate the effects of these potential confounders, and identified posttreatment decrease in cd8 + t cells and b cells and increase in cd4 + /cd8 + ratio as independent predictors for poor clinical efficacy, especially cd8 + t cells. moreover, we also found that corticosteroid treatment increased total lymphocytes significantly in comparison to the patients without corticosteroid treatment. the anti-inflammatory effects of corticosteroid might have contributed to the posttreatment increase in lymphocytes, which outweighed the lympholytic effects of corticosteroid. importantly, cd8 + t cells have been shown to play a critical role in mediating viral clearance after acute respiratory infections of respiratory syncytial virus (rsv), influenza a virus (iav), and human metapneumovirus [18, 19] . in animal experiments, the transfer of rsv-or iav-immune cd8 + t cells into athymic mice significantly reduced viral titers [20, 21] . as in our results, cytotoxic immunity was involved in antiviral processes and the recovery of cytotoxic immune function (particularly cd8 + t cells) might be a reliable indicator of disease severity and recovery. in conclusion, peripheral lymphocyte subset alteration showed a clear association with the clinical characteristics of covid-19. cd8 + t cells tended to be an independent predictor for covid-19 severity and treatment efficacy. these findings might help elucidate the pathogenesis and develop novel biomarkers and therapeutic strategies for covid-19. financial support. this study was supported by zhongnan hospital of wuhan university program of excellent doctoral (postdoctoral) research (grant number znyb2019003). potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of refractory covid-19 pneumonia in wuhan, china [published online ahead of print 16 a novel coronavirus from patients with pneumonia in china the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 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analysis coronavirus as a possible cause of severe acute respiratory syndrome relation between cortisol metabolism and its lympholytic effect in p1798 lymphosarcoma recovery from a viral respiratory infection. ii. passive transfer of immune spleen cells to mice with influenza pneumonia clearance of persistent respiratory syncytial virus infections in immunodeficient mice following transfer of primed t cells respiratory syncytial virus infection in c57bl/6 mice: clearance of virus from the lungs with virus-specific cytotoxic t cells the cd8 t cell response to respiratory virus infections key: cord-291486-5h96msv1 authors: kistler, amy; avila, pedro c.; rouskin, silvi; wang, david; ward, theresa; yagi, shigeo; schnurr, david; ganem, don; derisi, joseph l.; boushey, homer a. title: pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity date: 2007-09-15 journal: j infect dis doi: 10.1086/520816 sha: doc_id: 291486 cord_uid: 5h96msv1 background. between 50% and 80% of asthma exacerbations are associated with viral respiratory tract infections (rtis), yet the influence of viral pathogen diversity on asthma outcomes is poorly understood because of the limited scope and throughput of conventional viral detection methods. methods. we investigated the capability of the virochip, a dna microarray—based viral detection platform, to characterize viral diversity in rtis in adults with and without asthma. results. the virochip detected viruses in a higher proportion of samples (65%) than did culture isolation (17%) while exhibiting high concordance (98%) with and comparable sensitivity (97%) and specificity (98%) to pathogen-specific polymerase chain reaction. a similar spectrum of viruses was identified in the rtis of each patient subgroup; however, unexpected diversity among human coronaviruses (hcovs) and human rhinoviruses (hrvs) was revealed. all but one of the hcovs corresponded to the newly recognized hcov-nl63 and hcov-hku1 viruses, and >20 different serotypes of hrvs were detected, including a set of 5 divergent isolates that formed a distinct genetic subgroup. conclusions. the virochip can detect both known and novel variants of viral pathogens present in rtis. given the diversity detected here, larger-scale studies will be necessary to determine whether particular substrains of viruses confer an elevated risk of asthma exacerbation. tory syncytial virus (rsv) (reviewed in [3] ). in older children [2] and adults [4, 5] , human rhinoviruses (hrvs) are implicated in the majority of cases, with variable contributions from human coronaviruses (hcovs), influenza viruses, parainfluenza viruses, rsv, and human metapneumovirus (hmpv) (reviewed in [6] ). however, the factors that determine the clinical outcomes associated with rtis in persons with asthma are not well understood. host factors governing inflammatory and immune responses have been demonstrated to influence whether a host with asthma experiencing a viral rti will develop an exacerbation of symptoms [7] [8] [9] [10] . in contrast, it remains unresolved whether variation in viral pathogens may also influence this outcome. for example, despite the prominent association between hrvs and asthma exacerbation, deliberate inoculation of a laboratory-adapted hrv-16 isolate into subjects with asthma did not produce wheezing, despite efficient infection and the generation of upper respiratory tract symptoms [11] [12] [13] [14] [15] [16] . although these experimental hrv inoculation studies have not demonstrated clinically significant exacerbations, a small decline in pulmonary function has been observed [17, 18] . likewise, not every cold in subjects with asthma is associated with exacerbation of asthma symptoms. these findings raise the possibility that some respiratory viral pathogens may have greater potential than others of triggering asthma exacerbations in susceptible hosts. if this is so, then the spectrum of viral isolates recovered from rtis in persons with asthma who experience exacerbations should differ from that in persons with asthma whose rtis do not trigger exacerbations. few prior studies have attempted to make this comparison, and those that did have generally not used methods that can differentiate among the different subtypes of viruses within a family. estimating the number of hrv isolates, rather than identifying them by serotype or genotype, may be insufficient given that certain isolates of hrv are demonstrably inefficient at triggering asthma on inoculation. part of the reason for the paucity of prior studies of this type derives from the limitations of conventional viral detection methods. viral culture is insensitive, and antigen testing, while reasonably sensitive, is (1) unavailable for several important classes of pathogen, notably hrvs and hcovs, and (2) not designed to discriminate among isolates of a given viral species. polymerase chain reaction (pcr) methods have high sensitivity but are limited to the detection of previously characterized or highly conserved viral sequences. we have developed an alternative, comprehensive strategy for viral detection that uses the virochip, a dna microarray bearing the most conserved sequences of all known viruses of humans, animals, plants, and microbes [19, 20] . in addition to the ability of the virochip to detect known viruses, it can also detect new members of known virus families by crosshybridization-a significant advantage over pcr-based methods [20] [21] [22] [23] . here, we have applied the virochip to an ongoing prospective study designed to analyze the diversity of viruses that cause rtis in adults with and without asthma, with particular attention given to those that provoke asthma attacks. we recruited adults with and without asthma by advertising on a community web site (craigslist; http://sfbay.craigslist.org/), by posting flyers on campus, and by sending e-mail messages to subjects who had responded to previous advertisements for research studies. participant recruitment started in the fall of 2001 and extended through the end of december 2004. the announcements requested that subjects contact us within 48 h of the onset of "cold" symptoms. we evaluated subjects first within 1-3 days of cold onset (visit 1), then again between days 4 and 7 of cold symptoms (visit 2), and at 6 weeks or longer thereafter to assess baseline status (visit 3). eighty-three subjects were enrolled; 53 had asthma, and 30 did not. asthma was defined as a history of asthma symptoms (recurrent dyspnea, wheezing, and chest tightness) associated with a positive methacholine test result [8] . subjects without asthma had neither a history of asthma symptoms nor a positive methacholine test result. all were informed of the purposes, procedures, and risks of participating, and all signed informed-consent forms approved by the university of california, san francisco, committee of human research. study procedures. at the first visit, participants completed questionnaires on demographics, medical history, asthma history, and date of onset of cold symptoms. at each visit, participants provided information on the severity of current upper and lower respiratory tract symptoms by use of a validated cold questionnaire [24] and diary form [8] that have been described elsewhere. spirometry measurements (forced expired volume in 1 s [fev 1 ] and forced vital capacity [fvc]) were obtained, and the percentage predicted was calculated using crapo equations. nasal lavage (nl) was then performed by instilling 5 ml of warmed normal saline into each nostril and, after a 20-s dwell time, having the subject expel the nasal contents into a plastic cup. subjects with asthma recorded symptom severity and a.m. and p.m. peak flow measurements (airwatch; imetrikus) in a diary twice daily for 7 days after visit 1 and 7 days before visit 3. the visit procedures were repeated at day 5-7 and at baseline (6 weeks or longer) after the onset of cold symptoms. definition of asthma exacerbation. before initiating the study, we defined asthma exacerbation as either a worsening of asthma symptoms that required corticosteroids (systemic or topical) or a worsening of symptoms on the basis of study measurements. the latter was defined by an increase in daily asthma symptom score of 10 points or more (range, 0-50 points) above the baseline daily average for at least 2 days together with at least 1 of the following: (1) decrease in fev 1 by 10% or more at any of the first 2 visits; (2) decrease in peak flow measurements by 20% or more for at least 2 days during the cold week; or (3) increase in daily albuterol use of 4 puffs or more for at least 2 days during the cold week. specimen processing. nl specimens from visit 1 (days 1-3 after the onset of cold symptoms) were analyzed for the presence of virus by pcr, viral culture, and virochip microarray analysis. for microarray analysis and pcr, 1 ml of homogenized nl specimen was mixed with 3.5 ml of rlt buffer (qiagen) containing 2-mercaptoethanol and then frozen at bedside in dry ice and stored at ϫ80њc. a 0.1-ml nl aliquot was cultured in duplicate with a combination of 5 different cell lines (hela, wi38, mrc5, primary monkey kidney, and hfdl, an in-house line of human fetal diploid lung cells [california department of health services viral and rickettsial disease laboratory, richmond]). some specimens were inoculated into all 5 cell types, and all were cultured in at least 3, including primary monkey kidney and human fetal diploid. if cytopathic effect occurred, viral antigen detection tests (respiratory viral antigen detection kit; chemicon) were performed to identify antigens from adenovirus, rsv, influenza viruses a and b, and parainfluenza viruses 1, 2, and 3. enteroviruses and rhinoviruses were differentiated by reactivity with the monoclonal panenterovirus reagent from chemicon and the enterovirus monoclonal antibody from dako cytomation (enterovirus clone 5-d8/1). rna extraction. samples were masked and total rna was extracted with an on-column recombinant dnase treatment step using the rneasy miniprep kit (qiagen), in accordance with the manufacturer's instructions. amplification and microarray analysis. rna was randomly amplified and labeled and was then hybridized to the virochip microarray as described elsewhere (protocol s1 in wang et al. [19] ). microarrays were scanned using the axon 4000b scanner and genepix software (version 3; axon instruments). a.k., who was blinded to the viral culture isolation and hrv pcr results, interpreted the hybridization signatures. epredict, a computational tool developed for the virochip array hybridization signature, was used to determine the virochip results, with the p value cutoff for positivity set at .05, as described elsewhere [25] . samples for which multiple viruses met this threshold were further evaluated by hierarchical cluster analysis of sum-normalized background-subtracted array hybridization intensities from all nl specimens, by use of cluster software (version 3) [26] . co-occurrence of samples within clusters was used to make calls for specimens that either had multiple significant epredict scores or had no significant epredict scores. all viral-positive calls were confirmed by recovery of viral sequence. specific pcr for hrv detection. for each sample, 3 ml of the randomly amplified material was used for independent pcr to detect and sequence the hrv vp4/vp2 capsid gene junction, as described elsewhere [27] . the vp4/vp2 pcrs were performed in a blinded manner. primers 9656-reverse (5 -gcatciggyar-yttccaccaccancc-3 ; positions 1083-1058 of hrv-1b; national center for biotechnology information [ncbi] accession number d00239) and 9895-forward (5 -gggaccaactactt-tgggtgtccgtgt-3 ; positions 534-560 of hrv-1b) were used for pcr (35 cycles of 94њc for 30 s, 58њc for 30 s, and 72њc for 30 s). comparative sequence analysis of recovered hrv vp4/vp2 pcr products. clustalw (version 1.82) was used to align the vp4/vp2 capsid gene junction sequences obtained for the clinical isolates of hrv for all 102 reference hrv serotypes [27] . neighbor-joining phylogenetic trees were generated from the resulting alignment using the phylip package (version 3.2) [28] . specific pcr for hcovs. for each of the 8 hcov-positive samples identified by virochip microarray analysis, 3 ml of the randomly amplified material was used for pcr to detect a 440bp region of the polymerase gene using pan-cov primers (5 -ggttgggactatcctaagtgtga-3 and 5 -ccatcatca-gatagaatcatcata-3 ) that have been described elsewhere [29] ; amplification was with 40 cycles of 94њc for 60 s, 48њc for 60 s, and 72њc for 60 s. sequence analysis of hcov pcr products amplified from clinical isolates. pcr products were extracted using qiaquick (qiagen) and were either sequenced directly using path-ogen-specific primers or subcloned into pcr2.1 topo vector (invitrogen) and sequenced using m13 forward and m13 reverse primers with the bigdye cycle sequence kit on an abi 3130 automated sequencer (applied biosystems). the identity of each of the hcovs in the present study was inferred on the basis of the highest scoring match from blast analysis (version 2.2.13) [30] of the resulting sequences. amplified cdna was subcloned into pcr2.1 topo plasmid (invitrogen). three hundred eighty-four colonies were picked, and dna was purified by magnetic bead isolation followed by dna sequencing using the bigdye cycle sequence kit/abi 3730xl sequencer. sequence reads were assembled by use of consed for linux (version 13.4) [31] . assemblies were screened by blast analysis [30] to remove any contigs with human or bacterial sequence similarity. gaps in assemblies were filled by synthesis of oligonucleotides with at least 100 bp of overlapping sequence with available contigs. accession numbers. genbank accession numbers for the sequenced viruses presented here are ef077237-ef077281. the geo database (http://www.ncbi.nlm.nih.gov/geo/) series accession number for all virochip microarray data presented here is gse8053. the first goal of our analysis was to assess the performance of the virochip relative to conventional viral detection methods. to do this, we used a set of nl specimens from an ongoing prospective study of naturally acquired upper rtis (naturis) in adults with and without asthma. a total of 82 cold events captured from this study were available for analysis. breakdown of participants, specimens, associated clinical outcomes, and corresponding virochip results are summarized in figure 1 . each naturi specimen was analyzed independently in a blinded manner by 3 distinct viral detection methods: virochip analysis and culture isolation for 9 common respiratory pathogens (hrv; rsv; influenza viruses a and b; human parainfluenza viruses 1, 2, and 3; adenovirus; and human enterovirus) and pcr for hrv. where the virochip detected viruses, follow-up pcr and sequence recovery was performed to confirm the presence of the detected viral species. a high proportion of specimens tested positive for virus by virochip analysis (65%). reflective of the outpatient setting of our study, the 2 most prevalent virus families detected by the virochip corresponded to hrvs and hcovs (figure 2a and table 1 ). four additional viruses generally thought to be associated with lower rtis-rsv and the closely related hmpv, influenza virus, and human parainfluenza virus-were also detected. we also detected double infections (rsv plus hcov and rsv plus influenza virus) ( figure 2a and table 1 ). in contrast, virus was detected by culture isolation in only 17% of samples. three different viral pathogens (hrv, rsv, and influenza virus) and 1 double infection were detected by this method (table 1) . head-to-head comparison of these 2 assays yielded an overall low concordance (55%). virtually all of the discordant results (34/35) corresponded to results that were positive by the virochip but negative by culture isolation. these results were not surprising, given the known limitations of viral culture isolation [32] . we also compared the performance of the virochip to pathogen-specific pcr, which is known for its high sensitivity. to address this in a statistically significant manner, we focused on pcr detection of hrv [27] , the most common pathogen detected in the study population. we found excellent concordance (98% agreement) between virochip and hrv-specific pcr results, indicating that virochip analysis is a highly sensitive (97%) and specific (98%) viral detection method in comparison to pathogen-specific pcr (table 2). analysis of virochip hybridization signatures for the 8 hcovs detected among the study subjects revealed 3 distinct signatures ( figure 2b) . pcr recovery and sequence analysis of a fragment of the hcov polymerase gene [29] revealed that each of these signatures corresponded to a distinct hcov subtype: (1) hcov-oc43, (2) hcov-nl63, and (3) hcov-hku1. each of these isolates shared between 97% and 100% sequence identity with the corresponding hcov polymerase gene sequences present in the ncbi database. surprisingly, the bulk of the hcovs detected in this outpatient population did not correspond to the more common hcov types (oc43 and 229e) historically detected in us adult rtis [33] . of the 8 hcovs detected in this study population, 4 were hcov-nl63, 3 were hcov-hku1, and only 1 was hcov-oc43. the different hcov types were detected in samples collected at different times during the study. all 4 of the hcov-nl63 isolates were detected in persons with colds that occurred during the early winter of 2002, whereas the hcov-hku1 isolates were detected during the late fall of 2001 (1 isolate) and the early winter of 2004 (2 isolates). the sole hcov-oc43 isolate was identified during the mid-fall of 2004. no hcovs infections were detected in this study population in 2003. interestingly, hcov-nl63 was detected only in persons with asthma, and 3 of the 4 infections were accompanied by an exacerbation of asthma symptoms. the most commonly detected virus in our study population was hrv (figures 1 and 2a and table 1). to investigate hrv diversity, we sequenced the junction of the vp4/vp2 capsid genes of the hrv isolates and performed phylogenetic analysis of these sequences and of the published sequences from all 102 hrv serotype reference strains [27] . we found that the virochip hybridization signatures we observed corresponded to ∼29 different hrvs: 16 hrva serotypes, 8 hrvb serotypes, and a novel third set of 5 divergent hrvs (referred to as hrv'x'; figure 3), which possessed slightly more sequence similarity to hrva than to hrvb reference serotypes. none of the divergent hrv'x' isolates were culturable; thus, unambiguous detection and classification of these isolates by conventional serotyping [34] , drug susceptibility [35] , or receptor-type usage assays [36] was not possible. recovery of complete coding sequence from 2 of the hrv'x' isolates (hrv'x'-1 and hrv'x'-2) and analysis of their sequence identity with a representative subset of 27 fully sequenced hrva subgroup genomes and 7 fully sequenced hrvb subgroup genomes [37] indicated that these hrv'x' isolates were indeed hrvs. scanning pairwise identity revealed that the differences between the hrv'x' and the hrva subgroup genomes were not confined to a single locus but spanned the entire genome ( figure 4) . although the vp4/vp2 phylogenetic analysis indicated that the hrv'x' isolates were more similar to hrva than hrvb reference serotype strains, comparison of the level of genomewide sequence identity shared within the fully sequenced subset of hrva genomes to the levels of sequence identity shared between the hrva and the hrv'x' isolates showed that the hrv'x' isolates were almost as genetically distinct from hrva as the hrvb subgroup genomes ( figure 4) . moreover, pairwise sequence identity between the hrv'x' genomes was much lower than that detected among the fully sequenced hrva or hrvb subgroup genomes (data not shown). taken together, these data demonstrate that these hrv'x' isolates correspond to a novel divergent subgroup of hrv and suggest that this divergent branch of hrv'x' isolates may possess a higher level of genetic diversity than seen previously in the hrva and hrvb subgroups. this is the first prospective study to use a pan-viral detection strategy to investigate the influence of viral pathogens on clinical outcome in rtis in persons with asthma. we find that, like pcr, the virochip technology is superior to standard culture isolation methods for detection of viral pathogens. moreover, the virochip exhibits comparable sensitivity and specificity to pathogen-specific pcr. on the whole, the distribution and proportion of distinct viral pathogens detected by the virochip agrees with previous pcr-based analyses of viral pathogens associated with upper rtis and those accompanied by exacerbation of asthma symptoms [3, 38] . however, virochip analysis has allowed us to uncover a remarkable amount of diversity among the viral pathogens in this relatively small study population. this diversity indicates that future studies that seek to link a particular virus or set of viruses to a discrete clinical outcome, such as exacerbation of asthma symptoms, will need to include large numbers of subjects and use pan-viral detection methods (such as the virochip) that can differentiate among such isolates. two observations of viral diversity uncovered by virochip analysis of nl specimens derived from this study are particularly noteworthy. first, the diversity and distribution of hcovs detected in the present study were surprising. the 2 more recently described hcov-hku1 and hcov-nl63 were the predominant hcovs rather than hcov-oc43 and hcov-229e, which have been traditionally implicated in up to 15% of common colds in the us adult population [32] . here, instead we see hcov-nl63 and hcov-hku1 making up approximately that same proportion of colds detected in our study population. given that hcov-nl63 and hcov-hku1 have not been implicated previously as significant players in outpatient respiratory tract illnesses among immunocompetent adults in the united states [39, 40] , these results were unexpected. no hcov-229e isolates and only a single hcov-oc43 isolate were detected in this study group, despite the fact that independent studies with the virochip have demonstrated that, when present, both hcov-oc43 and hcov-229e are readily detectable (c. y. chiu, a. urisman, t. l. greenhow, submitted). further analysis will be required to determine whether the patterns of hcovs detected here reflect an increased susceptibility of adults with asthma to contract these hcovs, the arrival of a local outbreak of these hcov types, or an actual shift in the prevalence of the distinct hcovs circulating in the us adult population. second, the virochip detected remarkable and unanticipated diversity among hrv isolates. in addition to detecting almost 30 distinct hrv species closely related to known reference hrv serotypes, we also identified a subgroup of genetically distinct hrvs in a significant fraction (5/37) of the clinical isolates of hrv. although the clinical significance of hrv diversity remains incompletely understood, the detection of such a high level of genetic divergence among hrv strains captured in this relatively small study population indicates that the standard hrv reference serotypes (which were characterized almost 30 years ago) do not adequately describe the diversity of currently circulating hrvs. we do not believe that these hrv'x' strains are an anomalous subgroup of hrvs unique to this study population because (1) a similar proportion of divergent hrv strains were detected by virochip analysis in an unrelated cohort of pediatric subjects with rtis (c. y. chiu, a. urisman, t. l. greenhow, submitted); (2) divergent hrvs have also been recently identified in a study of pediatric respiratory infections in australia [41] ; (3) based on the 5 noncoding region sequence alone, virtually identical isolates of hrv have been reported in independent analyses of hrv infections among europeans [42] ; and (4) recent independent application of a distinct pan-viral detection tool, masstag pcr analysis, has also documented a set of highly diverged hrvs circulating in the us population [43] . comparison of the vp4 sequences of the hrv'x' strains identified here suggests that one of the hrv'x' strains (hrv'x'-2) possesses high sequence similarity (98% identity with vp4 sequence dq875926) to one of the divergent hrvs recently reported by lamson et al. [43] . however, the vp4 sequences in the other 4 hrv'x' strains identified here possess !85% nucleotide sequence identity with the set of divergent hrvs identified by lamson et al. [43] . the identification of 2 distinct sets of genetically divergent hrv clinical isolates indicates that the set of previously unrecognized hrvs currently circulating in the united states may be quite large. a deeper knowledge of the extent of the current hrv diversity should inform future studies of the role played by hrv strains in asthma exacerbations, given that a high proportion of such events are attributable to infection by these agents. in sum, the present data demonstrate that the virochip captures the entire spectrum of known respiratory viral pathogens in a single test, exhibits excellent sensitivity, and provides the capacity to identify as-yet-undiscovered agents. the application of the virochip to prospective clinical studies should enable us to develop a comprehensive picture of the diversity of viral pathogens present during infection, a critical missing piece of the puzzle required to advance our understanding of how different viral pathogens influence the course and 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during rhinovirus colds in normal and in asthmatic subjects effects of experimental rhinovirus 16 infection on airway hyperresponsiveness to bradykinin in asthmatic subjects in vivo experimental rhinovirus 16 infection: effects on cell differentials and soluble markers in sputum in asthmatic subjects effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo exacerbations of asthma in adults during experimental rhinovirus infection peak expiratory flow changes during experimental rhinovirus infection experimental rhinovirus 16 infection causes variable airway obstruction in subjects with atopic asthma microarray-based detection and genotyping of viral pathogens viral discovery and sequence recovery using dna microarrays microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult identification of a novel gammaretrovirus in prostate tumors of patients homozygous for r462q rnasel variant diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a panvirus microarray the wisconsin upper respiratory symptom survey is responsive, reliable, and valid e-predict: a computational strategy for species identification based on observed dna microarray hybridization patterns cluster analysis and display of genome-wide expression patterns genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70 phylip-phylogeny inference package (version3.2) characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia basic local alignment search tool consed: a graphical tool for sequence finishing the common cold coronavirus infections in working adults: eight-year study with 229 e and oc 43 antigenic groupings of 90 rhinovirus serotypes two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity many rhinovirus serotypes share the same cellular receptor genome-wide diversity and selective pressure in the human rhinovirus rhinovirus and the lower respiratory tract coronavirus hku1 infection in the united states human coronavirus nl63, a new respiratory virus characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season masstag polymerase-chainreaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during we are grateful to shoshannah beck for providing technical support with sample processing and microarray analysis for a subset of the specimens and to anatoly urisman, kael fischer, charles chiu, and patrick tang for assistance, advice, and technical input throughout the course of this study. key: cord-315328-8g40ukml authors: clementi, nicola; ferrarese, roberto; criscuolo, elena; diotti, roberta antonia; castelli, matteo; scagnolari, carolina; burioni, roberto; antonelli, guido; clementi, massimo; mancini, nicasio title: interferon-β-1a inhibition of severe acute respiratory syndrome–coronavirus 2 in vitro when administered after virus infection date: 2020-06-19 journal: j infect dis doi: 10.1093/infdis/jiaa350 sha: doc_id: 315328 cord_uid: 8g40ukml the ongoing coronavirus disease 2019 pandemic has forced the clinical and scientific community to try drug repurposing of existing antiviral agents as a quick option against severe acute respiratory syndrome–coronavirus 2 (sars-cov-2). under this scenario, interferon (ifn) β-1a, whose antiviral potential is already known, and which is a drug currently used in the clinical management of multiple sclerosis, may represent as a potential candidate. in this report, we demonstrate that ifn-β-1a was highly effective in inhibiting in vitro sars-cov-2 replication at clinically achievable concentration when administered after virus infection. the ongoing coronavirus disease 2019 pandemic has forced the clinical and scientific community to try drug repurposing of existing antiviral agents as a quick option against severe acute respiratory syndrome-coronavirus 2 (sars-cov-2). under this scenario, interferon (ifn) β-1a, whose antiviral potential is already known, and which is a drug currently used in the clinical management of multiple sclerosis, may represent as a potential candidate. in this report, we demonstrate that ifn-β-1a was highly effective in inhibiting in vitro sars-cov-2 replication at clinically achievable concentration when administered after virus infection. keywords. sars-cov-2; ifn-β-1a; covid-19 clinical trial. the current severe acute respiratory syndrome-coronavirus 2 (sars-cov-2) pandemic is severely affecting global health, putting an unprecedented strain on health facilities worldwide. the lack of effective direct-acting antiviral drugs and of immune modulatory therapies, validated through large population studies, worsens the scenario. while waiting for specific antivirals and vaccines to be developed, the biomedical community are also focused on drug repurposing: this is the case with hydroxychloroquine, viral protease inhibitors, and several immunomodulatory drugs already in clinical use for other indications [1] . in this scenario, interferons (ifns) may also be considered, including ifn-β-1a, which has been widely used, and is still applied in some settings, for the management of relapsingremitting multiple sclerosis [2] . at this time, several articles have already suggested that type i ifns can interfere with coronavirus infections [3, 4] . in particular, the activity of ifnβ-1a has been described against sars-cov-1 both in vitro and in vivo, showing a protective effect on acute lung injury in a macaque model of infection [5, 6] . in the current study, we assessed its anti-sars-cov-2 activity in vitro to give a preclinical background to clinical trials evaluating the possible therapeutic role of ifn-β-1a in patients with coronavirus disease 2019 (covid-19). vero e6 cells (vero c1008; clone e6-crl-1586; american type culture collection) were cultured in dulbecco's modified eagle medium supplemented with nonessential amino acids, penicillin/streptomycin, hepes buffer, and 10% (vol/vol) fetal bovine serum (fbs). a clinical isolate of sars-cov-2 (hcov-19/ italy/unisr1/2020; gisaid accession no. epi_isl_413489) was obtained and propagated in vero e6 cells. virus stocks were titrated using both plaque reduction (plaqueforming units per milliliter) and end-point dilution ( median tissue culture infective dose per milliliter) assays. in plaque reduction assays, confluent monolayers of vero e6 cells were infected with eight 10-fold dilutions of virus stock. after 1 hour of adsorption at 37°c, the cell-free virus was removed. cells were then incubated for 48 hours in dulbecco's modified eagle medium containing 2% fbs and 0.5% agarose. cells were fixed and stained, and viral plaques were counted. in end-point dilution assays, vero e6 cells (4 × 10 5 per well) were seeded into 96-well plates and infected with base 10 dilutions of virus stock. after 1 hour of adsorption at 37°c, the cell-free virus was removed, and complete medium was added to cells. after 48 hours, cells were observed to evaluate the cytopathic effect (cpe). vero e6 cells were seeded into 96-well plates 24 hours before the experiment, and when at 95% confluency for each well, infected for 1 hour with sars-cov-2 at a multiplicity of infection (moi) of 0.001 [7, 8] . cells were washed with phosphate-buffered saline 1× to remove cell-free virus particles, and 200 μl of fbs-free medium containing different concentrations (5000 to 0.01 iu/ml) of ifn-β-1a (avonex; biogen idec) was added to cells. the experiment ended 96 hours after infection. the possible drug toxicity of ifn-β-1a at a concentration of 5000 iu/ml was also tested on uninfected cells. two experiments were performed in quadruplicate; live images were acquired (with an olympus ckx41 inverted phase-contrast microscope) at 48, 72, and 96 hours after infection, and cell supernatants were collected for real-time quantitative reverse-transcription polymerase chain reaction (qrt-pcr) analysis at 48 and 72 hours after infection. the sars-cov-2 rna relative amounts detected in each experimental condition as a cycle threshold (ct) value were compared, with a mean ct value determined for the positive infection control. the viral rna was purified from 140 μl of all cell-free culture supernatant, using the qiaamp viral rna mini kit (qiagen) and following the manufacturer's instructions. the purified rna was subsequently used to perform the synthesis of first-strand complementary dna, using the superscript first-strand synthesis system for rt-pcr (thermo fisher scientific), following the manufacturer's instructions. real-time pcr, using the sybr green dye-based pcr amplification and detection method, was performed to detect the complementary dna. we used the sybr green pcr master mix (thermo fisher scientific), with the forward primer n2f (tta caa aca ttg gcc gca aa), the reverse primer n2r (gcg cga cat tcc gaa gaa), and the following pcr conditions: 95°c for 2 minutes, 45 cycles of 95°c for 20 seconds, annealing at 55°c for 20 seconds and elongation at 72°c for 30 seconds, followed by a final elongation at 72°c for 10 minutes. rt-pcr was performed using the abi-prism 7900ht fast real time instrument (applied biosystems) and opticalgrade 96-well plates. samples were run in duplicate, with a total volume of 20 μl. cpe cells observed were normalized to corresponding virus infection control and used to fit a curve with nonlinear regression for half-maximal effective concentration (ec 50 ) interpolation. the qrt-pcr results were analyzed, calculating the difference in ct (δct) as the difference between ct values obtained for tested drug concentrations and for the infection control. then 2-way analysis of variance and dunnett multiple comparisons tests were performed to evaluate differences in δct means evaluated for each group. vero e6 cells were treated with concentrations ranging from 5000 to 0.01 iu/ml of ifn-β-1a 1 hour after inoculation with sars-cov-2 and monitored for cytopathic effect and real-time-pcr quantitative evaluation at 48, 72, and 96 hours after infection. inhibition of the sars-cov-2 by ifn-β-1a was dependent on both time and drug concentration. no morphological alterations related to drug toxicity was observed in uninfected cells treated with ifn-β-1a at 5000 iu/ml. in particular, cpe was assessed at 48, 72, and 96 hours after infection ( figure 1a ). first signs of cpe were already observed at the first time point, when cells were treated with low drug concentrations. marked cpe was evident at 72 hours, showing that 10 iu/ml of the drug gave full protection from virus infection, while it was inhibited only partially with lower concentrations (5 to 0.1 iu/ml). as expected, 96-hour images showed that only higher concentrations of ifn-β-1a (5000 to 50 iu/ml) completely protected cells from sars-cov-2 infection. lower tested concentrations (0.05 and 0.01 iu/ml) had no effect on hindering virus replication. data were used for ec 50 calculations at different time points, resulting in 1.971 iu/ml (95% confidence interval, .3969-4.891 iu/ml) at 48 hours, 2.071 iu/ ml (.5982-5.819 iu/ml) at 72 hours, and 4.682 iu/ml (3.505-6.018 iu/ml) at 96 hours after infection ( figure 1b) . cell supernatants collected 48 and 72 hours after infection from different cells treated with all drug concentrations were analyzed using rt-pcr. the results were fully comparable with cpe data ( figure 1c) for both time points, as ct levels detected were inversely proportional to the amount of target nucleic acid in the sample. the δct values were reported as the differences between ct values for treated and untreated cells. significant δct values were observed down to the ifn-β-1a concentration of 5 iu/ml, at 48 hours (p < .05) and especially at 72 hours (p < .001). the ct for 10 iu/ml was higher at 72 hours than at 48 hours (both p < .001), and results obtained with both 500 and 50 iu/ml concentrations were significantly different from the infection control at both time points (p < .001). several clinical trials on the administration of ifn to patients with covid-19 are currently ongoing, even without experimental preclinical evidence of anti-sars-cov-2 potential [9] (https://www.hra.nhs.uk/covid-19-research/approved-covid-19-research/281317/). among the ifns currently available for clinical use, ifn-β-1a represents an interesting option, because its pharmacological features are well known. a very recent article, just released as a preprint during the submission of the current manuscript, describes the effect of ifn-β-1a when used before infection of cells with sars-cov-2 [10] . our in vitro observations shed light for the first time on that antiviral activity of ifn-β-1a against sars-cov-2 when administered after the infection of cells, highlighting its possible efficacy in an early therapeutic setting. to this point, we detected that ifn-β-1a effectively inhibits both infectious virus particles and viral rna on treated cells, when compared to viruspositive infection control without toxicity at its highest tested concentration. moreover, the drug ec 50 evaluated at 48, 72, and 96 hours after infection can be easily accessed in the clinical setting and could therefore help in addressing drug administration regimens in vivo [11] . from this perspective, it is important to note that, in our experimental setting, ifn-β-1a activity is retained up to 96 hours after its use on the infected cells. we are aware of the limitations of this preliminary study, such as the lack of a parallel evaluation of the activity ifnβ-1a on other viruses, such as vesicular stomatitis virus, whose clinical sensitivity to the drug is well known, to establish the level of susceptibility of sars-cov-2 to type i ifn. moreover, owing to the lack of standardized phenotypic tests for this novel coronavirus, we have preferred to set the virus amount used for all assays on the cpe observed at the 3 time points (48, 72, and 96 hours after infection), rather than using a predetermined moi. hence, the antiviral activity of ifn-β-1a against sars-cov-2 was evaluated only at a single low moi in a multiple-cycle replication condition, as previously reported for sars-cov-1 [7, 8] . it would also be interesting to test the activity of ifn-β-1a on other sars-cov-2 isolates featuring different phenotypic behaviors and possibly on animal models of covid-19 to further assess, and dissect, the clinical potential of this therapeutic approach [12, 13] . this would have certainly have allowed a more complete evaluation of the clinical potential of ifn-β-1a activity in the clinical setting of covid-19. moreover, further in vitro testing on other cells of different ifns, such as ifn-λ, may complement our preliminary results, it being of extreme importance to continue supporting ifn-based clinical trials [14] . finally, we are fully aware that the preclinical evaluation of the antiviral activity of a drug, such as ifn-β-1a, is only a partial assessment of its possible clinical role in a disease such as covid-19, in which the beneficial or detrimental effect of type i ifn is still to be established and in which immune-mediated damage is probably extremely important in determining the development of the worst outcomes of the infection [15] . nonetheless, while we are surprised by the current lack of data on ifn-β-1a against sars-cov-2 in the literature, it is both urgent and clinically important to deliver data indicating whether type i may display direct antiviral activity against this virus. obviously, its antiviral potential deserves further investigation in such an atypical setting. targeting sars-cov-2: a systematic drug repurposing approach to identify promising inhibitors against 3c-like proteinase and 2'-o-ribose methyltransferase the interferon beta therapies for treatment of relapsing-remitting multiple sclerosis: are they equally efficacious? a comparative review of open-label studies evaluating the efficacy, safety, or dosing of different interferon beta formulations alone or in combination treatment of sars with human interferons type 1 interferons as a potential treatment against covid-19 interferon-β 1a and sars coronavirus replication exacerbated innate host response to sars-cov in aged non-human primates severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasome-independent inhibition of m-calpain severe acute respiratory syndrome-related coronavirus is inhibited by interferon-α interferon-a2b treatment for antiviral activities of type i interferons to sars-cov-2 infection recombinant leukocyte a interferon: pharmacokinetics, single-dose tolerance, and biologic effects in cancer patients sars-coronavirus-2 replication in vero e6 cells: replication kinetics, rapid adaptation and cytopathology animal models for emerging coronavirus: progress and new insights the effectiveness of antiviral agents with broad-spectrum activity against chikungunya virus varies between host cell lines type i interferon and hiv: subtle balance between antiviral activity, immunopathogenesis and the microbiome key: cord-007375-hqmyund4 authors: tang, yi-wei; li, haijing; wu, huiyun; shyr, yu; edwards, kathryn m. title: host single-nucleotide polymorphisms and altered responses to inactivated influenza vaccine date: 2007-10-01 journal: j infect dis doi: 10.1086/521370 sha: doc_id: 7375 cord_uid: hqmyund4 we analyzed the relationship between host gene polymorphisms and responses in recipients of inactivated influenza vaccine, who were classified into poor, normal, or adverse response groups. the frequency of the mannose-binding lectin-2 codon 54 allele was significantly different among the 3 types of responders, with a decreased odds ratio for the development of poor or adverse responses (p = .033). there was no statistical relationship between responses and either tumor necrosis factor-α or interleukin (il)-10 promoter polymorphisms among the 3 response groups. when poor and normal responses were combined, the -1082 a allele in the il-10 promoter conferred a significantly decreased risk of the development of adverse responses (p = .041). these data indicate that host polymorphisms play a role in determining responses to influenza vaccine. populations, occasional serious adverse events, in addition to local pain and tenderness at the injection site, have been noted [2] . vaccine responses are determined not only by the chemical nature of the antigen and the manner in which it is delivered but also by environmental factors and host genetic factors. identifying the predictors of individual variability in immune responses to vaccination and the factors that contribute to an increased risk for adverse reactions would enhance our understanding of vaccine responses. it has been demonstrated that host polymorphisms affect the development and progression of certain diseases and disorders by either encoding altered gene products or causing changes in transcriptional regulation. the mannose-binding lectin (mbl)-2 gene encodes a calcium-dependent protein that plays an important role in innate immunity; circulating mbl-2 levels are largely the result of several single-nucleotide polymorphisms (snps) in the exon 1 gene and promoter region. variant mbl-2 alleles have been associated with increased susceptibility to several infections [3] [4] [5] . tumor necrosis factor (tnf)-a and interleukin (il)-10 are 2 important cytokines that are associated with the regulation of cellular immune responses. several polymorphisms in their promoter regions have been shown to directly affect their gene transcription and are associated with the development and progression of autoimmune and infectious diseases [6, 7] . furthermore, the imbalance in th1/th2 cytokine production may contribute to the adverse responses induced by vaccines when natural infection occurs [8] . the present study focused on 8 host snps in 3 immunogenetic genes: codons 52 (rs503078), 54 (rs1800450), and 57 (rs1800451) in the mbl-2 exon 1 gene; ϫ238 (rs36152) and ϫ308 (rs1800629) in the tnf-a promoter region; and ϫ592 (rs1800872), ϫ819 (rs1800871), and ϫ1082 (rs1800896) in the il-10 promoter region. using immune responses and adverse events assessed in an earlier influenza vaccine trial that enrolled 5210 subjects [9] , we defined poor and normal immune responses and the presence or absence of adverse events as the variables of interest. archived serum specimens were retrieved, genomic dna was extracted, and 8 snp alleles were determined by either (1) polymerase chain reaction (pcr) followed by restriction fragment-length polymorphism (rflp) analysis or (2) real-time allele-discrimination taqman pcr. subjects who experienced either poor influenza virus-specific antibody responses or adverse events to vaccines were compared with those who had brisker immune responses and no adverse reactions. subjects, materials, and methods. a large number of healthy volunteers had been enrolled in a national institutes of health-funded, double-blind randomized controlled trial at vanderbilt university to compare the safety, immunogenicity, and efficacy of standard trivalent inactivated influenza vaccine (h1n1, h3n2, and b) with experimental bivalent cold-adapted live attenuated vaccine (h1n1 and h3n2) in the 1990s [9] . only recipients of the inactivated vaccine were included in the present study (institutional review board approval number 990325). the recipients were categorized on the basis of their vaccine-induced responses into the following groups: (1) poor responders, defined as vaccine recipients with a prevaccination hemagglutination-inhibition (hi) antibody titer of !1:16 who achieved a !4-fold increase in hi titer to both the h1n1 and h3n2 vaccine components after the first vaccination; (2) adverse responders, defined as vaccine recipients with a temperature у38.3њc after the first vaccination; and (3) normal responders, defined as vaccine recipients with a у4-fold increase in hi antibody titer to both the h1n1 and h3n3 vaccine components and a temperature of р38.3њc after the first vaccination. all subjects categorized as poor and adverse responders who had archived serum specimens available were included in the present study. approximately the same number of subjects was selected from normal responders, who were matched for age, sex, and race to the poor and adverse responders. genomic dna was extracted from serum by use of a qiaamp blood kit (qiagen), in accordance with the manufacturer's instructions [5] . dna extracted from 200 ml of serum ranged from 150 to 1000 ng (on the basis of measurement of optical density at 260 nm), and ∼20 ng of dna was used in pcr amplification. pcr was used to amplify a 119-bp region of exon 1 that contains codons 52, 54, and 57 and to detect and determine the alleles in these codons. polymorphisms were determined by rflp analysis through the digestion of pcr products with the restriction enzymes bani, mboii, and mlui (new england biolabs), followed by separation on 4% gel with ethidium bromide staining [10] . a real-time allele-discrimination pcr assay was used to detect and discriminate alleles ϫ238 and ϫ308 snp in the tnf-a promoter region and ϫ592, ϫ819, and ϫ1082 snp in the il-10 promoter region, by use of the abi prism 7700 sequence detection system (applied biosystems). pcr amplification was performed by denaturation at 95њc for 10 min, followed by 40 cycles of denaturation at 92њc for 15 s, and then annealing and extension at 62њc for 1 min. after pcr, the genotype of each sample was attributed automatically by measuring allele-specific fluorescence with the abi prism 7900 sequence detection system, by use of the sds software for allelic discrimination (version 2.2.2; applied biosystems). nucleotide sequences of primers and fluorophore taqman mgb probes were designed using primer express software (version 1.5; applied biosystems) (for tnf-a ϫ238, 5 -aaatcagtcagtggcccagaa-3 , 5 -tcattcaaccagc-ggaaaact-3 , 5 -fam-ctccctgctccgatt-mgb-3 , and 5 -vic-ctccctgctctgatt-mgb-3 ; for tnf-a ϫ308, 5 -gaaatggaggcaataggttttgag-3 , 5 -gtaggaccct-ggaggctgaac-3 , 5 -fam-ccgtccccatgcc-mgb-3 , and 5 -vic-ccgtcctcatgcc-mgb-3 ; for il-10 ϫ592, 5 -agctgaagaggtggaaacatgtg-3 , 5 -caagcagcccdifferences in age, sex, and race among the poor, normal, and adverse responders were examined using epiinfo software (version 6; centers for disease control and prevention). because very few subjects were determined to be homozygous for all 8 tested snps, all alleles with heterozygous and homozygous mutations were combined for statistical analysis. a multinomial logistic regression was performed for a global test in which the 3 response groups were analyzed simultaneously to obtain an overall p value for the 3 groups. unconditional univariate logistic regression was used to test the association between host polymorphisms and varied vaccine-induced responses as well as to estimate odds ratios (ors) and 95% confidence intervals (cis). the wild type of the snps was treated as the reference category for the or calculation. the probability of the logistic regression was modeled for an abnormal response (either poor or adverse) for mbl-2 and for an adverse response for tnfa and il-10. all p values presented in this report are 2-sided, and was considered to indicate statistical significance. p р .05 all of the calculations were done using sas software (version 9.1; sas institute). results. vaccinees enrolled in the parent vaccine trial who had received inactivated influenza vaccine and who fell into 1 of the 3 above-defined response categories were re-reviewed. all poor and adverse responders with available archived serum specimens were included. approximately the same number of subjects was selected for the normal response group, who were matched by age, sex, and race with the other 2 groups. a total of 298 subjects were included in the present study-99, 101, and 98 in the poor, normal, and adverse response groups, respectively. no significant differences were noted in age ( , , , and mean ‫ע‬ sd 30.6 ‫ע‬ 14.2 30.9 ‫ע‬ 14.3 32.3 ‫ע‬ 14.9 years), sex (proportion male, 45.5%, 48.5%, and 49.0%), or race (proportion white, 94.9%, 92.1%, and 92.9%) among the poor, normal, and adverse response groups, respectively. we first analyzed the 3 snps in the mbl-2 exon 1 gene; the frequency of each allele in codons 52, 54, and 57 is listed in table 1. the differences in each allele among the poor, normal, and adverse responders were analyzed by multinomial logistic regression. there were no significant differences in codon 52 and 57 alleles; however, a significant difference in allele frequency in codon 54 was detected ( ; ). an two host snps in the tnf-a (ϫ238 and ϫ308) and 3 in the il-10 (ϫ592, ϫ819, and ϫ1082) promoter regions were determined by real-time allele-discrimination taqman pcr. the multinomial logistic regression analysis did not discern any significant differences in allele frequency among the poor, normal, and adverse responders (table 2) . we then focused on testing our hypothesis that imbalanced th1/th2 cytokine production is associated with an adverse response, indicated by a temperature у38.4њc. we combined the subjects in the poor and normal response groups and compared them with adverse responders by unconditional univariate logistic regression analysis. in comparison to the poor/normal response group, the gra polymorphism in the il-10 promoter ϫ1082 allele indicated a significantly decreased risk for the development of adverse responses (or, 0.558 [95% ci, 0.319-0.976]) (table 2) . these data suggest that il-10 promoter polymorphisms may be associated with adverse systemic responses to influenza vaccine. discussion. in humans, the immune response to vaccination is heterogeneous despite the use of a constant formulation, route of administration, and dosage. although the majority of vaccinees generate brisk immune responses with no adverse reactions, ∼5% experience either hyporesponsiveness or adverse events [2] . the present study explored the possibility that host gene polymorphisms influence inactivated influenza vaccineinduced immune responses by comparing the frequencies of 8 snps in the mbl-2 gene and in the tnf-a and il-10 promoter regions among different groups. we found a significant difference in allele frequency in the mbl-2 codon 54 among the poor, normal, and adverse responders, suggesting that the allele polymorphism is independently associated with poor and adverse responses to influenza vaccination. mbl-2 is a member of the collectin family and is important in the initiation of the lectin pathway of complement activation and in opsonization [11] . the variant alleles in the mbl-2 gene are associated with mbl-2 deficiency, especially in individuals homozygous for the variant alleles [3] . host polymorphisms can affect the development and progression of certain diseases and disorders by encoding altered gene products, resulting in poor immune responses. the variant codon 54 allele is more prevalent than codons 52 and 57 in white populations. individuals with allele a in codon 54 demonstrate an even lower mbl-2 protein concentration than individuals with the 52 t allele, and the mbl-2 protein produced is incapable of activating the classic complement pathway [12] . of the 3 polymorphisms within the mbl-2 gene, codon 54 has independently been found to be associated with increased susceptibility to infection [4, 13] . when natural disease occurs after receipt of inactivated vaccine, th1/th2 cytokine imbalances have been demonstrated for respiratory syncytial virus infection and measles [8] . inflammatory responses must be finely tuned: too strong a response produces adverse events after vaccination, whereas too weak a response attenuates the immune responses. the th1-like tnfa is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis and development of various infectious diseases. in contrast, the th2-like il-10 is a potent anti-inflammatory cytokine that plays a role in down-regulating cell-mediated and cytotoxic inflammatory responses. several polymorphisms in the promoter regions of the genes for these 2 cytokines have been reported that affect the transcriptional regulation of the 2 genes. il-10 responses to influenza vaccination have been reported to vary significantly among age groups and vaccines [8] . we hypothesized that snps in promoter regions result in imbalanced th1/th2 cytokine production, which leads to the occurrence of adverse events after the administration of inactivated influenza vaccine. although a multivariate analysis did not show that the allele frequencies of 5 snps in the tnf-a and il-10 promoter regions among the poor, normal, and adverse response groups altered responses, a specific analysis between adverse and nonadverse (poor or normal) responses indicated that the ϫ1082 gra polymorphism in the il-10 promoter region conferred a significantly decreased risk for the development of adverse responses. previous studies have indicated that the gra snp at ϫ1082 is important in il-10 regulation; homozygous individuals (g/g) had higher il-10 expression after in vitro stimulation [11] . the snp at ϫ1082 has been associated with increased susceptibility of infection, severity of illness, organ dysfunction, and mortality [14, 15] . these findings support the present data by suggesting that the ϫ1082 allele polymorphism in the il-10 promoter region may be associated with adverse responses induced by influenza vaccine. by use of genetic sequencing of the human genome, scientists are relating polymorphisms in various host gene alleles to variable host immune responses to infectious agents and vaccines. because most previous vaccine trials have focused on humoral antibody responses, archival serum specimens are available for study. several earlier studies as well as the present one have demonstrated that substantial quantities of human genomic dna are present in such clinical samples as serum and cerebrospinal fluid [5] . with appropriate approval by institutional review boards, retrospective studies using the large quantity of archived serum specimens from previous vaccine trials and prospective studies can be conducted to assess many genetic factors. there were several limitations to the present study, including (1) the small quantities of dna available from serum specimens, (2) only 1 adverse event (fever) was assessed, and (3) a small number of host snps and not the entire genome were assessed. in spite of these limitations; however, this study suggests that additional studies should be conducted to confirm these findings. influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly guillain-barre syndrome and the 1978-1979 influenza vaccine association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis mannose-binding lectin in severe acute respiratory syndrome coronavirus infection analysis of candidate-host immunogenetic determinants in herpes simplex virus-associated mollaret's meningitis variation in the tnf-alpha promoter region associated with susceptibility to cerebral malaria association of tnf2, a tnf-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study responses to influenza vaccination in different t-cell subsets: a comparison of healthy young and older adults a randomized controlled trial of cold-adapted and inactivated vaccines for the prevention of influenza a disease mannose-binding lectin (mbl) deficiency: variant alleles in a midwestern population of the united states hutchinson iv. an investigation of polymorphism in the interleukin-10 gene promoter distinct and overlapping functions of allelic forms of human mannose binding protein mannose-binding lectin gene polymorphism is a modulating factor in repeated respiratory infections association of il-10 polymorphism with severity of illness in community acquired pneumonia pneumococcal septic shock is associated with the interleukin-10-1082 gene promoter polymorphism we thank jennifer doersam, jamie rickmyre, sandra yoder, shufang meng, jody peters, amondrea blackman, pam palmer, sharon tollefson, and william dupont for their excellent technical assistance and barney graham, david persing, and bonnie lafleur for their helpful suggestions and review of the research proposal and/or manuscript. key: cord-296197-ohfhnpma authors: deborggraeve, stijn; laurent, thierry; espinosa, diego; van der auwera, gert; mbuchi, margaret; wasunna, monique; el-safi, sayda; al-basheer, ahmed almustafa; arévalo, jorge; miranda-verástegui, césar; leclipteux, thierry; mertens, pascal; dujardin, jean-claude; herdewijn, piet; büscher, philippe title: a simplified and standardized polymerase chain reaction format for the diagnosis of leishmaniasis date: 2008-11-15 journal: j infect dis doi: 10.1086/592509 sha: doc_id: 296197 cord_uid: ohfhnpma background. definite diagnosis of leishmania infections is based on demonstration of the parasite by microscopic analysis of tissue biopsy specimens or aspirate samples. however, microscopy generally shows low sensitivity and requires invasive sampling. methods. we describe here the development of a simple and rapid test for the detection of polymerase chain reaction-amplified leishmania dna. a phase 1 evaluation of the text was conducted in clinical samples from 60 nonendemic and 45 endemic control subjects and from 44 patients with confirmed cutaneous leishmaniasis (cl), 12 with mucocutaneous leishmaniasis (mcl), and 43 with visceral leishmaniasis (vl) from peru, kenya, and sudan. results. the lower detection limits of the assay are 10 fg of leishmania dna and 1 parasite in 180 µl of blood. the specificity was 98.3% (95% confidence interval [ci], 91.1%–99.7%) and 95.6% (95% ci, 85.2%–98.8%) for nonendemic and endemic control samples, respectively, and the sensitivity was 93.2% (95% ci, 81.8%–97.7%), 91.7% (95% ci, 64.6%–98.5%), and 86% (95% ci, 72.7%–93.4%) for lesions from patients with cl or mcl and blood from patients with vl, respectively. conclusions. the leishmania oligoc-test showed high specificity and sensitivity in clinical samples and was able to detect the parasite in samples obtained by less invasive means, such as blood, lymph, and lesion scrapings. the assay is a promising new tool for simplified and standardized molecular detection of leishmania parasites. leishmaniasis is a vectorborne disease caused by protozoa of the genus leishmania and is endemic in many areas of the tropics, subtropics, and mediterranean basin [1] . clinical presentations, depending on the infecting species and the immune status of the patient, range from self-healing skin sores to devastating cutaneous and mucocutaneous ulcers and the lethal visceral form [2] . the disease is re-sponsible for important public health and economic problems in affected regions [3] . definite diagnosis of leishmania infections is currently based on microscopic demonstration of the parasites in skin biopsy specimens or mucosal aspirate samples for cutaneous leishmaniasis (cl) and mucocutaneous leishmaniasis (mcl) and in spleen or bone marrow aspirate samples for visceral leishmaniasis (vl) [1] . however, this conventional method is hampered by its low and variable sensitivity and the need for invasive sampling techniques. sensitivity may be increased by prior in vitro cultivation of the parasite, but this technique is cumbersome and time consuming. serologic tests, such as the direct agglutination test (dat) [4] , support the clinical diagnosis of vl, but antibodies remain detectable for years after successful treatment [5, 6] . furthermore, serologic tests are less useful in patients with vl coinfected with hiv [7] and in those with cl or mcl [8] . amplification of the parasite dna by the polymerase chain reaction (pcr) has evolved into one of the most specific and sensitive methods for leishmania detection [9, 10] . despite its promising features, pcr is restricted to wellequipped laboratory settings, partly due to technical complexity, from dna extraction to pcr and product detection. there is a need for simplification, and a first step concerns the visualization of amplified dna, which is usually identified using electrophoresis followed by ultraviolet transillumination in the presence of the carcinogenic ethidium bromide. safer nucleic acid stains based on fluorescent dyes are much more expensive and not routinely used in most laboratories. alternative methods for pcr product detection, such as real-time pcr [11] , pcr-elisa [12, 13] , and mass spectrometry [14] , have been developed but remain complex, expensive, and highly dependent on specific equipment. along with the need for simplification of the pcr assay, there is a demand for standardization and optimization [10] . at present, standardization of pcr for leishmania detection is largely neglected, and the abundance of in-house pcr assays may lead to diagnostic inconsistencies. oligochromatography provides a simple and rapid dipstick format for detection of pcr products (coris bioconcept; patent wo 2004/099438a1) [15] . pcr products are visualized on the dipstick by hybridization with a gold-conjugated probe, allowing sequence-specific pcr product detection. this detection format takes only 5-10 min and requires no equipment other than a water bath and a pipette. internal controls for amplification and chromatographic migration are incorporated in the assay. the technique has already been successfully applied for the diagnosis of human african trypanosomiasis [16] , toxoplasmosis [17] , and severe acute respiratory syndrome [18] , and high sensitivity and specificity were reported. we present here the development and evaluation of a leishmania-specific pcr-oligochromatographic test (leishmania oligoc-test), targeting a short sequence within the leishmania 18s rrna gene. after providing proof of concept in experimental samples, we evaluated (phase 1) the leishmania oligoc-test in a larger series of clinical samples from nonendemic and endemic control subjects and patients with cl, mcl, or vl. dna from leishmania species (l. donovani, l. amazonensis, l. infantum, l. braziliensis, l. peruviana, l. guyanensis, l. panamensis, l. mexicana, l. major, l. lainsoni, l. tropica, and l. aethiopica) and from trypanosoma brucei gambiense and trypanosoma cruzi was obtained from the dna reference bank at the institute of tropical medicine antwerp (itma). dna from other pathogens (sporothrix schenckii, plasmodium falciparum, mycobacterium tuberculosis, and schistosoma mansoni) was obtained from other research groups. informed consent was obtained from patients or guardians and from persons without disease. the human and animal experimentation guidelines of the itma were followed. ethical clearance for the study was obtained from the institutional review boards in belgium, kenya, sudan, and peru. spiked blood. l. donovani promastigotes were grown in glucose-lactalbumin-serum-hemoglobin medium [19] with 10% fetal calf serum at 26°c. a 10-fold dilution series of parasites, ranging from 10,000 parasites to 1 parasite per 180 l of blood, was made in fresh blood obtained on edta from a healthy volunteer. nonspiked blood was used as a negative control. nonendemic controls. dna extracts from the blood of 20 persons with confirmed t. brucei gambiense infection from the democratic republic of the congo (drc), 20 with confirmed p. falciparum infection from zambia, and 20 with confirmed t. cruzi infection from chile were obtained from other research groups. t. brucei gambiense and p. falciparum infections were confirmed by direct parasite detection with microscopy, and t. cruzi infections were confirmed by kinetoplast dna pcr followed by southern blot hybridization [20] . patients with cl or mcl and healthy endemic controls (peru). lesion biopsy specimens from 36 patients with cl, mucosal aspirate samples from 12 patients with mcl, and lesion scrapings from 8 patients with cl were collected in 2006 and 2007 at the instituto de medicina tropical alexander von humboldt, lima, peru. cl and mcl were confirmed by means of microscopy and/or cultivation in 3 ml of novy-macneal-nicolle medium (difco laboratories) containing 15% defibrinated rabbit blood. forty-one of the 56 cl and mcl lesion samples had a positive direct microscopy result, and the remaining 15 became positive after culture. all biopsy specimens were obtained from the active edge of the lesion with a sterile 4-mmdiameter biopsy puncher, and simple sterile lancets were used for the lesion scrapings. dental biopsy specimens from 8 healthy endemic control subjects were collected in 2007 in a dental clinic in lima, peru. biopsy specimens and lesion scrapings were stored in absolute ethanol at ϫ20°c until dna was extracted. patients with vl and healthy endemic controls (kenya and sudan). sample collections in kenya and sudan were performed by the kenya medical research institute and khartoum university, respectively. blood samples from patients with confirmed vl and from healthy endemic control subjects were collected in 2007 in the baringo district in kenya (25 vl and 18 control samples) and gedarif state in sudan (18 vl and 19 control samples). a patient was classified as having confirmed vl if parasites were observed during microscopic analysis of lymph or bone marrow aspirate samples for sudan and of spleen aspirate samples for kenya. a person was classified as a healthy endemic control if there was no clinical suspicion for vl and if the dat titer was below 1:3200 for sudan and 1:12,000 for kenya (threshold values used routinely at the 2 institutions). along with the blood samples, bone marrow from 12 and lymph from the remaining 6 of the 18 sudanese patients with confirmed vl were collected for subsequent analysis with the leishmania oligoc-test; 200 l of blood, bone marrow, or lymph was used for dna extraction. dna from spiked blood samples and from biopsy specimens and lesion scrapings was extracted using the qiaamp dna blood mini kit and the qiaamp dna mini kit (qiagen), respectively. dna from blood, bone marrow, and lymph node samples from patients with vl and healthy endemic controls was extracted according to the method of boom et al. [21] . dna was eluted in 50 l of pure water or tris-edta (te) buffer and stored at ϫ20°c, except for the sudanese clinical samples, which were eluted in 100 l of te buffer. primers, probes, and ic dna were synthesized by biomers.net (figure 1). sequences of the 18s rrna gene of the trypanosomatid parasites l. donovani (genbank accession number x07773), t. brucei gambiense (genbank accession number aj009141), and t. cruzi (genbank accession number af303660) were aligned and leishmania-specific primers were designed using dnaman software, version 5.0 (lynnon). the sense primer 18s-l-f and the antisense primer 18s-l-r amplify a 115-bp sequence within the 18s rrna gene of leishmania. the ic dna has the same length as the leishmania target sequence. both sequences are identical except for a 17-bp central part. the detection probe was designed, synthesized, and conjugated with gold particles using the procedure described in patent wo 2004/099438a1 [15] . the leishmania capture probe and the ic capture probe, which hybridize to the central part of the leishmania amplicon and the ic dna amplicon, respectively, were synthesized and biotinylated at the 5' end. the 5'-acg and 5'-a linkers were added to avoid steric hindrance by the biotinavidin binding during hybridization. the migration control se-quence is the reverse complement of the detection probe sequence. the 50-l pcr mixture was prepared by adding 5 l of sample dna and 1 u of hotstar taq polymerase (qiagen) to 44.8 l of leishmania ampli-mix (coris bioconcept). this premade pcr mix contains all components to allow pcr amplification, the primers 18s-l-f and 18s-l-r and the ic dna at a concentration of 3.2 ϫ 10 ϫ18 mol/l. the commonly used dttp was replaced by dutp to allow elimination of carryover contamination with uracil-dna n-glycosylase (ung). an initial denaturation step at 94°c for 15 min to activate the hotstar taq polymerase was followed by 40 cycles of 94°c for 30 s, 60°c for 1 min, 72°c for 1 min, and a single final extension at 72°c for 5 min. amplification was done in 200-l thin-wall pcr tubes (abgene) in a t3 thermocycler 48 (biometra). preparation. the leishmania oligo-strip was constructed as described by deborggraeve et al. [16] but with the following modifications. the lower absorbent pad on the test side was impregnated with the detection probe and the leishmania capture probe. the lower absorbent pad on the control side was impregnated with the detection probe and the ic capture probe. on both sides of the nitrocellulose membrane, 2 lines were coated, a line with neutralite avidin (belovo sa) and a line with the migration control probe. principle. a schematic overview of the leishmania oligoc-test principle is presented in figure 2 . the assay was repeated when the control lines indicated migration or pcr failure. assay procedure. after pcr amplification, the pcr product was denaturated at 94°c for 30 s and transferred immediately to ice. then, 40 l was mixed with an equal volume of migration buffer preheated at 55°c, and the leishmania oligo-strip was dipped into the solution. test results were read after 10 min. (1) is performed using a pcr mix containing single-stranded internal control (ic) template (2) . this ic template is amplified with the same primers as the leishmania dna target but contains a specific internal sequence (dotted line). when the pcr and subsequent denaturation is completed, the pcr product solution contains single-stranded leishmania and ic dna (3) and is mixed with an equal volume of migration buffer preheated at 55°c. the leishmania oligo-strip is dipped into the solution, and test results are read after a 10-min migration at 55°c (4). during migration, the solution takes up the impregnated probes: the gold-labeled detection probe (5) hybridizes with both types of amplicons, while the biotinylated leishmania capture probe on the test side (6) and the biotinylated ic capture probe on the control side (7) hybridize with their respective amplicons. gold and biotin labeling is indicated by white circles and black circles, respectively. the biotinylated capture probes accumulate the hybridized complex on the neutralite avidin lines on both sides of the strip, resulting in visible red lines (8) . for a negative sample, only the ic amplicon is present and is captured on the control side (9) . the excess detection probes migrate further and hybridize on the complementary probes coated on both sides of the dipstick as a control for migration (10) . the ic line and the migration control lines determine whether the test is valid or invalid. an invisible migration control line indicates an invalid detection step, while an invalid pcr is indicated by a negative ic line in combination with a negative test line. the latter is possibly due to inhibitory factors in the extracted dna. when a sample contains high concentrations of leishmania dna, competition between the leishmania dna and the ic template dna during pcr can result in an invisible ic control line combined with a strongly visible leishmania test line. in this case, the test is considered valid. accurate detection of the parasite is of utmost importance in the diagnosis of leishmania infections and in disease control. we here report on the development, proof-of-concept analysis, and phase 1 evaluation of a simplified molecular test for detecting leishmania organisms in clinical samples. the leishmania oligoc-test is based on pcr amplification of leishmania dna followed by simple and rapid detection of the pcr product in dipstick format. the lower detection limits of the assay were evaluated in l. donovani promastigotes, and 10 fg of purified dna and 1 parasite in 180 l of human blood could be detected. fluctuations in the analytical sensitivity between different isolates might be expected. in 1998, inga et al. [23] reported a lower copy number of the rrna genes in l. (viannia) peruviana from southern peru compared with l. (v.) peruviana from northern peru. in our study, however, we did not observe a significantly lower sensitivity of the leishmania oligoc-test in 13 patients originating from l. (v.) peruviana-endemic regions in southern peru, because only one of these patients had a negative test result. no cross-reaction with nontarget human pathogens was observed, and all leishmania species tested had a positive test result. considering the high similarity of the 18s rrna gene sequence in leishmania and endotrypanum, crithidia, wallaceina, and leptomonas organisms [24] , our assay might have positive results with these nonpathogenic lower trypanosomatids. although the chances of finding such protozoa in immunocompetent patients are negligible, they may show up as opportunistic infections in immunocompromised patients [25] . if needed, confirmation could be provided by direct sequencing of the pcr product, because leishmania-specific point mutations are present (authors' unpublished data). the leishmania oligoc-test showed a high diagnostic specificity when tested in 60 nonendemic control blood samples from persons infected with t. brucei gambiense, p. falciparum, or t. cruzi. one t. brucei gambiense-infected person from the drc had a positive leishmania oligoc-test result. we cannot exclude the possibility that this could be due to a leishmania infection, because vl has been occasionally reported in the drc [26, 27] . when evaluated in 56 clinical samples from patients with confirmed cl or mcl (definition based on microscopy and/or culture) collected in peru, the leishmania oligoc-test showed an overall diagnostic sensitivity of 92.9%, whereas all 8 dental biopsy specimens from healthy control subjects were negative. this finding is similar to the observed sensitivities of other pcr assays reviewed by vega-lópez [8] . direct microscopy of the lesion showed a lower sensitivity (78.8%). the lack of a positive pcr result in some of the confirmed cl and mcl lesion samples might be explained by (1) heterogeneity in parasite distribution in the lesion, (2) delay between sample collection and testing, or (3) false-positive microscopic results caused by staining artifacts. this is supported by the fact that 3 of the 4 leishmania oligoc-test negative samples were also negative in a second pcr analysis performed in peru targeting the leishmania kinetoplast dna [28] (authors' unpublished data). leishmania dna could be detected in all lesion scrapings, which is encouraging, because this offers a less invasive sampling procedure compared with biopsy punctures. the higher sensitivity for lesion scrapings can be explained by the lower amount of pcr-inhibiting factors. this is confirmed by the observation of garcia et al. [29] , who reported a higher sensitivity of pcr for lesion scrapings than for biopsy specimens in 44 bolivian patients with cl or mcl. the similar sensitivities of our method for cl and mcl samples is encouraging, considering the generally low parasite load in the latter. the sensitivity of 86% in blood from patients with confirmed vl is promising and suggests that the oligoc-test can contribute significantly to less invasive vl diagnosis. patients with clinically suspected infection who have positive serologic results and positive leishmania oligoc-test results in blood might not need to undergo the invasive bone marrow or spleen sampling. previous pcr studies found the same range of sensitivity in blood [9, 30] . the lower sensitivity of the test in blood samples from sudan (77.8%) versus those from kenya (92%) could be due to bias in parasite load or experimental differences during dna extraction. further evaluations with different types of extraction methods might be recommended. despite the low number of available lymph node samples, the observation that all of them showed positive leishmania oligoc-test results is encouraging. indeed, if molecular diagnosis in lymph nodes proves to have high sensitivity, this would be a major step forward in the search for a noninvasive method for diagnosing vl. previous studies showed similar findings in a larger number of lymph node samples [31, 32] . further studies of molecular diagnosis in the lymph would be interesting, especially for east african vl, for which the majority of patients have enlarged lymph nodes [33] . two of 37 dat-negative blood samples from kenyan and sudanese endemic control subjects were positive with the leishmania oligoc-test. although pcr contamination can never be 100% excluded and low sensitivity of the dat in sudan has been reported [34] , these positive results in the healthy control group are probably due to asymptomatic infections. in vl-endemic regions, there is a high prevalence of asymptomatic infections, which are generally not treated because the available drugs are very toxic. therefore, the interpretation of pcr results should always take into account possible asymptomatic infections [35] . multiple attempts to simplify and speed up the detection of pcr products have been reported, such as pcr-elisa [12, 13] and fluorescent self-probing amplicons [36] , but none of them merges simplicity and speed with "low-tech" approaches. the leishmania oligoc-test combines the sensitivity and specificity of pcr with the simplicity and speed of membrane oligochromatography. detection of pcr products is performed in 10 min and does not require any equipment except for a water bath and a pipette. the cost of the leishmania ampli-mix components is similar to that of conventional pcr mix, and the cost of the oligo-strip is much lower than that of commercial agarose for the same number of samples. the global cost of diagnosis is reduced with the oligoc-test, because the internal amplification control eliminates the need for additional pcrs to check inhibition. a common drawback of pcr in the diagnostic process is carryover contamination leading to false-positive results. the implementation of the dutp/ung decontamination system and the single-step detection format of the leishmania oligoc-test facilitate contamination control. presentation of the leishmania oligoc-test as a self-containing kit with a ready-to-use pcr mix and dipsticks will allow quality control and standardization. however, sampling and dna extraction are also important steps for accurate molecular diagnosis and need to be standardized. the described tool may help in the rapid and simple molecular diagnosis of leishmaniasis and in epidemiological studies for which a sensitive marker of infection is required, although the current format is not directly designed for high-throughput applications. there is a need for further phase 2 and 3 studies to estimate the diagnostic accuracy of the leishmania oligoc-test in patients with suspected infection and with positive serologic test results, healthy endemic controls, and patients with other pathologic conditions. furthermore, a multicentric study in which different pcr methods are evaluated on the same samples would be highly valuable. in addition, a standardized oligochromatographic format for identification of leishmania species would be welcome, given the high importance of accurate species identification in patient management, disease control, and epidemiological studies. recently, a first prototype to discriminate between l. infantum and l. donovani by means of the oligochromatographic approach has been developed (t. laurent, g.v.d.a., m. hide, et al., unpublished data). the combination of the dipstick with novel isothermal nucleic acid amplification techniques, such as nucleic acid sequence-based amplification [37, 38] and loop-mediated isothermal amplification [39] , may circumvent the need for thermocycling reactions and therefore further simplify molecular diagnosis. clinical spectrum of leishmaniasis leishmaniasis and poverty operational validation of the direct agglutination test for diagnosis of visceral leishmaniasis immunologic tests in patients after clinical cure of visceral leishmaniasis pre-and post-treatment antibody levels in visceral leishmaniasis contribution of serological tests to the diagnosis of visceral leishmaniasis in patients infected with the human immunodeficiency virus diagnosis of cutaneous leishmaniasis clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in hiv-infected and hiv-uninfected patients: a single-center, 8-year experience in italy and review of the literature molecular diagnosis of leishmaniasis: current status and future applications real-time pcr in clinical microbiology: applications for routine laboratory testing the high sensitivity of a pcr-elisa in the diagnosis of cutaneous and visceral leishmaniasis caused by leishmania infantum a new pcr-elisa for diagnosis of visceral leishmaniasis in blood of hiv-negative subjects rapid identification of emerging pathogens: coronavirus one step oligochromatographic device and method of use molecular dipstick test for diagnosis of sleeping sickness dna extraction and pcr assays for detection of toxoplasma gondii detection of sars-cov by oligochromatography of rt-pcr amplicons structures antigéniques de trypanosoma brucei (protozoa, kinetoplastida) use of polymerase chain reaction (pcr) and hybridization assays to detect trypanosoma cruzi in chronic chagasic patients treated with itraconazole or allopurinol rapid and simple method for purification of nucleic acids probable inference, the law of succession, and statistical inference relation between variation in copy number of ribosomal rna encoding genes and size of harbouring chromosomes in leishmania of subgenus viannia phylogeny of trypanosomatidae and bodonidae (kinetoplastida) based on 18s rrna: evidence for paraphyly of trypanosoma and six other genera lower trypanosomatids in hiv/aids patients case of leishmaniasis in the congo autochtonous visceral leishmaniasis in zaire diagnosis of leishmania via the polymerase chain reaction a simplified procedure for field work american tegumentary leishmaniasis: direct species identification of leishmania in non-invasive clinical samples leishmaniasis in sudan. visceral leishmaniasis evaluation of pcr for diagnosis of visceral leishmaniasis molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification kala-azar in displaced people from southern sudan: epidemiological, clinical and therapeutic findings diagnostic tests for kala-azar: a multi-centre study of the freeze-dried dat, rk39 strip test and katex in east africa and the indian subcontinent diagnostic accuracy of a new leishmania pcr for clinical leishmaniasis in nepal and its role in diagnosis of disease detection of pcr products using self probing amplicons and fluorescence nasba and other transcription based amplification methods for research and diagnostic microbiology quantitative nucleic acid sequence-based assay as a new molecular tool for detection and quantification of leishmania parasites in skin biopsy samples loop-mediated isothermal amplification of dna we kindly acknowledge drs. c. grevelding, f. portaels, d. mumba, u. d'alessandro, and a. solari for providing dna from schistosoma mansoni, mycobacterium tuberculosis, trypanosoma brucei gambiense, plasmodium falciparum, and trypanosoma cruzi, respectively. we thank dr. h. schallig and g. schoone for providing the material for the extraction method of boom et al. and dr. alejandro llanos-cuentas and hamad el-neel el-taib for assistance with the projects in peru and sudan, respectively. key: cord-279725-d82sj80v authors: ströher, ute; dicaro, antonino; li, yan; strong, james e.; aoki, fred; plummer, frank; jones, steven m.; feldmann, heinz title: severe acute respiratory syndrome-related coronavirus is inhibited by interferon-α date: 2004-04-01 journal: j infect dis doi: 10.1086/382597 sha: doc_id: 279725 cord_uid: d82sj80v current treatment schemes for severe acute respiratory syndrome (sars) include broad-spectrum antibiotics, glucocorticoids, and ribavirin. we evaluated the susceptibility of the sars-related coronavirus (sars cov) to ribavirin and interferon (ifn)-α in vitro by use of cytopathic effect, plaque assay, and immunoblot analysis. ribavirin did not inhibit viral growth at concentrations attainable in human serum. in contrast, ifn-α showed an in vitro inhibitory effect starting at concentrations of 1000 iu/ml. in conclusion, ribavirin alone is unlikely to be beneficial in the prophylaxis or treatment of sars cov infections. clinical trials with ifn-α might be justified to determine a beneficial effect on the outcome of sars. lishing an antiviral treatment for patients with sars. sars is primarily diagnosed by a process of clinical/epidemiological exclusion. despite improvement in the recent past, laboratory diagnosis, particularly molecular detection of the virus by polymerase chain reaction, remains unreliable, especially in the first few days of the disease; in addition, the tests are not yet validated. serologic analysis is thought to be the confirmatory laboratory test, but most patients with sars develop detectable igg antibody levels 3-4 weeks after the onset of symptoms. thus, differential diagnosis remains a problem for the clinician at the time of initial presentation, particularly in individuals without known exposure to other patients with sars or history of travel to a region where sars is endemic. the case-fatality rate of 9.5% reflects, in part, the lack of an effective specific treatment for this viral infection. broad-spectrum antibiotics to treat community-acquired bacterial pneumonia, glucocorticoids, and ribavirin have been administered to patients with sars, but their efficacy is unknown [3] [4] [5] . recently, glycyrrhizin has been demonstrated to inhibit the sars-related cov (sars cov) in vitro [6] . ribavirin has been used on the basis of its ability to inhibit other covs [7] and also showed an inhibitory effect on several other rna viruses, such as bunyaviruses and arenaviruses [8] . to support the search for effective antiviral treatments, we evaluated the susceptibility of sars cov isolates (detailed studies were performed with the tor2 isolate [toronto, canada]) to ribavirin and interferon (ifn)-a-2b in vitro. our data indicate that ribavirin does not inhibit the virus at concentrations attainable in human serum but that ifn-a-2b may be useful and deserves further evaluation as a therapeutic agent. materials and methods. studies were performed with the recently sequenced tor2 isolate, obtained from a toronto patient [1, 4] . the general findings were confirmed by use of 3 additional independently obtained isolates from toronto patients (tor3, tor7, and tor684). all isolates originated from nasopharyngeal swabs. vero e6 cells (atcc 1568) were used for the susceptibility studies. the cells were maintained in dulbecco's modified eagle's medium (dmem) containing 10% fetal bovine serum (gibco brl). vero e6 cells were infected at an moi of 0.001 with the sars cov. after 1 h of adsorption, the supernatants were replaced with dmem containing various amounts of ribavirin (0-2000 mg/ml) or ifn-a-2b (0-5000 iu/ml) (schering-plough) for 72 or 96 h. for immunoblot analysis, cell lysates were analyzed by sds-page on 10% tris gels. protein was electrotransferred to polyvinylidene difluoride membrane (immobilon p membrane; millipore). viral antigen was detected by use of enhanced chemiluminescence, using a patient serum sample and horseradish peroxidase-conjugated anti-human igg. for plaque assay, confluent vero e6 cells were infected in 10fold dilutions of sars cov; 45 min after infection, the inoculum was removed, and cell monolayers were overlaid with 0.9% lowmelting-point agarose in dmem, with or without ifn-a-2b. titers were determined 72 h after infection, following crystal violet staining. for microassay, vero e6 cells were infected with sars cov at an moi of 0.001 for 45 min at 37њc and then were incubated in dmem containing various amounts of ifn-a-2b (0-5000 iu/ml); 72 h after infection, cells were formalin fixed, removed from biocontainment, and analyzed by use of phase-contrast microscopy, using an axiovert 200 microscope (zeiss). results. in vitro studies were conducted in vero e6 cells, which is one of only a limited number of cell lines susceptible to sars cov. vero e6 cell monolayers were treated with ribavirin at various concentrations (0, 20, 50, 100, 200, 1000, and 2000 mg/ml) for 96 h. treatment was initiated concurrently with inoculation of the cell cultures with virus at an moi of 0.001. inhibition of viral cytopathic effect (cpe) was used to determine the antiviral effect of the drug. no reduction in cpe was detected after 4 days, compared with untreated control samples. viral titers determined from tissue culture supernatant were identical for treated and untreated cells and amounted to ∼10 6 pfu/ml (data not shown). ribavirin was tested up to concentrations 10 times greater (2000 mg/ml) than those that inhibit the replication of ribavirin-sensitive viruses, such as arenaviruses and bunyaviruses [9, 10] . there was no evidence that the sars cov was susceptible to the action of ribavirin. ifn susceptibility has been demonstrated for several coronaviruses, such as mouse hepatitis virus, porcine transmissible gastroenteritis virus, feline infectious peritonitis virus, and human coronavirus [11] [12] [13] [14] . inhibitory concentrations ranged from 2 iu/ml [12] to 1000 iu/ml [14] . therefore, we tested the susceptibility of the tor2 isolate to ifn-a-2b (schering-plough). in brief, vero e6 cells were infected at an moi of 0.001, either 20 h after or at the time of treatment with ifna-2b (250-5000 iu/ml). treatment was continued for 72 h. the cpe was used to demonstrate an antiviral effect. as indicated in figure 1 , the cpe on day 3 after infection was significantly decreased in tor2-infected cultures treated with 1000 iu/ml of ifn-a-2b in both treatment groups, compared with that in the untreated control group. this indicated that pretreatment was not necessary for the protective effect in tissue culture (data not shown). almost complete protection was achieved when infected vero e6 cells were treated with 5000 iu/ml of ifn-a-2b. to quantify the effect of ifn-a-2b on the replication of the sars cov, vero e6 cells were infected at an moi of 0.001 and were incubated in the presence ifn-a-2b (0-5000 iu/ml), as described above. after 72 h, cells and supernatants were harvested to determine protein expression by immunoblot analysis and virus yield by plaque titration on vero e6 cells. as indicated in figure 2a , a concentration of 1000 iu/ml of ifn-a-2b substantially reduced virus yields, and ∼1 log decrease in virus yield was achieved at a dose of 2000 iu/ml of ifn-a-2b. the reduction in virus yield seems to be related to a decrease in virus growth, as supported by the change in the plaque morphology ( figure 2b ). inhibition of viral protein expression was investigated by use of immunoblot analysis after sds-page of the cell lysates ( figure 2c ). a serum sample from a patient with sars who seroconverted to the sars cov was used to detect viral proteins. earlier studies had shown that this serum sample reacted strongly with the nucleocapsid protein of the sars cov [15] . viral protein expression was reduced at a concentration of 1000 iu/ml and abolished at concentrations of 2000 and 5000 iu/ml of ifn-a-2b. for confirmation, the effect of ifn-a-2b on 3 additional independent sars cov isolates (tor3, tor7, and tor684) obtained from canadian patients with sars was determined. all isolates showed the same pattern of inhibition by ifn-a-2b as . b, analysis of cell-to-cell spread of sars cov in the presence and sd absence of ifn-a-2b. vero e6 cell monolayers were infected with 10fold dilutions of the sars cov. after 1 h of virus adsorption, the inoculum was removed, and cell monolayers were overlaid with 0.9% low-meltingpoint agarose in dmem (b2) or dmem containing 5000 iu/ml of ifna-2b (b1). on day 3 after infection, cells were stained with crystal violet. c, protein analysis. cell lysates were subjected to 10% sds-page. protein was electrotransferred to polyvinylidene difluoride membrane (immobilon p membrane; millipore). viral antigen was detected by use of a patient serum sample and horseradish peroxidase-conjugated anti-human igg, by use of enhanced chemiluminescence. np, nucleoprotein. d, comparison of the effect of antiviral activity on different sars cov isolates. vero e6 cells were infected with 4 independently isolated sars covs (isolates tor2, tor3, tor7, and tor684) and were analyzed for their sensitivity to ifn-a-2b, as described above. the tor2 isolate ( figure 2d ). for all isolates, the ic 50 of ifna-2b was ∼500 iu/ml. the present study has demonstrated that the sars cov is not susceptible to ribavirin at high concentrations, even when added at the time of infection. in contrast, the virus is inhibited in tissue culture by ifn-a-2b at concentrations у1000 iu/ml. although the relationship between in vitro and serum ifn concentrations and biological effect is not known, peak serum ifn concentrations of at least 500 iu/ml are observed after intramuscular (im) injection of iu/ml 7 3.0 ϫ 10 [16] . similar ifn serum concentrations (100-750 iu/ml) after im injection were reported from a study in patients with hepatitis c virus (hcv)-associated systemic vasculitis [17] . doses up to iu/ml have been infused intravenously, which 7 3.6 ϫ 10 might achieve serum concentrations in the range observed to inhibit the sars cov. at present, there is no data on the serum levels of ifn-a in patients with sars, which would be helpful before treatment. the in vitro studies have thus far only been performed in vero e6 cells. this is significant, because vero e6 cells have been shown to have an ifn i gene deficiency and thus are unable to express endogenous ifn i [18] . however, the ifndependent pathways are functional and can be activated by exogenously provided ifn. we have tested a limited number of different cell lines (hela, 293t, 3t3, and crfk) for susceptibility to the sars cov; thus far, we have failed to identify another line that supports viral replication (data not shown). the observation that the vero cell clone e6 propagates the virus may indeed be related to the lack of a functional ifn system and, thus, supports the findings in the present study. despite efforts by our group and others to grow the virus in different rodent species, a small animal model has not yet been established. recently, cynomolgus macaques (macaca fascicularis) have been infected with the sars cov, which induced similar clinical symptoms and pathological findings to those of humans with sars [19] . because only a limited number of animals have been used in the present study, it remains to be determined in the future whether the cynomolgus macaque is a suitable model for sars and, thus, for antiviral in vivo studies. until an animal model is definitively established, it might be justified to consider clinical trials with ifn, which is already approved for human use, to determine whether ifn has a beneficial effect on the outcome of sars cov infection. combined with the observation of a lack of efficacy of ribavirin in patients with sars, our in vitro data further support the conclusion that ribavirin, at least alone, is unlikely to be beneficial in the prophylaxis or treatment of sars cov infection. whether combined therapy with ifn-a-2b and ribavirin would inhibit the replication of the sars cov in vitro has not yet been evaluated; the combination is more effective than either agent used alone for the treatment of hcv infection in humans. the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada a cluster of cases of severe acute respiratory syndrome in hong kong glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug effect of ribavirin on bunyavirus reproduction in cell culture and in an experiment on white mice inhibitory effect of selected antiviral compounds on arenavirus replication in vitro the biological relationship of mouse hepatitis virus (mhv) strains and interferon: in vitro induction and sensitivities comparative susceptibility of respiratory viruses to recombinant interferons-alpha 2b and -beta inhibitory effects of recombinant feline interferon on the replication of feline enteropathogenic viruses in vitro antiviral action of interferon-alpha against porcine transmissible gastroenteritis virus mass spectrometric characterization of proteins from the sars virus: a preliminary report clinical pharmacokinetics of interferons plasma exchange and interferon-alpha pharmacokinetics in patients with hepatitis c virus-associated systemic vasculitis transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis koch's postulates fulfilled for sars virus we thank the severe acute respiratory syndrome task force at the canadian science centre for human and animal health for valuable discussions. key: cord-275355-4izc5jxs authors: hayden, frederick; croisier, alice title: transmission of avian influenza viruses to and between humans date: 2005-10-15 journal: j infect dis doi: 10.1086/444399 sha: doc_id: 275355 cord_uid: 4izc5jxs nan reports of seropositivity for different avian influenza a viruses in exposed poultry workers, including the new findings reported by puzelli et al. in this issue of the journal of infectious diseases [1] , and the recent instances of cross-species transmission that caused human disease [2] raise fundamental questions regarding the routes of transmission of avian viruses to and between humans, possible differences in transmission patterns between human and avian influenza viruses, and implications for prevention in those occupationally exposed to infected animals and also in health care, household, and community settings. documentation of seropositivity for avian influenza viruses in farm workers is not a new finding [3] , and previous studies have assessed human susceptibility by intranasal inoculation of selected avian influenza viruses [4] . however, the outbreak in europe of h7n7 virus infec-tion that led to many cases of conjunctivitis and 1 death resulting from viral pneumonia [5, 6] , as well as the unprecedented epizootic caused by highly pathogenic avian influenza h5n1 virus in southeast asia, emphasize the importance of these issues. one strength of the study by puzelli et al. is the multiplicity of the serological tests used, which included a microneutralization assay with infectious virus, a hemagglutination inhibition (hi) assay, and a confirmatory western blot analysis with purified h7 hemagglutinin to exclude the possibility of nonspecific cross-reactions with antibodies to human influenza viruses. differential absorption with human influenza virus has also been utilized to confirm the presence of avian influenza virus-specific antibodies [7] . such methods are essential to document seropositivity for an avian influenza virus, particularly when concerns about extensive transmission to humans are raised, as was reported elsewhere for h7n7 virus infection in the netherlands [8] . the possibility that the latter virus caused widespread subclinical infections in poultry workers and household contacts-and, hence, manifested efficient human-tohuman transmission-was raised by 1 study that used a modified hi assay to measure antibody [8] . similarly, the recent report of asymptomatic infection by h5n1 virus in northern vietnam, as de-termined by the detection of h5n1 rna in household contacts, requires substantiation by confirmatory serological testing [9] , although culture-confirmed h7n3 illnesses have occurred without an apparent detectable serologic response [10] . even in those individuals with proven seropositivity for an avian influenza virus, it is uncertain whether they have been only exposed to antigen or are productively infected. the findings that seropositivity occurs in small numbers of poultry workers exposed during outbreaks of illness in poultry caused by some avian strains (h7n7, h7n3, and h5n1) but not others (h7n1 and h5n2) argue for actual infection and support the notion that some avian influenza viruses are more likely than others to infect humans [1] . definitive evidence for active infection would include detection of virus or viral rna at the time of exposure or illness. in any case, the recent reports of the apparently greater adaptation of h7n7 [7] and h5n1 [9] avian influenza viruses to humans than was previously recognized and the potential for reassortment during dual infection with a low or highly pathogenic avian influenza virus and a conventional human influenza virus mandate careful laboratory documentation involving multiple assays. transmission of human influenza virus occurs by inhalation of infectious droplets or airborne droplet nuclei and, perhaps, by indirect (fomite) contact followed by self-inoculation of the upper respiratory tract or conjunctival mucosa. the relative importance of these routes is debated, and there is evidence to support each of them, including transmission within health care facilities [11, 12] , in human influenza. it is likely that each route contributes to transmission under appropriate circumstances and that the manifestations of illness, respiratory tract viral loads, and, perhaps, the type of infecting influenza virus influence the likelihood of transmission by a particular route. of course, the use of measures to prevent infection, such as personal protective equipment (e.g., masks and eye protection), hand hygiene, and specific chemoprophylaxis or immunization modalities, by potentially exposed persons will alter the observed risks. transmission of avian influenza virus likely encompasses these routes, as well as others. human conjunctiva [13] and ciliated nasal epithelial cells [14] contain cellular receptors that are recognized preferentially by the hemagglutinin of avian (a2,3 linkages between the terminal sialic acid residues and galactose), rather than human (a2,6 linkages), influenza viruses. the distribution of avian-type receptors in the lower airways and other tissues of humans requires study. however, it is particularly concerning that perhaps only 2 amino acid changes in the viral receptor binding site may be required to change the tropism of the h5 hemagglutinin from avian-to human-type receptors [15] . clinically apparent infections due to avian influenza viruses of the h7 subtype typically cause conjunctivitis and demonstrate higher viral loads in the eye than in the pharynx [5, 6, 10] . the importance of the eye or nose as a site of initial infection and the importance of subsequent replication with non-h7 avian influenza viruses are unknown. in h5n1infected patients, conjunctivitis has not been a feature, and rhinorrhea has been inconsistently reported. in contrast, the frequent occurrence of diarrhea and the detection of viral rna in most fecal samples tested suggest that h5n1 virus may replicate in the human gastrointestinal tract and raise the question of whether human feces could be a source of transmission [16] . most cases of human infection due to avian influenza viruses have involved close contact with infected poultry, particularly ill or dying chickens. during the outbreak in hong kong in 1997, 1 case-control study [17] found that exposure to live poultry within a week before the onset of illness was associated with human disease, but no significant risk was related to traveling, eating or preparing poultry products, or being exposed to persons with disease caused by h5n1 virus. another study in hong kong [18] found that exposure to ill poultry and butchering of birds were associated with seropositivity for h5 avian influenza viruses. four workers who culled infected birds in japan [19] and 2 animal attendants who cared for infected tigers in thailand [20] were found to have antibodies to h5n1 virus during the outbreaks in 2004; seroconversion indicating recent infection was found in only 1 of the japanese workers. during the first wave of human infections in 2003-2004, a history of direct contact with poultry was found in 8 of 10 h5n1-infected patients in vietnam [21] and with dead chickens in 8 of 12 h5n1-infected patients in thailand [22] , whereas no clinical cases of illness were noted in those involved in mass culling of poultry. it has been estimated that 12%-61% of rural thai residents have regular contact with birds [22] . however, ∼30% of h5n1-infected patients in vietnam have not reported exposure to sick poultry [16] , which leaves the issue open to speculations about more frequent human-tohuman transmission than has been found. however, this finding might be biased, because retrospective notification of animal disease has important consequences in some countries. infection after consumption of fresh duck blood and undercooked poultry products has been suspected in some cases of illness. indeed, transmission to felids was observed after experimental feeding of infected chickens to domestic cats [23] , and feeding tigers raw infected chicken led to outbreaks of illness in thai zoos, in which felid-to-felid transmissions were also implicated [20, 24] . infected birds shed high concentrations of virus in feces [25] . direct intranasal or conjunctival inoculation while swimming in contaminated water or, perhaps, inhalation or ingestion of water could have been potential modes of transmission to some h5n1-infected patients. as for human influenza, hand contamination from fomites and self-inoculation into the eye or upper respiratory tract remain possible modes. greater adaptation of avian influenza viruses to human hosts could alter the routes of transmission and increase the likelihood of human-to-human spread. in addition to sporadic bird-to-human and suspected environment-to-human transmission, human-to-human transmission of h5n1 avian virus has been implicated by epidemiological findings in several household clusters in which similar illnesses were reported in relatives [21] and in 1 well-documented situation in which there was child-to-mother and likely childto-aunt transmission in thailand [26] . these probable human-to-human transmissions involved close contact during the critical phase of illness and were inefficient without additional chains of transmission. several household contacts also developed symptomatic h7n7 avian virus infections after exposure to ill family members in the netherlands in 2003 [6] . however, in contrast to the studies of human influenza viruses [27] , molecular epidemiological studies to rigorously establish human-to-human transmission of h5 avian viruses have not been completed. cohort studies in 1997 found that human-to-human transmission might have occurred through close physical contact but not through social contact [28] . intimate, face-to-face contact without the use of measures to prevent infection was implicated in these circumstances, and no evidence to date indicates that there has been human-to-human transmission of h5n1 avian virus by small-particle aerosol exposure. recent serosurveys in southern vietnam and thailand have not found evidence for inapparent infections in family contacts [16] . although viral rna was detected by polymerase chain reaction in swab samples from asymptomatic family contacts of ill patients in vietnam in 2005, these infections remain to be confirmed by serological testing. nosocomial transmission of h5n1 virus to health care workers (hcws) was found by serological assessment in hong kong in 1997 [29] and is suspected in a nurse exposed to an infected patient in vietnam in 2005 [16] . to date, the risk of infection in health care settings appears low, even when appropriate isolation measures have not been used [7, 30, 31] . in 2004, no illness occurred in exposed hcws or laboratory workers in vietnam [21] or in 35 exposed and unprotected hcws in thailand [30] . no serological evidence of infection was present in 83 exposed and masked hcws in hanoi [7] , and another study of 64 unprotected hcws in ho chi minh city found no illness or seroconversion [31] . however, given the potential threat and changing transmissibility of avian influenza viruses, isolation precautions within health care facilities should encompass the measures used for potentially pneumoenteric pathogens, such as severe acute respiratory syndrome-associated coronavirus [32] . in summary, observations made to date suggest that differences in the routes of transmission between human and avian influenza viruses exist. the multiple potential routes for the spread of avian influenza viruses, particularly h5n1, indicate that, in addition to protection for the respiratory tract and eyes, proper hand hygiene may be especially important in preventing infection. this applies also in emergency departments and clinics where patients with febrile illnesses who are from areas with documented h5n1 virus infections in poultry or people may be evaluated. in households in which illness has occurred, additional specific protective measures-that is, postexposure chemoprophylaxis with oseltamivir-would be advisable for known household contacts. in affected countries, public education regarding simple precautionary measures for food preparation, poultry handling, and avoidance of contaminated water are essential until effective human vaccines for h5n1 viruses become available. serological analysis of serum samples from humans exposed to avian h7 influenza viruses in italy between 1999 and avian influenza. geneva: world health organization pandemic influenza: a zoonosis? replication of avian influenza viruses in humans avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands world health organization international avian influenza investigation team, vietnam. lack of h5n1 avian influenza transmission to hospital employees final analysis of netherlands avian influenza outbreaks reveals much higher levels of transmission to humans than previously thought world health organization. who intercountry-consultation: influenza a/h5n1 in humans in asia human illness from avian influenza h7n3, british columbia influenza in the acute hospital setting transmission of influenza: implications for control in health care settings avian influenza and sialic acid receptors: more than meets the eye? human and avian influenza viruses target different cell types in cultures of human airway epithelium restrictions to the adaptation of influenza a virus h5 hemagglutinin to the human host world health organization. who consultation on case management and research on human influenza a/h5 case-control study of risk factors for avian influenza a (h5n1) disease, hong kong risk of influenza a (h5n1) infection among poultry workers, hong kong, 1997-1998 japan: serological investigation among humans involved in the mass culling operation probable tiger-to-tiger transmission of avian influenza h5n1 avian influenza a (h5n1) in 10 patients in vietnam human disease from influenza a (h5n1) avian h5n1 influenza in cats avian influenza h5n1 in tigers and leopards laboratory study of h5n1 viruses in domestic ducks: main findings probable person-to-person transmission of avian influenza a (h5n1) assessment of hemagglutinin sequence heterogeneity during influenza virus transmission in families antibody response in individuals infected with avian influenza a (h5n1) viruses and detection of anti-h5 antibody among household and social contacts risk of influenza a (h5n1) infection among health care workers exposed to patients with influenza a (h5n1), hong kong atypical avian influenza (h5n1) avian influenza h5n1 and healthcare workers world health organization. who interim guidelines on clinical management of humans infected by influenza a (h5n1) key: cord-297432-2edncbgn authors: helleberg, marie; niemann, carsten utoft; moestrup, kasper sommerlund; kirk, ole; lebech, anne-mette; lane, clifford; lundgren, jens title: persistent covid-19 in an immunocompromised patient temporarily responsive to two courses of remdesivir therapy date: 2020-07-23 journal: j infect dis doi: 10.1093/infdis/jiaa446 sha: doc_id: 297432 cord_uid: 2edncbgn the antiviral drug remdesivir has been shown clinically effective for treatment of covid-19. we here demonstrate suppressive but not curative effect of remdesivir in an immunocompromised patient. a man in his fifties treated with chemoimmunotherapy for chronic lymphocytic leukemia experienced a 9-week course of covid-19 with high fever and severe viral pneumonia. during two 10-day courses of remdesivir starting 24 and 45 days after fever onset, pneumonia and spiking fevers remitted, but relapsed after discontinuation. kinetics of temperature, c-reactive protein, and lymphocyte counts mirrored the remitting/relapsing sars-cov-2 infection. combination therapy or longer treatment duration may be needed in immunocompromised patients. the pandemic of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused a huge burden of morbidity and mortality worldwide. recently, preliminary results of the adaptive covid-19 treatment trial (actt), a multicenter randomized controlled trial of remdesivir versus placebo for treatment of coronavirus disease 2019 (covid-19) in hospitalized patients, demonstrated that remdesivir reduced time to recovery, in particular for those not yet having experienced respiratory failure with need for assisted ventilation [1] . despite clinical benefit, there was still a substantial mortality rate, especially among patients with underlying risk factors. we here report the clinical course and findings in an immunocompromised patient with remission of covid-19 during treatment with remdesivir but relapse soon after discontinuation. this pattern was repeated during and after a second course of remdesivir treatment. data were obtained from electronic health records. the diagnosis of covid-19 was confirmed by polymerase chain reaction (pcr) of throat swabs using the sars-cov-2 assay from roche and the cobas 6800/8800 system, apart from the first test where an in-house assay was used. plasma samples were analyzed using chemiluminescent immunoassays-based kits for sars-cov-2 igm/igg and chemiluminescence analyzer iflash from yhlo. the patient gave informed consent. a caucasian man in his fifties, diagnosed with chronic lymphocytic leukemia (cll) 2 years prior and treated with 6 cycles of fludarabine, cyclophosphamide, and rituximab ending 3 months prior, presented with spiking fevers, muscle pain, and loss of taste and smell. pcr for sars-cov-2 on a throat swab was positive (cycle threshold value 13). the cll disease was in complete remission according to the international workshop on chronic lymphocytic leukemia 2018 criteria, with a remaining low lymphocyte count of 0.8 × 10 9 /l. he had no other comorbidities, a normal body mass index of 25, and he did not smoke or drink alcohol in excess. until he presented with the acute infection, he had been in good general condition and worked full time. covid-19 was diagnosed the day he started having fevers. at the time of diagnosis, he had no respiratory distress and selfisolated at home. in the following description, days are counted from the onset of fever. the clinical course and key findings are summarized in figure 1 . on day 14 he was admitted to hospital with a 1-week history of dry cough and a 4-day history of continuous spiking fevers and dyspnea. pcr for sars-cov-2 on a throat swap remained positive (quantitation not available). vital signs and laboratory test results are summarized in table 1 . a chest x-ray showed bilateral interstitial infiltrates. he was treated empirically with piperacillin/tazobactam. during the following days his condition worsened. he needed supplemental oxygen, increasing to 8 l/minute, and blood tests showed severe lymphopenia and elevated biomarkers of inflammation. on day 22 a pcr for sars-cov-2 on a throat swap was still positive and a chest x-ray showed progression of the interstitial infiltrates and new consolidating infiltrates. on day 24 he was enrolled in the actt trial and was randomized to treatment with placebo or remdesivir for 10 days. after completion of the trial, unblinding showed that he was in the remdesivir arm and had been treated with remdesivir 200 mg intravenous (iv) loading dose followed by 100 mg iv every day. two days after the initiation of remdesivir, the fever abated and his general condition improved. he continued to improve and was discharged on day 35 after finishing the 10-day course of remdesivir. three days after the last dose (day 36), high fevers and dyspnea recurred, and he was readmitted to hospital. sars-cov-2 pcr on a throat swab was positive. cultures of blood, airway secretions, and urine showed no growth and he did not improve on empiric treatment with meropenem. another course of remdesivir was initiated on day 45 (after completion of the trial). the next day he was afebrile and the following day his general condition improved. markers of inflammation that had increased after discontinuation of remdesivir decreased again. he received 10 days of treatment with remdesivir. sars-cov-2 pcr on a throat swab at the end of treatment (day 54) was negative and he was discharged. the day after discontinuation of remdesivir, the fever recurred once again, and he was readmitted (day 55). sars-cov-2 pcr on a throat swab was again positive. this time he was not in respiratory distress and did not need supplemental oxygen. however, he had fever with temperatures > 40°c and complaint of general malaise and on day 58 he received an infusion of 2 × 270 ml convalescent plasma iv. from day 60 he was afebrile and biomarkers of inflammation were improving. on day 65 he was in good general condition and discharged from hospital. the patient did not receive corticosteroids during the course of the disease. tests for sars-cov-2 antibodies on days 38, 52, and 58 were negative. he had received a 7-valent conjugate pneumococcal vaccine followed by a 23-valent polysaccharide vaccine in 2018, but a test for pneumococcal antibodies during the second admission showed that he had no antibodies and thus no vaccine response. sars-cov-2 can cause a range of disease manifestations, from asymptomatic infection through mild to severe disease, but there is typically resolution of the infection within 1-3 weeks after the onset of symptoms in the immunocompetent host [2] . the pathogenesis of severe disease is not well elucidated. critical illness in covid-19 is characterized by high fever and significantly elevated biomarkers of inflammation and is thought to be caused by an aberrant hyperactivated immune response and cytokine storm [3] , but there is also emerging evidence that preexisting immunocompromise is associated with a significantly increased risk of severe disease [4, 5] . the role of ongoing viral infection versus a secondary hyperreactive immune response in the aftermath of viral replication for the development of severe covid-19 is not completely understood [6] . we present a case of severe covid-19 in a patient with b-and t-lymphocyte impairment secondary to cll treated with chemoimmunotherapy 3 months prior to the sars-cov-2 infection. biomarkers and repeated positive pcr tests for sars-cov-2 indicated continuous viral replication for up to 9 weeks. both the spiking fever, dyspnea, and abnormalities in biomarkers of inflammation resolved during treatment with the antiviral drug, remdesivir, but there was relapse soon after discontinuation of treatment. when treatment was resumed, the symptoms and signs of inflammation abated once again (supplementary figure 1) . only a day after the second course of remdesivir had finished, the fever recurred, and abnormalities of blood tests worsened but this time the disease was milder. these observations indicate that remdesivir suppressed viral replication but was unable to eradicate the infection. the convincing effect of remdesivir also argues for ongoing viral replication being an important driver of disease manifestations in this immunocompromised patient. however, due to the lack of quantitative viral load measurements, analyses of viral outgrowth, and sampling from the lower respiratory tract we cannot draw any firm conclusions. a large multicenter randomized controlled trial of remdesivir versus placebo for covid-19 in hospitalized patients showed that remdesivir reduces time to recovery significantly, with the largest effect size among patients requiring supplemental oxygen but not invasive or noninvasive mechanical ventilation [1] . in this subgroup, there was also a significant reduction in mortality. the patient we here describe belonged to this subgroup at the time of randomization. the optimal duration of treatment with remdesivir in immunocompetent as well as immunocompromised patients remains to be defined. of note in the actt study, a suggestion of a survival benefit for patients with advanced disease dissipated in the days following completion of an initial 10-day treatment course [1] . therapies combining remdesivir with another antiviral drug or an immune modulator may reduce risk of treatment failure and improve treatment outcomes. randomized controlled trials employing this treatment strategy are underway. our patient was treated with convalescent plasma when he experienced the second relapse after treatment discontinuation, because remdesivir was not available at that time. he recovered in the following days. whether he recovered spontaneously or due to treatment with convalescent plasma is unknown. the immunological mechanisms for control of sars-cov-2 infection in humans have not yet been clearly elucidated. both cytotoxic t cells and antibody-mediated immune responses may be important for clearance of the viral infection [7] . convalescent or hyperimmune immunoglobulins for treatment of covid-19 are currently being tested in clinical trials, although the clinical beneficial effects hereof remain uncertain [8] . the patient described in this report had not developed sars-cov-2 antibodies 8 weeks after the infection was diagnosed by pcr. cll is associated with b-lymphocyte impairment as well as a general immune dysfunction, leading to increased risk and severity of infections [9] . solid organ transplant recipients, who primarily have compromised t-lymphocyte function, are also at significantly increased risk of severe covid-19 [10] . but in general, comprehensive data on the outcome of covid-19 in patients with various types of immune deficiencies are still scarce. it is difficult to determine the duration of ongoing viral infection using routine clinical laboratory methods. viral rna can be detected in biological samples by pcr for several weeks after resolution of symptoms when replication-competent virus can no longer be detected by viral cell cultures [11, 12] . there is correlation between the viral load as determined by quantitative pcr and results of viral cell cultures [11, 12] . however, viral culture assays may have suboptimal sensitivity and a negative culture does not necessarily rule out that replication-competent virus is present [13] . we did not have access to a validated quantitative pcr for sars-cov-2 and therefore only qualitative pcr was performed. viral cultures were not available. also, in patients with covid-19 pneumonia, the virus may only be detected in the lower respiratory tract [14] , suggesting that the sampling we did from the pharynx may not have focused on the location of viral replication (ie, the lungs). there was close correlation between subjective symptoms, kinetics of body temperature, levels of c-reactive protein, and the lymphocyte and platelet counts in relation to treatment with remdesivir. these dynamic changes were most likely causally related to the provision of the antiviral medication. the patient was unresponsive to empiric antibacterial therapy and extensive and repeated microbiological examinations failed to identify other pathogens. decreased lymphocyte counts have consistently been reported in sars-cov-2 and other viral infections [15] . the rapid recovery of lymphocyte counts during and immediately after antiviral therapy seen in this case suggests that the lymphopenia is caused by redistribution of lymphocytes from the peripheral blood to inflamed tissues and not by lymphocytes being killed as a result of the infection. the course and findings in this clinical case suggest that remdesivir has a rapid onset of action and can suppress, but may not eradicate, sars-cov-2 in immunocompromised patients. there is a need for development of additional treatments to improve outcomes as well as consideration for longer treatment courses with remdesivir in certain subgroups of patients. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. remdesivir for the treatment of covid-19-a preliminary report clinical course of 2019 novel coronavirus disease (covid-19) in individuals present during the outbreak on the diamond princess cruise ship hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression cancer patients in sars-cov-2 infection: a nationwide analysis in china covid-19 infection in kidney transplant recipients immunosuppression for hyperinflammation in covid-19: a double-edged sword? the many faces of the anti-covid immune response convalescent plasma or hyperimmune immunoglobulin for people with covid-19: a rapid review infectious complications in patients with chronic lymphocytic leukemia: pathogenesis, spectrum of infection, and approaches to prophylaxis opensafely: factors associated with covid-19 death in 17 million patients virological assessment of hospitalized patients with covid-2019 viral rna load as determined by cell culture as a management tool for discharge of sars-cov-2 patients from infectious disease wards molecular diagnosis of respiratory virus infections laboratory diagnosis of emerging human coronavirus infections-the state of the art covid-19 patients' clinical characteristics, discharge rate, and fatality rate of metaanalysis we thank mikkel gybel-brask and henrik ullum, capital region blood bank, department of clinical immunology, rigshospitalet, copenhagen university hospital for provision of convalescent plasma to treat the patient. disclaimer. the funding source had no role in writing the manuscript or the decision to submit it for publication.financial support. this work was supported by the danish national research foundation (grant number 126).potential conflicts of interest. m. h. reports speaker honoraria from msd, gsk, and gilead; travel grants from gilead, bms, and gsk; and advisory board activity for gsk outside the submitted work. c. u. n. has received consultancy fees and/or travel grants from janssen, abbvie, novartis, roche, sunesis, gilead, astrazeneca, and csl behring; and research support from novo nordisk foundation, abbvie, astrazeneca, and janssen. a.-m. l. reports personal fees/travel grants from gilead, gsk, and msd; and advisory board activity for gilead outside the submitted work. all other authors report no potential conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-315476-7rdiesav authors: peret, teresa c. t.; boivin, guy; li, yan; couillard, michel; humphrey, charles; osterhaus, albert d. m. e.; erdman, dean d.; anderson, larry j. title: characterization of human metapneumoviruses isolated from patients in north america date: 2002-06-01 journal: j infect dis doi: 10.1086/340518 sha: doc_id: 315476 cord_uid: 7rdiesav human metapneumovirus (hmpv) was recently identified in the netherlands and was linked to acute respiratory tract illness. in this study, 11 isolates from 10 patients with respiratory disease from quebec, canada, were tested by a reverse-transcriptase polymerase chain reaction based on the fusion protein gene. identified sequences were consistent with hmpv. the patients were 2 months to 87 years of age (median age, 58 years) and presented with acute respiratory tract illness during the winter season. sequence studies of the nucleocapsid, fusion, and polymerase genes identified 2 main lineages of hmpv and cocirculation of both lineages during the same year. these findings support a previous finding that hmpv is a human respiratory pathogen that merits further study. human metapneumovirus (hmpv) was recently identified in the netherlands and was linked to acute respiratory tract illness. in this study, 11 isolates from 10 patients with respiratory disease from quebec, canada, were tested by a reverse-transcriptase polymerase chain reaction based on the fusion protein gene. identified sequences were consistent with hmpv. the patients were 2 months to 87 years of age (median age, 58 years) and presented with acute respiratory tract illness during the winter season. sequence studies of the nucleocapsid, fusion, and polymerase genes identified 2 main lineages of hmpv and cocirculation of both lineages during the same year. these findings support a previous finding that hmpv is a human respiratory pathogen that merits further study. a new paramyxovirus associated with respiratory illness, human metapneumovirus (hmpv), was described recently [1] . data from that report suggest that hmpv is similar to respiratory syncytial virus (rsv), in that infection usually occurs during winter months and is common during childhood (most children have serologic evidence of infection by age 5 years). the genomic organization of hmpv resembles that of the avian pneumovirus [2] and has been tentatively assigned to the subfamily pneumovirinae, genus metapneumovirus. in the present article, we describe polymerase chain reaction (pcr) and sequencing studies done on 11 isolates from respiratory specimens from 10 canadian patients with acute respiratory tract illness. isolates and patients. we studied 11 unidentified isolates from 10 patients with acute respiratory tract illness that were recovered from 1997 through 1999 in quebec city, quebec, canada. the specimens from which the isolations were made were from endobronchial lavage (1 specimen), pharyngeal swabs (2 specimens), and nasopharyngeal aspirates (5 specimens); the origin of 3 specimens was not determined. patients were 2 months to 87 years of age (median age, 58 years). of the 10 patients, 7 were hospitalized for respiratory illness in an acute care hospital, and 3 were residing in a long-term care facility at the time of illness. all 11 isolates were recovered in llc-mk2 cells (rhesus monkey kidney cells) and had focal rounding and cell destruction without apparent syncytia formation. no cytopathic effect was noted in mdck or nci-h292 cells. the original specimens had negative results of testing for influenza viruses when they were inoculated into embryonated eggs. the isolates did not adsorb erythrocytes and had negative results of testing by indirect immunofluorescence assays for influenza viruses a and b; parainfluenza viruses 1, 2, and 3; adenovirus; and rsv (bartels; chemicon). results of reverse-transcriptase (rt) pcr or pcr assays for adenovirus, coronavirus, influenza viruses a, b, and c, parainfluenza viruses 1, 2, and 3, rhinovirus, and rsv were negative for all 11 isolates. electron microscopy (em). the isolation material was prepared for negative-stain em examination by use of 2% phosphotungstic acid negative staining. we adjusted phosphotungstic acid ph to 6.5 with 1 n potassium hydroxide and used formvar-carbon grids pretreated with glow discharge [3] . samples were mixed 1:1 with catalase crystals and prepared for negative-stain em examination, as described elsewhere [4] , to determine the dimensions of virus particles and nucleocapsid structures. oligonucleotide primer design for hmpv. published nucleocapsid (n) and fusion (f) gene sequences of hmpv and avian pneumovirus were used to develop primers for detection and sequencing of hmpv at the respiratory virus section (centers for disease control and prevention, atlanta). the f primer set was used for hmpv identification by rt-pcr. polymerase (l) gene primers, used in the initial hmpv studies [1] , were later provided to corroborate our findings. primer pair mpvf1f (5 0 -ctttggacttaatgaca-gatg-3 0 )-mpvf1r (5 0 -gtcttcctgtgctaactttg-3 0 ) and primer pair mpvn3f (5 0 -gagaagagctgggtagaag-3 0 )-mpvn3r (5 0 -caaacaaactttctgct-3 0 ) were used to amplify regions in the f (450 bp) and n (377 bp) genes, respectively. rt-pcr and nucleotide sequencing. total rna was extracted by use of a guanidium-isothiocyanate-phenol method (rnazol ls; tel-test). viral rna was amplified in a 1-step rt-pcr (access rt-pcr; promega). the pcr assay was carried out in a mix containing 10 ml of 5£ reaction buffer, 10 mm dntps, 1.25 mm forward and reverse primers, 2 ml of 25 mm mgso 4 , 5 u of avian myeloblastosis virus rt, 5 u of thermus flavus dna polymerase, and 5 ml of rna; nuclease-free water was added until the volume of the mix was 50 ml. amplification conditions consisted of 45 min at 42 c; 2 min at 94 c; 35 cycles of pcr for 1 min each at 94 c, 54 c, and 72 c; and a final extension at 72 c for 7 min. each rna sample was run against a housekeeping gene to verify rna integrity. the pcr products were purified with either the qiaquick gel extraction kit or qiaquick pcr purification kit (qiagen). both strands were sequenced on an abi 377 sequencer, using a fluorescent dye terminator kit (applied biosystems). the nucleotide sequences were edited with sequencher version 3.1.1 for the power macintosh (gene codes). phylogenetic analysis. partial nucleotide sequences of n, f, and l genes were aligned by use of clustal w, version 1.7, for unix [5] . n and l sequences were aligned with published hmpv partial and full gene sequences. f sequences were aligned with the single full hmpv f gene sequence available. phylogenetic trees for group a and b alignments were computed by maximum parsimony-, distance-, and maximum likelihood-based criteria analysis with paup* version 4.0.d8 [6] . for the bootstrap analysis, sequences were added randomly, and 1 tree was held at each step (100 bootstrap replicates). nucleotide sequences were translated with translate in the wisconsin package, version 10.2 for unix (genetics computer group). pairwise nucleotide and amino acid distances were calculated, respectively, as the proportion of differences (uncorrected p value) and mean character difference, using mega (molecular evolutionary genetics analysis; mega software) [7] . em studies. the 11 isolates initially recovered in llc-mk2 cells were also successfully passaged in vci-h292 cells. em examination showed that all 11 specimens contained viruses with morphologic characteristics that were consistent with paramyxovirus. the particles were pleomorphic, spherical, and filamentous (figure 1). the mean length of the projections on the particles was 15 nm (se, 0.27), the nucleocapsid diameter was 17 nm (se, 0.36), and the nucleocapsid pitch spacing was 7 nm (se, 0.24). the nucleocapsid length was , 200 nm to 1000 nm. spherical particles varied considerably in size, but the mean diameter was 209 nm (se, 27.8). filamentous particles averaged 282 £ 62 nm in size, but too few were available for us to obtain a satisfactory statistical representation. the measure-ments are in accordance with the dimensions of members of the metapneumovirus and pneumovirus genera [2, 8] . sequencing studies. phylogenetic analyses based on the n (300 nt), f (405 nt), and l (102 nt) genes gave comparable results (i.e., they identified 2 major groups or lineages; figure 2 ). an overall nucleotide comparison for the isolates revealed 93%-100% similarity between isolates in the same group and 83%-85% similarity between the 2 groups. the predicted amino acid sequences were less distinct: they showed 95%-97% similarity between the 2 distinct groups and 97%-100% similarity between isolates in the same group. of note, several sequences from isolates from patients in the netherlands (designated "nld"), reported elsewhere [1] , clustered in those 2 main groups, along with the 11 isolates from patients in canada (designated "can") described in the present article. despite the limited data available, we observed cocirculation of both groups (can97-82 and can97-83) during the same year. some sequences clustered with published hmpv sequences identified in different years, as seen on subclusters (can97-82, nld99. of note, 2 hmpv isolates, can98-75 and can99-80, each belonging to a different group, came from the same child. the second isolate was found 10 months after the first. this demonstrates that the child was reinfected, rather than persistently infected, with hmpv. our data support the findings of van den hoogen et al. [1] that a new virus, tentatively named "human metapneumovirus," is associated with acute respiratory disease. in addition, our data demonstrate that this virus is present in north america and likely worldwide. our findings are also consistent with the aforementioned findings [1] : we identified 2 major groups or lineages and sequence diversity within these 2 major groups. human rsv, a paramyxovirus in the same taxonomic subfamily, has been classified into 2 major groups, a and b. concurrent circulation of both human rsv groups and variants has been identified [8] . one could speculate that hmpv might also follow these trends. we found some hmpv isolates that, despite close genetic relatedness, did not originate from a single outbreak but were from discrete and unrelated cases of respiratory illness, as also occurs with human rsv. further sequencing studies that examine more isolates and different hmpv genes, including the surface glycoprotein g gene, should be conducted to confirm and refine these observations. we detected virus in isolates from children with acute respiratory tract infection, as described in the first report of hmpv [1] . we also detected virus in isolates from adults with acute respiratory tract infection. if the serologic studies from the netherlands [1] are indicative of the epidemiologic features of infection in canada (i.e., most children are infected by age 5 years), then hmpv likely can reinfect an individual later in life, possibly repeatedly, as does human rsv. in fact, like human rsv infection, hmpv reinfection may well occur in the first years of life, as is illustrated by the isolation of 2 hmpvs from the same child in 2 consecutive winter seasons. it appears that the virus isolated in the second year was able to evade, at least partially, immunity induced 10 months earlier. the ability to detect hmpv in children and in adults suggests that hmpv disease should be studied in all age groups. given the very limited data available on infection with this virus in humans, etiologic studies should include testing of specimens from appropriate control patients. in conclusion, hmpv presents some exciting new opportunities and challenges in our efforts to understand human respiratory tract disease. a newly discovered human pneumovirus isolated from young children with respiratory tract disease characterization of a virus associated with turkey rhinotracheitis a glow discharge unit to render electron microscope grids and other surfaces hydrophilic an accurate measurement of the catalase crystal and its use as an internal marker for electron microscopy clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice phylogenetic analysis using parsimony (*and other methods) mega: molecular evolutionary genetics analysis respiratory syncytial virus we thank brian holloway and staff (centers for disease control and prevention biotechnology core facility, dna chemistry section) for the oligonucleotide synthesis. key: cord-317421-xzf723w2 authors: schieffelin, john s title: infectious disease outbreaks: the need for an all-in approach date: 2020-04-08 journal: j infect dis doi: 10.1093/infdis/jiaa167 sha: doc_id: 317421 cord_uid: xzf723w2 nan as of march 29, 2020, the 2018 to 2020 outbreak of ebola virus disease (evd) in the democratic republic of congo (drc) has led to 3453 cases and 2264 deaths [1] . this outbreak should be remembered for several major successes. first, the palm trial [2] represents a major milestone as a randomized, controlled, therapeutic trial during an outbreak that compared 4 different therapeutic approaches to optimized standard of care therapy. second, vaccination as a control measure of viral hemorrhagic fevers began almost immediately after the outbreak was recognized [3] . it is unfortunate that the outbreak will also be remembered for another reason. it is the first evd outbreak to occur in the midst of active, civil conflict. for the entire duration of the outbreak, both local and international responders have had to contend with the threat of violence, some of which was directed specifically at the evd response [4] . it is sad that the threat of violence against healthcare workers and ebola treatment units (etus) has been an issue in several different ebola virus outbreaks [5, 6] . however, the ongoing and pervasive violence instigated by armed militias and other actors during the current outbreak is unprecedented. such violence not only threatens the safety of healthcare workers and likely reduces their ability to work effectively in the etus, but it also interferes with the work of surveillance teams and may decrease the confidence of the local population in the response. in their article entitled "identifying mechanisms of violence that impact ebola virus disease transmission during the 2018-2019 outbreak in the democratic republic of the congo, " kelly et al [7] test the hypothesis that violent events directly targeting the ebola response result in greater transmission than violence that does not directly target the ebola response. specifically, they measure the impact of different types of violence on evd transmission. data on case counts were obtained through the daily situation reports produced by the drc ministry of health. violent events were obtained from 2 sources: the armed conflict location & event data project and world health organization situation reports. two thousand seven hundred seventy-four probable and confirmed evd cases were included, as well as 656 violent events. sixty-two violent events were ebola-targeted whereas the remaining 594 were not. their results indicate that ebola-targeted violence results in increased transmission to a greater degree than non-ebola-targeted. the control of evd outbreaks requires a multifaceted approach that includes early detection of suspected cases, isolation of those cases, identification and tracing of contacts, and the prevention of new infections [8, 9] . these goals can only be accomplished through social mobilization and risk communication, highly specialized medical care and infection control practices, support for safe burial practices, and vaccination of those most at risk [10, 11] . community mistrust of responders as well as social resistance to medical burials, quarantine rules, and the misunderstanding of strongly limited visitation rules at etus are also barriers to ebola virus control efforts [12] . a lack of institutional trust and belief in misinformation have been linked to reduced adherence to evd preventive measures. although it is intuitive that violence, especially ebola-targeted violence, will impact disease transmission, kelly et al [7] adequately estimate that degree of impact. however, despite the continuous threat of violence, progress in controlling the outbreak has been made. multiple international nongovernmental organizations are working hand in hand with each other and with local institutions as never before. thousands of people have been vaccinated. randomized, controlled trials comparing 4 different therapeutics have been successfully completed. the standard of care has been raised. how did this happen in the face of violence? effective social mobilization is based on trusted sources communicating critical risk mitigation strategies [13] . at some point during this outbreak, local and international partners gained the support of the local community. in other words, the local community did their part to end the outbreak by supporting responders and heeding to their advice to get tested if symptomatic and to practice social distancing. because the 2018-2020 evd outbreak in drc appears to be close to an end, the world faces another pandemic. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus threatens the healthcare systems of developed and developing healthcare systems alike. today, there are hundreds of thousands of cases of coronavirus 2019 (covid-19) in many countries. hospitals in developed nations, including the united kingdom, italy, and the united states, are struggling to treat patients in hospitals that are all too frequently filled beyond capacity. shortages of personal protective equipment are in short supply. in some hospitals, 2 patients must share the same ventilator. yet the same rules of outbreak control applied in the drc are being encouraged. major cities are ordering businesses to close. millions of people are ordered to stay home. testing is being ramped up to identify the sick so that they can be isolated and separated from those most at risk. hospitals are prohibiting people from visiting stricken family members. highly specialized medical care is being implemented to save as many of those with severe symptoms as possible. we are pleading with the general public to abide by these intrusive rules, which many of us became familiar with during the evd outbreaks of 2013-2016 and 2018-2020. unlike evd, sars-cov-2 appears to pose the greatest threat to a small portion of the population. we are asking everyone to make financial, educational, and social sacrifices for those most at risk. once again, outbreaks begin to wane when communities listen to trusted voices and commit as one to make these sacrifices. when it comes to outbreaks, we must convince communities to join together, trust our leaders' advice and be all in it together. financial support. j. s. s. reports other support from walters-kluwer outside of the submitted work. potential conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. palm writing group; palm consortium study team. a randomized, controlled trial of ebola virus disease therapeutics world health organization. ebola ring vaccination results ebola responders killed as violence flares fear of ebola breeds a terror of physicians fear of ebola drives mob to kill officials in guinea identifying mechanisms of violence that impact ebola virus disease transmission during the 2018-2019 outbreak in the democratic republic of the congo chains of transmission and control of ebola virus disease in conakry, guinea, in 2014: an observational study considerations for use of ebola vaccine during an emergency response rapid assessment of knowledge, attitudes, practices, and risk perception related to the prevention and control of ebola virus disease in three communities of sierra leone a multi-site knowledge attitude and practice survey of ebola virus disease in nigeria ebola virus epidemic in war-torn eastern dr congo institutional trust and misinformation in the response to key: cord-333411-hqtb4a2c authors: tan, tina q; kullar, ravina; swartz, talia h; mathew, trini a; piggott, damani a; berthaud, vladimir title: location matters: geographic disparities and impact of coronavirus disease 2019 (covid-19) date: 2020-09-17 journal: j infect dis doi: 10.1093/infdis/jiaa583 sha: doc_id: 333411 cord_uid: hqtb4a2c the covid-19 pandemic in the united states has revealed major disparities in the access to testing and messaging about the pandemic based on the geographic location of individuals, particularly in communities of color, rural areas, and areas of low income. this geographic disparity, in addition to deeply rooted structural inequities, have posed additional challenges to adequately diagnose and provide care for individuals of all ages living in these settings. we describe the impact that covid-19 has had on geographic disparate populations in the united states and share our recommendations to what might be done to ameliorate the current situation. coronavirus disease 2019 has emerged as the worst global pandemic in the last 100 years, with almost 27 million diagnosed cases and over 880,000 deaths as of september 6, 2020. 1 covid-19 has exerted a devastating toll across all communities. however, disparity exists between low and middle-high income geographies in the level of care available to patients with covid-19. an analysis of deaths in massachusetts found an increase in excess deaths in the early days of covid-19. 2 mortality was 40% greater in cities and towns "with higher poverty, higher household crowding, higher percentage of populations of color, and higher racialized economic segregation" compared to those with the lowest levels of those measures. 2 a striking example of the impact of geography on covid-19 disease is the navajo nation, which has reported the highest per capita covid-19 rate in the united states with a rate of 2,304 cases per 100,000 people compared to new york state, new jersey, or the united states with rates of 1,806 cases/100,000, 1,668 cases/100,000, and 605 cases/100,000, respectively. 1 in memphis, data reveal that most covid-19 testing occurs in the predominantly white and well-off suburbs, not the majority african american, lower-income neighborhoods. 3, 4 further, testing rates are lowest in states designated as being unhealthy (based on factors such as life expectancy, population living at or below the poverty level, and number of low birth weight infants). this approach is problematic because the populations in these states have higher rates of chronic conditions that increase the risk of serious complications and death from covid-19, such as heart disease, chronic respiratory diseases, and diabetes. 5 these disparities in testing rates are concerning because delays in testing increase the chance of a surge in silent spread and severe covid-19 cases in states among rural and lower-income populations. in this viewpoint, we will discuss the geographic disparities that exist during this pandemic and provide our recommendations on what can be done to change the current paradigm. a c c e p t e d m a n u s c r i p t there is a wide disparity in covid-19 testing by geographic area. such disparities may stem from a lack of: hospitals and health care facilities, walk-in testing sites, access to transportation to drive-up testing sites, and lack of information regarding availability of testing. although urban sites may have public transportation and walk-in testing facilities, communities in rural areas or low income settings are challenged by limited access to individual/private transportation or walk-in testing locations. 6 a recent analysis highlighted that geographic access to covid-19 testing sites is as uneven as access to overall health care, with higher travel times to covid-19 test sites in rural counties and in counties with a larger non-white or uninsured population, suggesting that covid-19 cases are potentially under-counted and under-reported in these areas. 7 some of these limitations may be unique to communities in specific geographic areas, including areas with a large amish or mennonite population (where access to healthcare facilities is limited), native american communities, and other socially isolated communities. testing for novel diseases and information regarding how to access testing needs to be made widely available with resources tailored to the needs of the persons residing in the community, to avoid stigma associated with a new disease. it is critical that jurisdictions expand public health and commercial laboratory testing in all communities regardless of geography. innovative methods of testing, such as mobile testing units or home testing kits, should also be considered. while transportation may pose unique challenges to accessing testing, accurate information about the pandemic may not be disseminated to communities further away from urban settings and in isolated communities. these populations may consider pandemics more of an "urban disease" and less relevant for rural settings with lower population density and access to covid-19 testing has been limited across the united states with testing practices varying across different local and regional areas and across age groups. in many areas of the country, prioritization for testing has been limited to persons who are symptomatic. in general, many studies report that persons ≤ 18 years of age are more likely to be asymptomatic or have milder symptoms. therefore, they may not meet the testing criteria even if there has been significant exposure, leading to limited number of tests provided to children relative to the 22% of the population that they comprise. although the risk of severe based on antibody seroprevalence studies, it is estimated that the total number of persons in the u.s. infected with covid-19 is at least 10 times higher than the current number of confirmed cases, indicative of significant undertesting in pediatric and adult populations alike. [18] [19] [20] because of the lack of testing, the proportion of infants, children, adolescent and young adults infected with covid-19 who are potentially at risk for developing serious or life-threatening illness remains unclear. the limited access to testing is significantly worse for those who live in rural areas and low income settings, inhabited by a disproportionate number of socio-economically disadvantaged whites, african americans, and latinx populations. programs focused on increased testing of children, utilizing community centers, places of worship, and schools, are needed, especially with schools reopening. the covid-19 pandemic initially impacted the most densely populated areas in the united states particularly large urban areas in which health systems endured waves of cases that overwhelmed resources including hospital beds, personal protective equipment, intensive care capacity, ventilators, and personnel. it appeared that more rural communities had initially been less directly impacted. however, as social distancing recommendations around the country have relaxed and as access to testing has started to increase, rural populations have become more vulnerable to new surges of infection. those living in rural communities may not have experienced the first hand perception of threat and may be less likely to maintain strict adherence to precautions such as social distancing and wearing of masks. 21 the major concern for covid-19 infections in rural populations, in which 14% of the u.s. a c c e p t e d m a n u s c r i p t 8 population reside, is that healthcare systems are less equipped for high capacity and high acuity care. non-metropolitan hospitals have fewer intensive care unit beds (1.7 versus 2.8 beds per 10,000 population), fewer intensive care resources, and less access to id subspecialists. 22 a study looking at the distribution of u.s. id specialists found that of the 3,142 u.s. counties, 2,499 counties (79.5%) do not have a single id physician. 23 furthermore, individuals living in rural areas are more likely to be older and have underlying conditions that place them at an increased risk for severe infection. 24 therefore, outbreaks in rural areas have the potential to overwhelm the limited healthcare resources available. potential action items to ease the burden include: equipping previously non-icu areas to function as icus, expansion of in-hospital tele-health capabilities for subspecialty consults, and having state licensing boards allow qualified physicians and nurses to temporarily staff rural hospitals in an outbreak. the covid-19 pandemic has accentuated longstanding racial/ethnic disparities in healthcare access and outcomes in the united states particularly in regard to geographic locations in rural and remote areas and low income settings. as rural and urban geographic areas remain interconnected, health policymakers and government authorities need to develop emergency and preparedness plans that address the limited access to covid-19 testing, effective means of communication, provider shortage, and lack of healthcare facilities and intensive care units in rural areas. these geographically distant and isolated spots of covid-19 transmission can become fertile grounds for resurgence of the pandemic. the authors have no conflicts of interest to disclose. financial support: no funding was received. m a n u s c r i p t covid-19 and the unequal surge in mortality rates in code measures of poverty, household crowding, race/ethnicity, and racialized economic segregation racial disparities emerge in tennessee's testing for covid-19 data on covid-19 cases in unhealthier states have lower covid-19 testing rates rural america could be the region hardest hit by the covid-19 outbreak geographic access to united states sars-cov-2 testing sites highlights healthcare disparities and may bias transmission estimates characteristics and outcomes of children with coronavirus disease 2019 (covid-19) infection admitted to us and canadian pediatric intensive care units hyperinflammatory shock in children during covid-19 pandemic. the lancet acute heart failure in multisystem inflammatory syndrome in children (mis-c) in the context of global sars-cov-2 pandemic multisystem inflammatory syndrome related to covid-19 in previously healthy children and adolescents multisystem inflammatory syndrome in u.s. children and adolescents covid-19 possible striking more children than expected covid-19 in children in the united states: intensive care admissions, estimated total infected, and projected numbers of severe pediatric cases in 2020 disease 2019 in children -united states hospitalization rates and characteristics of children aged ≤ 18 years hospitalized with laboratory-confirmed covid-19 -covid-net, 14 states state-level data report seroprevalence of antibodies to sars-cov-2 in 10 sites in the united states cdc los alamos national laboratory covid-19 confirmed and forecasted case data covid-=19 fears diminish in many rural areas american hospital capacity and projected need for covid-19 patient care | health affairs where is the id in covid-19? the authors would like to thank the staff of the infectious diseases society of america (idsa) for their assistance with preparing this manuscript. a c c e p t e d m a n u s c r i p t 9 a c c e p t e d m a n u s c r i p t 11 key: cord-253768-y35m3vh1 authors: springer, sandra a; barocas, joshua a; wurcel, alysse; nijhawan, ank; thakarar, kinna; lynfield, ruth; hurley, hermione; snowden, jessica; thornton, alice; del rio, carlos title: federal and state action needed to end the infectious complications of illicit drug use in the united states: idsa and hivma’s advocacy agenda date: 2020-10-01 journal: j infect dis doi: 10.1093/infdis/jiz673 sha: doc_id: 253768 cord_uid: y35m3vh1 in response to the opioid crisis, idsa and hivma established a working group to drive an evidenceand human rights-based response to illicit drug use and associated infectious diseases. infectious diseases and hiv physicians have an opportunity to intervene, addressing both conditions. idsa and hivma have developed a policy agenda highlighting evidence-based practices that need further dissemination. this paper reviews (1) programs most relevant to infectious diseases in the 2018 support act; (2) opportunities offered by the “end the hiv epidemic” initiative; and (3) policy changes necessary to affect the trajectory of the opioid epidemic and associated infections. issues addressed include leveraging harm reduction tools and improving integrated prevention and treatment services for the infectious diseases and substance use disorder care continuum. by strengthening collaborations between infectious diseases and addiction specialists, including increasing training in substance use disorder treatment among infectious diseases and addiction specialists, we can decrease morbidity and mortality associated with these overlapping epidemics. the epidemiology of the us opioid epidemic continues to evolve and presents new challenges. in recent years, the epidemic has shifted from prescription opioid pills to injection of illicitly produced opioids, including heroin and fentanyl, with concomitant increasing injection of stimulants including cocaine and methamphetamine [1] [2] [3] . as a result, the incidence of injection drug use (idu)-related infections such as human immunodeficiency virus (hiv), hepatitis c virus (hcv), hepatitis b virus (hbv), and invasive bacterial and fungal infections, including staphylococcus aureus bacteremia, endocarditis, and skin and soft tissue infections, is rising [2, [4] [5] [6] [7] [8] [9] [10] [11] . injection of fentanyl or heroin alone and in combination with stimulants have led to new hiv outbreaks among people who use drugs throughout the country [4, [10] [11] [12] . in addition to hiv, both acute hcv and hbv infection incidence has mirrored the rise in injection opioids [5, 13] and hospitalizations for injection opioid-related endocarditis have increased more than 12-fold in recent years [6, 8] . at the state of the union address in february 2019, president trump called for a plan to end hiv as an epidemic in the united states. this plan seeks to reduce new infections by 75% in the next 5 years and by 90% in the next 10 years. even amid the opioid epidemic, such ambitious goals can be achieved if policy changes occur and adequate resources are provided. thus, more than ever, addressing the hiv epidemic as well as hcv and other idu-related infections also requires a focus on the opioid and co-occurring stimulant epidemics. doing so will improve patients' outcomes and reduce the public health risk of infectious disease transmission. nevertheless, a number of barriers to care in people who use drugs need to be addressed to end the opioid and hiv epidemics in the united states as well as reduce the other infectious disease health outcomes. to address these barriers we recommend expanding medicaid, expanding access to harm reduction services, improving treatment and surveillance to enhance the continuum of care, and treating opioid and other substance use disorders (sud), including through lowbarrier hospital and community-based treatment, as well as in the criminal justice setting. the authors of this paper are members of a working group created by the infectious diseases society of america (idsa) and the hiv medicine association (hivma) in 2017 to enhance their efforts to educate and advocate on the urgent need to better prevent and treat serious infections linked to the opioid and stimulant epidemics and underlying sud. the working group developed a policy agenda reflecting issues raised by infectious diseases and hiv physicians and health care professionals working at the intersection of infectious diseases and opioid use disorder (oud) and other sud epidemics more broadly. in this paper, we outline practice and policy suggestions that are likely to positively impact the oud, stimulant epidemics, and infectious diseases epidemics, and that have been reviewed and approved by the idsa and hivma board of directors as a call to action for infectious diseases and hiv practitioners. medications for treatment of opioid use disorder (mouds, which is now the preferred term to medication-assisted therapy) are recognized as the most effective treatments for oud [14] . there are 3 food and drug administrationapproved mouds-methadone, buprenorphine, and extended-release naltrexone (xr-ntx). methadone is a full opioid agonist and buprenorphine is a partial opioid agonist, while xr-ntx is an opioid antagonist. all are successful in treating oud and in decreasing mortality. all reduce illicit opioid use, opioid craving, overdose, and hiv and hcv transmission [14, 15] ; and buprenorphine and xr-ntx also improve hiv viral suppression in people living with hiv, the gold standard of care in treatment of hiv that is associated with reduced mortality and reduced transmission [16, 17] . of the 3 mouds, access to methadone and buprenorphine are limited by regulations. prescribing requires special training outside postgraduate programs and either a waiver from the drug enforcement agency in the case of buprenorphine or treatment in a federally certified opioid treatment program in the case of methadone. unfortunately, many clinical settings lack physicians trained in oud treatment. only about 5% of the nation's physicians have waivers to prescribe buprenorphine and most substance use treatment programs do not have opioid treatment programs, which makes methadone treatment challenging to obtain [18] . therefore, the prevailing care for these patients typically consists of withdrawal management or detoxification and referral to outpatient resources for follow-up treatment. this asks patients with severe oud to tolerate withdrawal symptoms, risking premature exit from hospital, and relapse to opioid use after failure to connect with oud treatment referrals. such inadequate care results in prolonged hospitalizations due to concern about relapse and nonadherence if patients leave the hospital, readmissions after oud relapse, and, if concomitant infection is present, lack of antibiotic adherence and reinfection. ultimately, this cycle leads to poor clinical outcomes, high health care costs, and excess deaths. infectious disease specialists are at the frontlines in many hospitals treating infectious diseases in people who use drugs and have an opportunity to screen and treat co-occurring suds. in 2018, congress passed legislation offering opportunities to heighten the response to the opioid epidemic and its infectious diseases complications. on 24 october 2018, president trump signed into law the substance use disorder prevention that promotes opioid recovery and treatment (support act). this bill includes a range of prevention, care, workforce, and public health provisions to strengthen the response to the opioid epidemic (table 1 ) [19] . the bill was passed with strong bipartisan support from congressional members recognizing that the status quo was woefully inadequate to respond to the opioid epidemic. idsa and hivma supported the support act, including provisions that improved medicaid and medicare coverage of sud treatment and services, and that increased the patient cap for which physicians could prescribe moud. priority issues for idsa and hivma were provisions authorizing funding for the centers for disease control and prevention (cdc) to eliminate opioid related infections through improved surveillance and prevention for infections linked to idu and funding for the health resources and services administration to build workforce capacity through a new substance use treatment provider loan forgiveness program, offering up to $250 000 in loan repayment over 6 years for providers working in substance use treatment facilities [20] . both programs depend on congress to appropriate funding. five million dollars was appropriated for fiscal year 2019 for the cdc eliminate opioid related infections funding provision. the fiscal year 2020 appropriations bills were signed into law on december 20, 2019 and included $10 million for the cdc's eliminate opioid related infections program and $12 million for the new substance use disorder loan forgiveness program [21] . other legislative proposals supported by idsa and hivma that have been introduced in the 116th congress include: the medicaid re-entry act that would allow medicaid coverage for inmates during the 30-day period preceding release from a public institution [22] ; the comprehensive addiction resources emergency act modeled after the highly successful ryan white hiv/aids program and that would provide funding to states to support comprehensive prevention, care, and treatment programs [23] ; and the mainstreaming addiction treatment act that would eliminate the requirement for clinicians to obtain a waiver to prescribe buprenorphine [24] . as outlined in this paper, urgent policy action is needed to reduce illness and death due to our nation's substance use epidemics. in addition to state and jurisdictional bans or restrictions on syringe services programs (ssp), funding remains a significant barrier to expanding access to ssp services [25] . increased state and federal funding are needed to expand ssp and other harm reduction services, including access to moud and infectious diseases treatment services in order to decrease hcv, hiv, idu-related infections, and vaccine-preventable diseases, and improve oud-related outcomes [26, 27] . studies have demonstrated that incorporation of ssps combined with moud is associated with a decrease in hcv and hiv acquisition risk by 76% and 34%, respectively [28] . ssps also can facilitate vaccine uptake for hepatitis a virus (hav), hbv, influenza, and invasive pneumococcal disease, which disproportionately impact people who inject drugs or experience unstable housing or homelessness [29, 30] . ssps can offer a safe space without stigma for individuals with suds to also receive counseling regarding safe sexual practice, safe injection practice, and provision of hiv preexposure prophylaxis (prep) and contraception. ssps that also provide treatment for oud can facilitate linkage to care for effective evidence-based treatments [31] . furthermore, persons with suds may be reluctant to seek nonemergent care for skin and soft tissue infections and postpone medical evaluation until the need is more urgent. providing care for skin and soft tissue infections in a supported setting may reduce progression to serious infections and reduce complications like wound botulism or partial drainage of abscesses. given the increased incidence of idu-associated infections and overdose deaths [1, 2] , there is a need to provide ongoing support for and increased access to ssps. the hiv outbreak in scott county, indiana, in addition to other emerging hbv and hcv epidemics, has highlighted the need to expand ssps, particularly in nonurban areas [32] . although federal funds to support ssps was an important step, additional federal funding and flexibility are needed to fully cover services and costs associated with these programs, including purchasing of sterile syringes and to support delivery of moud at ssps [33] . in jurisdictions where ssps are prohibited or sparse, cities and states should be incentivized to modify their laws and to encourage uptake by local jurisdictions. in some states, there is a limit on the number or location of such programs, or ssps may only be allowed during certain circumstances (ie, public health emergencies) [34] . such limitations should be eliminated given the documented need for these programs and their potential to reduce infectious diseases [34, 35] . additionally, drug paraphernalia laws, which prohibit possession of syringes, pose barriers to ssp expansion and effectiveness [36] . state and local governments should be encouraged to employ innovative programming, including mobile delivery and contracting with communitybased organizations. additionally, states should be incentivized to eliminate 1-for-1 syringe exchange (ie, exchanging 1 used syringe for 1 sterile syringe) because they create barriers to individuals who inject drugs having an adequate supply of sterile 2. incentivize states to give more authority to local governments to establish ssps and to eliminate barriers to sterile syringes, such as one-for-one needle exchange requirements. 3. allow jurisdictions that have approved overdose prevention sites or supervised injection facilities to implement and evaluate the intervention in the united states. 4. urge all states to expand medicaid. 5. fund demonstration projects and pilot studies to identify effective care models for comanagement of infectious diseases and sud. 6. increase funding for national and regional warmlines and peer-to-peer mentoring, programs for prescribers of mouds, and for cotreatment of related infections. 7. eliminate the buprenorphine waiver, remove patient caps, and offer grant funding for case management and other support services to clinics and practices that prescribe mouds. 8. increase funding and reimbursement for telehealth and other low-barrier access care delivery models. 9. support implementation of universal hcv testing. 10 . develop a national surveillance system to report and track idu-related infections to predict and respond to emerging epidemics. 11. integrate moud and counseling services during incarceration. 12. integrate screening for oud and treatment with moud into jails and prisons. 13. expand access to harm reduction during and after incarceration. 14. allow states to initiate medicaid coverage 30 days prior to release from criminal justice settings to facilitate care initiation and coordination during the transition to the community. syringes. secondary exchange, or the distribution of sterile syringes from 1 person to a social network, is often necessary due to distance and transportation barriers. in addition to ssps, other harm reduction services are needed to address the expanding epidemics. overdose prevention sites (also known as supervised injection facilities or safe injection sites) are facilities in which persons can inject drugs in a safe, clean environment under medical supervision. overdose prevention sites enable rapid, life-saving intervention in the case of drug overdose and can also provide injection equipment and referrals to care for sud and other health care services. overdose prevention sites have existed for many years in europe, australia, and canada. studies of overdose prevention sites in vancouver and sydney have found an increase in withdrawal management or detoxification service referrals and a decrease in drug overdose rates, syringe sharing, public injections, and publicly discarded syringes [37] [38] [39] . several us municipalities have advocated for overdose prevention sites, but political opposition has so far impeded implementation. a recent modeling study in seattle estimated that an overdose prevention site would yield cost savings through prevention of overdose deaths, enrollment in mouds, prevention of emergency medical services deployments, and emergency department visits and hospitalizations [40] . although concerns have been raised about violation of federal and state drug laws, overdose prevention sites have been legally established successfully in areas outside of the united states. review of the processes and experience could facilitate implementation in us jurisdictions that have approved overdose prevention sites. jurisdictions that have approved overdose prevention sites should be allowed to implement and evaluate the intervention in the united states. significant work needs to be done to improve the care continuum for people with infectious diseases and co-occurring sud. the first step needs to be ensuring that everyone has access to health care. federal support for the medicaid expansion must continue and the 14 states that have not expanded medicaid should be incentivized to do so [19] . recent studies have shown that expansion of health care services, mostly via medicaid expansion, increased utilization of moud [3, 41, 42] . expanding access to health coverage is necessary to prevent and treat the infection, underlying sud, and improve overall mortality and quality of life as evidenced by studies finding an association between enrollment in an affordable care act qualified health plan and improved outcomes for people with hiv [43] . as a next step, treatment programs that integrate substance use care and treatment for infectious complications in order to improve outcomes are needed. treatment of both the sud and associated infections (eg, hiv, hcv) can be cost-effective and is associated with improved infection and sud outcomes [44] . previous studies have shown that patients with either hiv or hcv who receive mouds have improved viral suppression (hiv) [16, 17] , achieve sustained virologic response/ cure (hcv) [45, 46] , and have increased retention in care [47] . however, significant gaps remain in understanding the role substance use treatment plays in caring for people with other idurelated infections, such as endocarditis, deep tissue abscesses, skin and soft tissue infections, and bone and joint infections. one innovative care model combined outpatient parenteral therapy with buprenorphine treatment and showed similar clinical and drug use outcomes to completing inpatient therapy and resulted in reduced hospital length of stay by 24 days [48] . studies are needed to evaluate novel approaches to antimicrobial treatment for idu-associated infections such as the role of long-acting glycopeptides. increased funding is necessary for other demonstration projects and pilot studies to identify effective care models for comanagement of infectious diseases and oud as well as other suds. additionally, we need to expand the network of providers prescribing moud. most infectious diseases and hiv physicians receive little to no formal training in the management of oud and other suds. training to identify and treat oud and other suds should be increased in medical schools, nursing schools, physician assistant schools, residency programs, and within hospitals. while all infectious diseases and hiv physicians should become familiar with harm reduction principles and be able to counsel patients regarding safe injection practices, we need broader national support for physicians table 2 to comanage oud, suds, and co-occurring infectious diseases. lack of confidence has been identified as a major barrier preventing some physicians from integrating buprenorphine into their practice for the treatment of oud [49] . warmlines, such as the one run by the clinical consultation center at the university of california san francisco, and videoconferencingbased learning communities such as project echo, are excellent resources to provide support on a number of clinical aspects of disease management (table 2 ) [44] . increasing funding for national and regional warmlines, telehealth-based learning communities, peer-to-peer mentoring programs, and other technical assistance programs such as the opioid response network will help decrease barriers to providing substance use treatment. the opioid response network is a network of experienced clinicians that is funded by the substance abuse mental health and services administration to provide technical assistance to improve access to substance use treatment. in addition, a reorganization of the buprenorphine prescribing system is needed. in order to increase the number of providers who prescribe moud and improve patient access, we recommend eliminating the buprenorphine waiver requirement, removing the patient caps, and dedicating grant funding for case management and other support services to clinics that prescribe mouds. increased funding and reimbursement are also needed for low-barrier care delivery models such as telehealth. these innovative programs, which have already begun to be tested in infectious diseases/oud comanagement [50] , have the potential to increase medication uptake and improve outcomes by increasing access to treatment where people reside. in addition, multidisciplinary team meetings, including surgeons, sud specialists, inpatient internal medicine clinicians, nurses, social work, and case management, are being piloted in several hospitals across the country in order to make informed and collaborative decisions on complex patients, such as those with recurrent endocarditis following valve repair. evaluation of the impact of these types of collaborative efforts, both on patient outcomes and workplace satisfaction, can help inform best practice for all hospitals. we also need to address the requirements of particularly highrisk patients, including pregnant women and infants born to mothers with oud, and persons experiencing homelessness who may be unable to access traditional care. oud among pregnant women has increased significantly and there is an urgent need to build capacity to manage oud among pregnant women [51] . infants born to mothers with oud during pregnancy are at increased risk for hiv, hbv, and hcv. screening for hiv, hbv, and hcv is recommended for all pregnant women [52] and has been successfully integrated into most prenatal screening paradigms, allowing for perinatal management that decreases the risk of infant infection. in september 2006, the cdc recommended screening all sexually active persons 13-65 years old for hiv at least once, but this has not occurred and needs emphasis in order to end the epidemic. in august 2019, the us preventive services task force issued a draft recommendation for universal hcv screening [53] . given that overall incidence of hcv is increasing alongside the opioid epidemic [54] , strategies including provider education and increased resources are needed to ensure universal hcv testing is performed, particularly among women of child-bearing age and in prenatal care to prevent infant infection [55] [56] [57] . persons experiencing homelessness and unstable housing are similarly at increased risk for infections associated with sud. this is, in part, due to the high prevalence of concomitant untreated mental illness and sud among these individuals and sanitation issues [58, 59] . it is also due to our inability to implement effective management strategies for sud and infections in this vulnerable population. in addition to ensuring persons who experience homelessness receive treatment of their infectious diseases and sud through low-barrier and street-based medicine programs, expanding access to stable housing would also improve short-and long-term outcomes and should be part of a comprehensive strategy [60, 61] . finally, to monitor progress of these interventions, we need a standardized mechanism for reporting idu-related infections. other than for hiv and, in some states, for hcv infection, there is no national database of idu-related infections for surveillance, prevention activities, and program evaluation. this makes it difficult-if not impossible-to identify, predict, and prevent new infectious disease epidemics related to substance use in the united states. in addition, the majority of federal funding has been directed towards opioid overdose treatment and hiv resultant from idu, but not toward the bacterial and fungal infection complications, partly due to lack of integrated surveillance systems for serious idurelated infections, such as endocarditis. developing national surveillance systems to track and predict new epidemics before they happen and increasing national institutes of health funding for research into other infectious diseases related to the worsening sud epidemics in this country are urgently needed. over half of the criminal justice-involved population (cjip) have oud or sud, with a 10-fold higher prevalence than found in the general adult population [62] . as such, employing and enforcing evidence-based treatment guidelines that address the overlap of sud and infectious diseases in the criminal justice system has the potential to improve morbidity and mortality substantially. key components include evaluating new entrants for sud and idu-related infections, integrating moud and counseling services during incarceration, providing both appropriate medical care during incarceration and harm reduction during and after incarceration, care coordination, seamless referral to outpatient care for sud and chronic infections, and uninterrupted insurance coverage for cjip. intertwined with national increases in sud and incarceration rates, there have been substantial increases in hiv and hcv in cjip, as well as outbreaks of hav and hbv [63, 64] . inequities that exist in health care access in the community are amplified by criminal justice involvement, leading to premature deaths [65] . mortality rates are high following release, primarily driven by untreated oud leading to fatal overdose, progression of hiv, and hbv/hcv-induced liver disease [66] [67] [68] . additionally, overall infectious disease testing rates and rates of vaccination against hav and hbv are low [69, 70] . integration of infectious disease management with treatment for oud in cjip is an endorsed strategy for reducing these health inequities that will likely lead to improved infectious disease outcomes and facilitates linkage to care postrelease [16, 17, 71] . despite the evidence, however, few incarcerated settings offer moud. sud screening in jails and prisons with linkage to substance use treatment also decreases postrelease mortality [72, 73] and increases postrelease hiv viral suppression [16, 17] . clearly, prevention and treatment for oud and associated infections in this population can improve both individual outcomes and public health, especially when initiated during incarceration. time spent in prison or jail provides a reachable moment-an opportunity to engage a vulnerable population. screening for oud and treatment with moud need to be integrated into jails and prisons to improve substance use and infectious diseases outcomes. in addition to testing and treatment, access to harm reduction tools to prevent infection needs to be prioritized in the cjip. harm reduction tools like condoms and clean needles are not routinely available in prisons or jails despite several research studies demonstrating the need for such tools, and the consequences of not providing them [74, 75] . increasing awareness and availability of prep in jails and prisons-continued from the community, initiating while detained, or initiated before release-need to be urgently deployed, especially in communities deemed to be at high risk of hiv outbreak [76] . as evidenced by previous successful implementation of intensified harm reduction, expansion can be implemented in jails, effectively containing outbreaks [77] . substance use treatment coupled with uninterrupted health insurance is needed to improve outcomes among persons who are released from jail and prison. as a case study, expansion of hcv treatment during incarceration is feasible, cost-effective, and the best option to move closer to national hcv elimination [78, 79] . hcv diagnosis in jails with linkage postrelease is a feasible alternative if hcv treatment costs are deemed prohibitive [80] . a major barrier in hcv linkage to care postrelease is that 90% of states have policies that withdraw enrollment in insurance programs when people are incarcerated, outsourcing health care to medical corporations hired by criminal justice administrators [81] . prior to release, there are often attempts to reestablish health insurance, but this is complex because of uncertainty around the date of release and place the person will live. the process of re-entry is a vulnerable time for people who are incarcerated, with high mortality related to drug use but also associated with suboptimal postrelease management of chronic conditions like liver disease [82] . increased flexibility of medicaid, allowing initiation of insurance before release and sustained coverage prior to conviction, would improve health care transitions into and out of correction settings. finally, additional research funding is needed to develop and evaluate strategies to manage non-hiv/hcv-related infections secondary to idu, such as skin and soft tissue infections or infective endocarditis. despite increasing frequency of endocarditis in people with oud/sud [7] , and high rates of history of incarceration in people with skin and soft-tissue infections [7] , there are limited data on the epidemiology of disseminated bacterial and fungal infections in cjip. since the time this manuscript was accepted in december of 2019 the covid-19 pandemic has changed the world and has made the implementation of many of these recommendations even more urgent. we are at a pivotal moment in the opioid epidemic in the united states. as we desperately attempt to decrease the staggering number of overdose deaths, we must also grapple more broadly with idu in general, which is causing increases in hiv, hcv, and other idu-related infections. as a result, we as infectious disease specialists need a paradigm shift in our clinical approach, and we need broad and aggressive policy changes to support that shift. throughout history, infectious diseases clinicians have risen to the challenge. in 1998, dr jonathan mann said in an address, "when the history of aids and the global response is written, our most precious contribution may well be that, at a time of plague, we did not flee, we did not hide, we did not separate ourselves. " this time is no different-it is our epidemic too. disclaimer. the funders were not involved in the research design, analysis, or interpretation of the data or the decision to publish the manuscript. financial support. this work was supported by the national institute on drug abuse, national institutes of health (grant number k02 da032322 for career development to s. a. s.). supplement sponsorship. this supplement is sponsored by the centers for disease control and prevention. potential conflict of interests. s. a. s. has provided scientific consultation to alkermes inc. j. a. b. reports providing waiver training courses for providers to become buprenorphine prescribers through the providers clinical support system. a. w. is a consultant to gensler architectural firm. a. n. reports grants from gilead focus program outside the submitted work. r. l. reports royalties for a book on infectious disease surveillance donated to minnesota department of health. all other authors report no 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system filling the gap: the importance of medicaid continuity for former inmates public health implications for adequate transitional care for hivinfected prisoners: five essential components key: cord-266695-ktbgm0p9 authors: dawson, liza; earl, jake; livezey, jeffrey title: sars-cov-2 human challenge trials: too risky, too soon date: 2020-06-04 journal: j infect dis doi: 10.1093/infdis/jiaa314 sha: doc_id: 266695 cord_uid: ktbgm0p9 nan a c c e p t e d m a n u s c r i p t 2 eyal et al. have recently argued that researchers should consider conducting sars-cov-2 human challenge studies to hasten vaccine development [1] . we have conducted (jl) and overseen (ld) human challenge studies and agree that they can be useful in developing anti-infective agents. we also agree that adults can autonomously choose to undergo risks with no prospect of direct benefit to themselves. however, we disagree that sars-cov-2 challenge studies are ethically appropriate at this time, for three reasons: 1) current scientific knowledge of sars-cov-2 infection is insufficient to manage risks; 2) autonomous decision-making, while necessary, does not override concerns about risk; and 3) undertaking challenge studies now would imperil confidence in the research enterprise, potentially undermining the global response to the covid-19 pandemic. current scientific knowledge is insufficient to manage the risks of severe disease or death of volunteers in sars-cov-2 human challenge studies, especially in terms of selecting low risk volunteers [2] . new risks of covid-19 continue to emerge, such as unexpected cardiovascular events [3] and strokes in otherwise healthy, young people [4] . selecting a proper dose for a challenge study while protecting volunteers would be difficult given the high variability in patient responses [5] . there are no highly effective treatments, nor is there information about long-term health consequences of infection. eyal et al. allude to other research involving risks of severe disease or death, including human challenge studies for other diseases. but such studies, for example malaria challenge trials, minimize and manage risks to volunteers by using well-characterized pathogens with known clinical sequelae in painstakingly defined sub-populations [6] . malaria treatment with fda-approved a c c e p t e d m a n u s c r i p t 3 drugs is readily available and decades of research enable selection of low-risk volunteers. even so, unexpected events can happen: a genetic polymorphism affecting metabolism of the malaria treatment primaquine was found in a challenge study [7] . had the disease been poorly understood, the results could have been catastrophic. it is not obvious that the possible benefits of developing a successful vaccine in less time justify the risks sars-cov-2 challenge studies, as eyal and colleagues suggest. there is no guarantee that any trial, or series of trials, will produce a viable vaccine: consider vaccine research for hiv or hepatitis c. there is also little precedent for fda to license a vaccine primarily based on evidence from challenge studies (recent approval of a cholera vaccine is an exceptional case [8] ). even promising results in challenge studies may not correlate with population-level effects [9] , and additional field trials would be needed. if a vaccine is proven effective, obstacles to production and distribution might limit how many lives it saves [10] . autonomous authorization (informed consent) is essential for protecting research volunteers' rights, and eyal et al. emphasize the legitimacy of a mature person's choice to accept risk. however, people often make decisions in irrational or idiosyncratic ways-in life generally [11] , and in research. volunteers often believe that unproven experimental treatments will medically benefit them (therapeutic misconception [12] ) or that unproven vaccines will protect against infection (preventive misconception [13] ). altruistic volunteers who sign up for potential challenge studies [14] amidst the global covid-19 pandemic may also suffer from a misconception-an a c c e p t e d m a n u s c r i p t 4 overconfidence that the research will provide substantial societal benefit [15] . given the inherent uncertainty in vaccine development, this kind of optimistic bias could lead people to take risks without seeing the associated benefits, in conflict with their core values and interests. further, volunteers who have a change of heart after being infected with sars-cov-2 would have no opportunity to withdraw from the study that would reduce risk [16] . beyond concerns about decision-making, sars-cov-2 human challenge studies have the potential to be exploitative. there are disparities in power, information, and control between researchers and volunteers [17] . economically disadvantaged people are often willing to join trials despite discomforts and risks because financial compensation is offered [18] . thus, vulnerable members of the public might bear a disproportionate burden of risks that are unjustifiably high. eyal et al. compare volunteering in a sars-cov-2 human challenge study to firefighting and living kidney donation, activities that are permissible despite their risks [19] . but there are important differences between research and non-research activities. clinical research is a complex, fragile enterprise based on shared understanding of risks, burdens, benefits, and values among diverse stakeholders [20] . in addition to rigorous research oversight, the research enterprise depends on stakeholders' mutual trust and willingness to adhere to certain expectations, including that researchers will prioritize the safety of study volunteers [21] . the fragility of the enterprise is due in part to issues noted: idiosyncrasies of human decision-making, uncertain risks and benefits, and potential exploitation. a c c e p t e d m a n u s c r i p t 5 when study volunteers die or suffer serious harm at the hands of researchers, investigators themselves become complicit, potentially undermining the stakeholders' confidence in the research enterprise. one very bad outcome not only harms the individual volunteer, it harms the whole research process [22] , and public trust is likely to plummet [23] . violations of public trust have ripple effects on research, public health efforts, and clinical care. the current landscape facing the research and public health communities is fraught. mistrust of research and of vaccines in particular is rampant; conspiracy theories, misinformation, and antiscience attitudes are spreading. bad outcomes in a sars-cov-2 human challenge study could be devastating, as recent experience demonstrates that mistrust interferes with public health efforts in epidemic conditions [24] . although sars-cov-2 human challenge studies are not ethically acceptable at present, this may change if the following conditions are met: 1. better characterization of factors leading to severe disease and mortality in sars-cov-2 infection to definitively screen out high-risk volunteers. 2. availability of proven effective treatment to prevent severe morbidity and mortality. 3. clearer understanding of protective effects of immunity and the elucidation of the goal of a vaccine to guide dosing and endpoint selection. a c c e p t e d m a n u s c r i p t 6 4 . a public engagement strategy to address the challenge study and the risks to participants. we agree that solutions to the covid-19 pandemic must be expedited, and advocate for efficient research and regulatory processes to support that goal. however, conducting sars-cov-2 human challenge trials now unjustifiably threatens both the well-being of volunteers and confidence in the research enterprise. human challenge studies to accelerate coronavirus vaccine licensure presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the cardiovascular considerations for patients, health care workers, and health systems during the covid-19 pandemic large vessel stroke as a presenting feature of covid-19 in the young clinical and virological data of the first cases of covid-19 in europe: a case series meeting report: convening on the influenza human viral challenge model for universal influenza vaccines, part 1: value; challenge virus selection; regulatory, industry and ethical considerations; increasing standardization, access and capacity primaquine failure and cytochrome p-450 2d6 in plasmodium vivax malaria single-dose live oral cholera vaccine cvd 103-hgr protects against human experimental infection with vibrio cholerae o1 el tor development of the rts,s/as malaria vaccine candidate if a coronavirus vaccine arrives, can the world make enough? nudge: improving decisions about health, wealth, and happiness quality of informed consent in cancer trials: a cross-sectional survey preventive misconception: its nature, presence, and ethical implications for research covid-19 human challenge trials how informed is declared altruism in clinical trials? a qualitative interview study of patient decision-making about the quest trials (quality of life after mastectomy and breast reconstruction) the right to withdraw from controlled human infection studies: justifications and avoidance serial participation and the ethics of phase i healthy volunteer research exploiting a research underclass in phase 1 clinical trials limits to research risks on the alleged right to participate in high-risk research central challenges facing the national clinical research enterprise the oxford textbook of clinical research ethics trust in health research relationships: accounts of human subjects vaccines face same mistrust that fed ebola key: cord-288485-m3g88fl2 authors: lam, katherine w; chow, kenneth w; vo, jonathan; hou, wei; li, haifang; richman, paul s; mallipattu, sandeep k; skopicki, hal a; singer, adam j; duong, tim q title: continued in-hospital angiotensin-converting enzyme inhibitor and angiotensin ii receptor blocker use in hypertensive covid-19 patients is associated with positive clinical outcome date: 2020-07-23 journal: j infect dis doi: 10.1093/infdis/jiaa447 sha: doc_id: 288485 cord_uid: m3g88fl2 background: this study investigated continued and discontinued use of angiotensin-converting enzyme inhibitors (acei) or angiotensin ii receptor blockers (arb) during hospitalization of 614 hypertensive laboratory-confirmed covid-19 patients. methods: demographics, comorbidities, vital signs, laboratory data, and acei/arb usage were analyzed. to account for confounders, patients were substratified by whether they developed hypotension and acute kidney injury (aki) during the index hospitalization. results: mortality (22% vs 17%, p > .05) and intensive care unit (icu) admission (26% vs 12%, p > .05) rates were not significantly different between non-acei/arb and acei/arb groups. however, patients who continued acei/arbs in the hospital had a markedly lower icu admission rate (12% vs 26%; p = .001; odds ratio [or] = 0.347; 95% confidence interval [ci], .187–.643) and mortality rate (6% vs 28%; p = .001; or = 0.215; 95% ci, .101–.455) compared to patients who discontinued acei/arb. the odds ratio for mortality remained significantly lower after accounting for development of hypotension or aki. conclusions: these findings suggest that continued acei/arb use in hypertensive covid-19 patients yields better clinical outcomes. hypertension is a common comorbidity in patients with coronavirus disease 2019 (covid19) and has been associated with worse clinical outcomes [1] [2] [3] [4] [5] [6] . because the widely used antihypertensive medications angiotensin-converting enzyme inhibitors (acei) and angiotensin ii receptor blockers (arb) may upregulate ace2 receptors [7] [8] [9] , through which severe acute respiratory syndrome coronavirus 2 (sars-cov-2) enters the host cells [10] , concerns have been raised as to whether their use may result in increased morbidity and mortality [4, [11] [12] [13] . on the other hand, ace2 has also been shown to have vasodilatory, anti-inflammatory, and antifibrotic effects that could potentially alleviate disease severity [14] [15] [16] . several clinical studies have reported that acei/arb use in covid-19 patients with hypertension does not worsen covid-19 disease severity or mortality [16] [17] [18] [19] [20] [21] [22] . however, these studies have either focused on acei/arb use prior to hospitalization or did not control for potential clinical interactions for which acei/arb use might be discontinued, such as the development of hypotension or acute kidney injury (aki). despite limited data, a number of professional societies [23, 24] have released statements recommending that acei/arbs should be continued in hypertensive covid-19 patients [25, 26] . this is based, in part, on the belief that these agents serve a beneficial role in treating underlying cardiovascular disease and may preclude further clinical deterioration. stony brook university hospital, about 40 miles east of new york city, is the largest academic hospital in suffolk county, serving a population of approximately 1.5 million people and with over 39 000 laboratory-confirmed covid-19 patients at the time of this analysis. the goal of this study was to investigate the effects of in-hospital continuation and discontinuation of acei/arbs on the clinical outcomes of hypertensive covid-19 patients, controlling for newly developed hypotension or aki during hospitalization. this retrospective single-center study from stony brook university hospital was approved by the human subjects committee with an exemption for informed consent and a health insurance portability and accountability act (hipaa) waiver. the stony brook university hospital covid-19 persons under investigation registry consisted of 6235 patients clinically suspected of covid-19 infection from 7 february 2020 to 23 may 2020. confirmation of covid-19 infection was based on a positive real-time polymerase chain reaction test for sars-cov-2 on a nasopharyngeal swab specimen. clinical data at hospital admission, including demographic information, chronic comorbidities present on admission, vital signs, laboratory blood tests, and outcomes, were extracted individually from the patients' electronic medical records. the primary outcome was in-hospital mortality and the secondary outcome was intensive care unit (icu) admission. patients were divided into 2 groups; hypertensive patients: (1) that were not taking acei/arbs at home (group a), and (2) taking acei/arbs at home. the latter group was further divided into those who discontinued acei/arbs during their hospital stay (group b), and those who continued acei/arbs during their hospital stay (group c). frequencies and percentages for categorical variables between the acei/arb groups were compared using χ 2 tests. continuous variables, expressed as median (interquartile range [iqr]), were compared between groups using nonparametric mann-whitney u tests. bonferroni correction for multiple comparisons was used where appropriate. mortality and icu admission rates were compared between group b (acei/arb discontinued) and c (acei/arb continued) with χ 2 tests (unadjusted without covariates) and with logistic regression (adjusted with covariates). age, sex, and significantly different comorbidities between groups were included in logistic regression models as covariates for controlling confounding effects. analyses were also stratified by hypotension and aki status. p values < .05 were considered statistically significant. all statistical analyses were performed using spss version 26 (ibm corporation). the stony brook university hospital covid-19 persons under investigation registry consisted of 6235 patients clinically suspected of covid-19 infection who presented to the emergency department between 7 february and 23 may 2020, of which 2789 were confirmed to be covid-19 positive (figure 1 ). there were 875 patients in this cohort who had a history of hypertension. we excluded 211 patients who were discharged directly from the emergency department, 5 who were transferred to other hospitals, and 45 whose discharge status was still unknown as of 23 may 2020. this yielded a final sample size of 614 hospitalized covid-19 patients with a history of hypertension. group a consisted of 279 hypertensive patients who did not take acei/arbs prior to admission, of whom 224 did not require icu care (ie, general admission) and 55 required icu care at any point during hospitalization. there were 62 deaths in this group. group b consisted of 171 hypertensive patients who discontinued their home medications, acei/arbs, in the hospital, of whom 126 did not require icu care and 45 required icu care. there were 48 deaths in this group. group c consisted of 164 hypertensive patients who continued their home medications, acei/arbs, in the hospital, of whom 144 did not require icu care and 20 required icu care. there were 10 deaths in this group. the median age of patients in the non-acei/arb group was older than that of the acei/arb group (73 years [iqr, 62-83] versus 68 years [iqr, 58-79], p = .004; table 1 ). sex and ethnicity were not significantly different between groups (p > .05). the prevalence of diabetes mellitus was higher in those in the acei/arb group (p = .01) but lower in those with chronic kidney disease (ckd) at baseline (p = .001), whereas the prevalence of asthma, history of coronary heart disease, chronic obstructive pulmonary disease, and cancer were not significantly different between groups (p > .05). note that the prevalence of ckd in our hypertensive covid-19 patient cohort was higher than that for all covid-19 patients in general [27] as expected. hematocrit (p = .003), sodium (p = .001), d-dimer (p = .003), and troponin (p = .005) levels were significantly different between the non-acei/arb and acei/arb groups after correction for multiple comparisons ( table 2) . elevated d-dimer and troponin levels have been previously associated with a more severe covid-19 disease course [7, [18] [19] [20] . for hypertensive covid-19-positive patients, the mortality was not statistically different between the non-acei/arb and acei/arb groups (unadjusted p = .127 without covariates, adjusted p = .336 with covariates; figure 2a ). by contrast, patients in the acei/arb group who continued these medications in the hospital (group c) had a significantly lower mortality rate compared to those who discontinued acei/arb in the hospital (group b) (unadjusted p = .001, adjusted p = .001; figure 2b ). the odds ratio was 0.215 (95% confidence interval, .101-.455). the percentage of hospitalized patients who developed hypotension subsequent to the index hospitalization was higher in the discontinued acei/arg group than the continued acei/arg group (42.7%, 73/171 vs 15.69%, 26//164, respectively; p = .001). the percentage of hospitalized patients who developed aki subsequent to the index hospitalization was higher in the discontinued acei/arb group than the continued acei/arb group (59.1%, 101/171 vs 18.9%, 31//164, respectively; p = .001). thus, mortality was stratified with respect to development of hypotension and aki (table 3 ). in patients that developed in-hospital hypotension, there was no significant difference in mortality between continuing or discontinuing acei/arbs (p > .05). in contrast, patients who did not develop hypotension and continued acei/arbs had a significantly lower mortality rate than those who discontinued (unadjusted p = .001, adjusted p < .017). similarly, in patients who developed aki, there was no significant difference in mortality between continuing or discontinuing acei/arbs in the hospital (unadjusted p = .050, adjusted p = .117). in patients who did not develop aki, those who continued acei/arbs had a significantly lower mortality rate than those who discontinued (unadjusted p = .001, adjusted p = .011). the icu admission rates were not significantly different between the non-acei/arb and acei/arb groups (unadjusted p = .923, adjusted p = .391; figure 3a ). by contrast, the icu admission rate was twice as high in the group that discontinued acei/arbs compared to the group that continued (unadjusted p = .001, adjusted p = .001; figure 3b ). icu admission was also stratified with respect to the development of hypotension or aki (table 4 ). in the hypotension and nonhypotension groups, there were no differences in icu admission rates between continuation versus discontinuation of acei/arbs in the hospital (p > .05). similarly, in the group that developed aki, there was no difference in icu admission rates between continuing or discontinuing acei/arbs in the hospital (p > .05). however, in patients who did not develop aki, those who continued acei/arbs had a significantly lower mortality rate than those who discontinued these agents (unadjusted p < .041, adjusted p = .043). this study analyzed in-hospital acei/arb use in hypertensive covid-19 patients and the associated confounding variables that likely resulted in discontinuation of acei/ arb use. the major findings are: (1) hypertensive covid-19 patients who continued acei/arbs in the hospital had lower in-hospital mortality and icu admission compared to those who discontinued acei/arbs; (2) for in-hospital mortality, this conclusion remained true after controlling for confounders by excluding patients who developed hypotension or aki for which acei/arbs were withheld on clinical grounds; and (3) for icu admission, this conclusion remained true after excluding patients who developed aki. overall, patients who were able to continue their acei/ arb had markedly lower mortality and icu admission rates, suggesting that the ability to continue acei/arb use is associated with improved clinical outcomes in hypertensive covid-19-positive patients and acei/arb may themselves be beneficial. although there were initial concerns regarding the safety of acei/arb use in hypertensive covid-19 patients, the majority of recent clinical studies found neutral effects of acei/arb on covid-19 severity. in a propensity score-matched analysis of over 12 500 patients in a new york city health system, reynolds et al reported that home-use of acei/arbs conferred no significant increase in the likelihood of developing severe covid-19 [18] . yang et al conducted a retrospective case-control study in a single institution in wuhan, china [19] . similar to reynolds et al, they reported no significant difference in mortality and disease severity in patients taking acei/arbs, although they noted that patients taking acei/arbs had lower levels of inflammatory markers such as c-reactive peptide and procalcitonin [19] . if these medications are indeed associated with lower inflammatory markers, they could conceivably play a role in mediating the dysregulated inflammatory response seen in severe covid-19 infections. for the 2 aforementioned studies, it was unclear whether acei/arb use was continued throughout the hospital course. li et al accounted for inpatient use of acei/arbs in covid-19 patients, defining acei/arb use as use of the medications at admission and continued throughout hospitalization [20] . they did not compare the outcomes of patients who continued versus discontinued medication use in the hospital. they concluded that the percentages of patients with hypertension taking acei/arbs did not differ between those with severe and nonsevere infections nor did it differ between nonsurvivors and survivors [20] . meng et al reported potentially positive outcomes in patients taking acei/arbs [21] . in a small cohort of 51 hypertensive covid-19 patients from wuhan, china, they found that patients on acei/arbs included fewer severe cases [21] . zhang et al found similar positive associations [22] . they used propensity score matching for age, sex, symptoms, comorbidities, creatinine, and other variables. they reported that inpatient acei/ arb use was associated with lower mortality rates compared to non-acei/arb users [22] . however, this study did not account for confounding clinical factors for which acei/arbs may be discontinued, such as the development of aki or hypotension during hospitalization. in contrast to most previous studies discussed above, we found that in-hospital continuation of acei/arb use was associated with better outcomes compared to in-hospital discontinuation of acei/arb use. hypertensive sars-cov-2 patients not on acei/arbs, compared with those taking acei/arbs, did not show increased risk of development of hypotension or aki. acei/arbs were discontinued because of the development of hypotension or aki. patients who developed hypotension or aki had a higher mortality rate. it is important to control for these 2 confounders. in the absence of hypotension or aki, patients who continued acei/arbs had lower mortality and icu admission rates compared to those who discontinued them, further supporting that continued acei/arb use may have beneficial effects. to our knowledge, this study reports the largest cohort of hospitalized hypertensive covid-19 patients on acei/arbs from a large academic hospital in the united states. note that the prevalence of ckd in our hypertensive covid-19 patient cohort was higher than that for all covid-19 patients [27] . although the non-acei/arb group was older than the acei/arb group, our statistical analysis included age as a covariate with logistic regression to ensure our primary finding was not due to age per se. abbreviations: acei, angiotensin-converting enzyme inhibitor; arb, angiotensin ii receptor blocker; copd, chronic obstructive pulmonary disease; iqr, interquartile range; ns, not significant. acei/arb continued in the hospital (group c patients). p = .001 using χ 2 test. p = .001 with adjustment for age, sex, history of heart failure, chronic obstructive pulmonary disease, and asthma (or = 0.215; 95% ci, .101-.455). abbreviations: acei, angiotensin-converting enzyme inhibitor; arb, angiotensin ii receptor blocker; ci, confidence interval; or, odds ratio. the mechanisms by which acei and arbs may exert their beneficial effects in hypertensive covid-19 patients are unknown. the continuation of acei/arbs during hospitalization may blunt the adverse effects of hypertension, a well-known risk factor for mortality in covid-19 [26] . acei/arbs have cardioprotective effects in postmyocardial infarction and heart failure patients by reducing myocardial wall stress [28, 29] . additionally, acei/arbs have been shown to reduce microvascular complications in patients with cardiac, cerebrovascular, and renal comorbidities [30, 31] . experimentally, aceis and arbs have the ability to upregulate ace2, which leads to the degradation of angiotensin ii and the increased formation of angiotensin 1-7; the latter is thought to have beneficial vasodilatory, anti-inflammatory, and antifibrotic effects [14] . it is also interesting to speculate that acei/arbs may play a role in ameliorating the detrimental effects of the cytokine storm seen during the immune response to sars-cov-2 [32, 33] , a state that has been linked to the proinflammatory effects of angiotensin ii. finally, arbs have been shown to prevent aggravation of acute lung injury in mice infected with the closely related betacoronavirus sars-cov-1, suggesting a primary pulmonary protective role [34] . finally, it is worth noting that the reasons for stopping acei/arbs were undoubtedly complex with multiple documented and undocumented possibilities. in clinical practice, hypotension and aki are the most frequent justifications for abbreviations: acei, angiotensin-converting enzyme inhibitor; aki, acute kidney injury; arb, angiotensin ii receptor blocker; ci, confidence interval. a χ 2 tests. b logistic regression with adjustment for age, sex, history of heart failure, chronic obstructive pulmonary disease, and asthma (comorbidities that were significantly different between groups b and c). stopping acei/arbs and there did not appear to be any indication that the current pandemic altered that paradigm. however, this is a retrospective study and the decision to continue or discontinue acei/arb use was part of standard clinical care. this study has several limitations. it was a retrospective single-center study. laboratory values were not reassessed in patients who continued or discontinued acei/arbs. moreover, the difference between acei and arb use could not be resolved due to the limited sample size. although aceis and arbs have similar mechanisms of action, their effects may need to be studied separately. this study did not account for when acei/arbs were discontinued for individual patients during their hospital stay. the current sample size did not allow for substratification based on how long the patients were on these medications before they were discontinued. individual chart review indicated that hypotension and aki were the main documented reasons for acei/arb discontinuation. however, in some instances, justification could not be discerned. moreover, early on in the pandemic, uncertainty existed as to whether acei/arbs were harmful so individual practitioners may have reflexively held these medications without indicating their justification. this contribution is beyond the analysis of the present investigation. these findings not only confirm that acei/arb use does not worsen clinical outcomes in covid-19 patients with a history of hypertension, but also suggest that covid-19 patients who are on acei/arbs should continue these medications in the hospital as they may have beneficial effects, as long as these patients do not develop hypotension or aki. data are number of deaths/number of patients, n/n (%). significant values in which p < .05 are noted in bold. abbreviations: acei, angiotensin-converting enzyme inhibitor; aki, acute kidney injury; arb, angiotensin ii receptor blocker; ci, confidence interval; icu, intensive care unit. a χ 2 test. b logistic regression with adjustment for age, sex, history of heart failure, chronic obstructive pulmonary disease, and asthma (comorbidities that were significantly different between groups). clinical characteristics of coronavirus disease 2019 in china a novel coronavirus from patients with pneumonia in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area upregulation of angiotensin converting enzyme 2 by shear stress reduced inflammation and proliferation in vascular endothelial cells effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 cell type-specific expression of the putative sars-cov-2 receptor ace2 in human hearts sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? hypertension prevalence in human coronavirus disease: the role of ace system in infection spread and severity antihypertensive treatment with acei/arb of patients with covid-19 complicated by hypertension covid-19 and heart failure: from infection to inflammation and angiotensin ii stimulation. searching for evidence from a new disease renin-angiotensin system blockers and the covid-19 pandemic: at present there is no evidence to abandon renin-angiotensin system blockers potential harmful effects of discontinuing ace-inhibitors and arbs in covid-19 patients risks of ace inhibitor and arb usage in covid-19: evaluating the evidence reninangiotensin-aldosterone system inhibitors and risk of covid-19 effects of angiotensin ii receptor blockers and ace (angiotensin-converting enzyme) inhibitors on virus infection, inflammatory status, and clinical outcomes in patients with covid-19 and hypertension: a single-center retrospective study association of renin-angiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease 2019 (covid-19) infection in wuhan, china renin-angiotensin system inhibitors improve the clinical outcomes of covid-19 patients with hypertension association of inpatient use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 covid-19: an acp physician's guide and resources hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid-19 renin-angiotensin system inhibition in cardiovascular patients at the time of covid19: much ado for nothing? a statement of activity from the directors of the board and the scientific directors of the italian society of hypertension comorbidities in covid-19: outcomes in hypertensive cohort and controversies with renin angiotensin system blockers cohort of four thousand four hundred four persons under investigation for covid-19 in a new york hospital and predictors of icu care and ventilation long-term aceinhibitor therapy in patients with heart failure or leftventricular dysfunction: a systematic overview of data from individual patients. ace-inhibitor myocardial infarction collaborative group cardiac remodelling and ras inhibition effect of the angiotensin-converting-enzyme inhibitor benazepril on the progression of chronic renal insufficiency. the angiotensin-converting-enzyme inhibition in progressive renal insufficiency study group a novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation angiotensin ii and inflammation: the effect of angiotensinconverting enzyme inhibition and angiotensin ii receptor blockade inside the heart of covid-19 interactions of coronaviruses with ace2, angiotensin ii, and ras inhibitors-lessons from available evidence and insights into covid-19 we thank all health care professionals for their hard work at the front line of the pandemic. key: cord-300019-8vxqr3mc authors: shi, ting; arnott, andrew; semogas, indre; falsey, ann r; openshaw, peter; wedzicha, jadwiga a; campbell, harry; nair, harish title: the etiological role of common respiratory viruses in acute respiratory infections in older adults: a systematic review and meta-analysis date: 2019-03-08 journal: j infect dis doi: 10.1093/infdis/jiy662 sha: doc_id: 300019 cord_uid: 8vxqr3mc acute respiratory tract infections (ari) constitute a substantial disease burden in adults and elderly individuals. we aimed to identify all case-control studies investigating the potential role of respiratory viruses in the etiology of ari in older adults aged ≥65 years. we conducted a systematic literature review (across 7 databases) of case-control studies published from 1996 to 2017 that investigated the viral profile of older adults with and those without ari. we then computed a pooled odds ratio (or) with a 95% confidence interval and virus-specific attributable fraction among the exposed (afe) for 8 common viruses: respiratory syncytial virus (rsv), influenza virus (flu), parainfluenza virus (piv), human metapneumovirus (hmpv), adenovirus (adv), rhinovirus (rv), bocavirus (bov), and coronavirus (cov). from the 16 studies included, there was strong evidence of possible causal attribution for rsv (or, 8.5 [95% ci, 3.9–18.5]; afe, 88%), flu (or, 8.3 [95% ci, 4.4–15.9]; afe, 88%), piv (or, not available; afe, approximately 100%), hmpv (or, 9.8 [95% ci, 2.3–41.0]; afe, 90%), adv (or, not available; afe, approximately 100%), rv (or, 7.1 [95% ci, 3.7–13.6]; afe, 86%) and cov (or, 2.8 [95% ci, 2.0–4.1]; afe, 65%) in older adults presenting with ari, compared with those without respiratory symptoms (ie, asymptomatic individuals) or healthy older adults. however, there was no significant difference in the detection of bov in cases and controls. this review supports rsv, flu, piv, hmpv, adv, rv, and cov as important causes of ari in older adults and provides quantitative estimates of the absolute proportion of virus-associated ari cases to which a viral cause can be attributed. disease burden estimates should take into account the appropriate afe estimates (for older adults) that we report. acute respiratory tract infections (ari), including pneumonia, constitute a substantial disease burden in adults and elderly individuals. respiratory viruses are detected more frequently than bacteria in adults with pneumonia [1] . the substantial contribution of viruses to ari hospitalizations among adults is being increasingly recognized [2, 3] . although influenza virus (flu) is the most widely recognized viral infection associated with respiratory illness, >25 viruses have been linked to pneumonia, causing a substantial disease burden in adults and elderly individuals. these include common pathogens such as rhinovirus (rv), respiratory syncytial virus (rsv), flu, human metapneumovirus (hmpv), parainfluenza viruses (piv), and human coronaviruses (covs) [2] . rsv is associated with a substantial disease burden in adults, especially among older adults (aged ≥65 years) [4] . moreover, adults hospitalized with rsv disease can develop severe respiratory complications [5] . rv has been associated with severe respiratory disease outbreaks in adults in long-term care facilities in several settings [6] . despite advances in diagnostic technology, defining the specific causes of viral pneumonia is challenging, particularly among older adults who may have lower viral loads and for whom viral diagnosis is frequently not considered and/ or testing is not performed [7] . therefore, it is necessary to measure concurrently the background prevalence of nasopharyngeal viral infection in a control (asymptomatic) group, to investigate the etiological role of viruses in older adults with ari to help inform decisions on prevention and management strategies. previously, we have conducted a systematic review to understand the etiological role of common respiratory viruses, focusing on children aged <5 years [8] . to the best of our knowledge, similar estimates for adults are lacking. therefore, we aimed to conduct a similar systematic review to identify all case-control studies since 1996 investigating the potential role of respiratory viruses in the etiology of aris in older adults aged ≥65 years. we conducted a systematic review across 7 databases (including 3 chinese databases) following the approach detailed in the prisma (preferred reporting items for systematic reviews and meta-analyses) guidelines [9] . tailored search strategies were developed and used to search the medline, embase, global health, lilacs, china national knowledge infrastructure (cnki), wanfang data, and chongqing vip databases (appendix). we further searched the reference lists of relevant articles for eligible articles. all searches were limited to between january 1996 and august 2017. no publication status criteria or language restrictions were used. we included studies that fulfilled the following selection criteria (supplementary figure 1) . three investigators (t. s., a. a., and i. s.) conducted independent searches of the english-language literature and extracted data by using standardized data extraction templates. one investigator (t. s.), whose first language is chinese, searched and extracted data from chinese-language databases (ie, cnki, wanfang, and cqvip). the protocol of this review was published in the prospero database (no. crd42017083332). the case group was defined as older adults with ari or pneumonia aged ≥65 years, adapted from world health organization integrated management of adolescent and adult illness [10] . the details of the definitions are displayed in supplementary table 1 . the control group was defined as older adults aged ≥65 years who were either healthy or did not have any respiratory symptoms. we categorized countries as either industrialized or developing, on the basis of 2015 criteria from the united nations children's fund [11] . we calculated odds ratios (ors) as the ratio of the odds of detecting each virus in older adults with ari or pneumonia to the odds of detecting each virus in healthy or asymptomatic controls, with accompanying 95% confidence intervals (cis). we used a continuity correction of 0.0005 if a virus was detected in one group but not the other [12] . this allowed calculation of an or for these instances and enabled inclusion in subsequent meta-analyses. using stata (version 13.0), we performed a meta-analysis of virus-specific ors and reported pooled meta-estimates with corresponding 95% cis, using the random effects model (ie, the dersimonian-laird method) because the included studies are heterogeneous in various aspects and are thus assumed to have different effect sizes [13] . the virus-specific attributable fraction among the exposed (afe) was used to quantify the etiological role of each virus in patients with ari. this is an estimate of the percentage of aris that can be attributed to each virus, in absolute terms [14] , and was calculated as 100 * [or − 1]/or, with a 95% ci (from the corresponding 95% ci of the or). moreover, for a specific virus, if all included studies did not report any virus detection in one group consistently (usually the control group), we assumed that a strong association indicating a possible causal role for this virus in ari could be concluded. in these circumstances, we considered that there was no need to run a meta-analysis that would only result in an extremely high or point estimate and an afe approaching 100%. we identified 4327 (239 from chinese databases) records through the literature search and 5 records from the reference lists of relevant articles. among them, only 16 studies (including 2 from chinese databases) fulfilled our inclusion and exclusion criteria ( figure 1 ) [1, [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] . forty-three studies were excluded for a variety of reasons: no data specific to older adults ≥65 years old were available (n = 1), the case or control definitions were not fulfilled (n = 4), no applicable data for cases and controls were reported (n = 36), or serum was used as the clinical specimen (n = 2). seven studies were conducted within developing countries, while 9 were from developed countries (supplementary table 2 ). although the search was performed for articles published since 1996, all included studies were published since 2003. all included studies were case-control studies with adults who had ari or pneumonia in the case group and asymptomatic or healthy adults in the control group. methods varied among studies. among the case definitions used, 7 studies used ari or acute lower respiratory tract infection, while the others used (severe) pneumonia (n = 9). all studies investigated a control group, which had no respiratory symptoms, and in 3 studies healthy older adults (without acute illness) served as controls. of the case ascertainment methods used, 8 articles recruited the cases from inpatients; 1, from outpatients; 3, from general practices; 1, from the community; and 3, from mixed settings (outpatient settings and the emergency department, and both outpatient and inpatient settings). in 11 studies, controls were ascertained in hospital-based outpatient or clinic sites, whereas in 5 studies, controls were identified from the community. all studies collected a mixture of nasopharyngeal swab specimens, nasopharyngeal aspirates, nasopharyngeal washes, oropharyngeal swab specimens, and nasal/throat swab specimens as the clinical specimen. all studies used polymerase chain reaction analysis (pcr; in some, pcr was combined with serologic analysis or culture) as the diagnostic test. meta-analyses of virus-specific ors are reported as well as the corresponding attributable fractions among the exposed (supplementary table 3 ). rsv, flu (including flu a), hmpv, rv, and cov (also cov oc43 and 229e) were significantly more common in older adults with a diagnosis of ari or pneumonia than in asymptomatic or healthy controls (ors, 8 these viruses had statistically significant positive afes, which showed clear associations between these viruses and ari or pneumonia in older adults. moreover, piv (including piv1 and piv3; data for piv2 and piv4 were not available), flu b, and adv were only identified in cases consistently across all included studies (8, 5 , and 8 studies, respectively) and absent in control groups. thus, these viruses were all assumed to have strong associations with ari. only 2 studies had data available for bov, and although both reported virus detection in a greater proportion of cases than controls [18, 28] , the association remains a question for further research. a subgroup analysis was performed to explore the roles of viruses in ari with respect to region: developing countries and industrialized countries. the meta-estimate or was higher in industrialized countries as compared to developing countries in the case of flu (with overlapping 95% cis), while it was similar for piv, adv, and rv. there were insufficient studies to conduct a similar subgroup analysis for other viruses. a sensitivity analysis was performed to investigate the roles of these common viruses in older adults admitted to hospitals with ari or pneumonia [1, 16, 20, 22, 23, 25, 27, 28] . eight studies were included, and results are presented in supplementary table 4 . the meta-estimate or did not differ significantly from the previous estimate, in which cases from other settings (ie, outpatient and general practice settings) were also included. and cov in ari and pneumonia in older adults, thereby indicating a potential for substantive reductions in the number of ari cases if older adults were vaccinated against these viruses or treated with antivirals. for the other respiratory viruses studied, the role of bov in ari and pneumonia was uncertain because of the limited evidence available from the published literature, requiring more research to clarify its role in older adults with ari. a sensitivity analysis focusing only on older adults who were admitted to hospitals with ari or pneumonia did not differ significantly from our estimate, in which patients from all settings were considered. this might result from the limited number of studies available to provide a more robust sensitivity analysis. no studies calculated adjusted ors to account for confounding effects from age or season, which might compromise the actual association and should be considered in the study design in future research. these findings should inform the results of studies that seek to estimate the global, regional, and national burden of disease due to these viruses in older adults [30] . they show that rsv, flu, piv, hmpv, adv, rv, and cov are important causes of ari in older adults, and disease burden estimates should take into account the appropriate afe estimates (for older adults) that we report, rather than the afe estimates in children aged <5 years. there is considerable international attention on rsv-associated ari in older adults at this time, during which novel vaccine and antiviral strategies are being evaluated and prioritized [31, 32] , and more-accurate disease burden estimates (using these results) would help to inform future policies and interventions. the prevalence of virus detection from etiologic studies of pneumonia in adults is substantially lower than the detection rate in studies of children. the epic (etiology of pneumonia in the community) study team showed that the viruses were detected in 26% of adults who had been hospitalized with community-acquired pneumonia, compared with 73% of children who were admitted to the hospital [1, 33] . there are several reasons for such low levels of detection, such as the inability to obtain lower respiratory tract specimens, the use of diagnostic tests with insufficient sensitivity, the absence of appropriate diagnostic testing methods, the undetectability of the virus at the time of the study, and the presence of unknown pathogens that were not identified. the low rate of virus detection among adults who were hospitalized for pneumonia highlights the need for more-sensitive diagnostic approaches, innovative discovery of pathogens, and assessing viruses in the past history (weeks before the presence of disease) [34] . moreover, chronic obstructive pulmonary disease (copd) exacerbation is a very important cause of aris and hospital admissions [35] . only 7 of 16 studies included copd in the etiologic data, and this information was unclear in the remaining studies, which might have underestimated the role of viral infection in these patients. a previous etiological review focusing on young children aged <5 years [8] showed that rsv, flu (including flu a), piv, hmpv, and rv were significantly more common in children hospitalized with acute lower respiratory tract infection than asymptomatic controls. the associations of these viruses (except rsv) with ari and pneumonia were stronger among adults. this is in part because, in comparison to young children, the detection of viruses in the control group (ie, among individuals without respiratory symptoms or healthy controls) was less common in older adults, with the exception of rsv. several methodological issues could affect our results: sample size, age group, case ascertainment, clinical specimen, and diagnostic testing. although a thorough search has been performed across 7 databases, including 3 chinese-language databases, only 16 studies from the published literature were identified, which met our selection criteria. not every virus of interest was tested in each study. the number of studies available was even smaller when subgroup analyses and sensitivity analyses were performed. moreover, the sample size varied from 50 to 2320 adults in the case group and from 27 to 541 adults in the control group. the small sample size undoubtedly contributed to the imprecise 95% cis around the ors. thus, we may have failed to detect clinically significant ari-virus associations, owing to small sample sizes. we aimed to stratify the association between common respiratory viruses in adults with ari or pneumonia by age. however, most articles did not stratify and report data by age group. instead, they summarized the result for the entire age group, usually in adults aged >18 years. therefore, some of our meta-estimate ors may not be representative of older adults who are aged ≥65 years. since age might be a risk factor for ari in adults (the rate of severe ari increases as age advances), this could potentially affect the viral profile detected, introducing further heterogeneity [1] . fifteen of 16 studies used passive clinic or hospital based case ascertainment. among them, cases were recruited from inpatients, outpatients, emergency departments, or general practices, which might reflect different healthcare behavior and disease severity. also, since the episodes of ari and pneumonia were only diagnosed through routine care, this introduced bias, considering that testing was only done when the clinicians deemed it necessary to test. similarly, 5 studies used community-based controls, while another 11 studies recruited older adult controls from hospitals or general practices. hospital or clinical ascertained controls may not reflect the general population and may have other health conditions potentially affecting their viral carriage. moreover, recruiting controls who were selected as healthy or without respiratory symptoms could favor those who were not exposed to the respiratory virus (yielding a falsely high or). therefore, we consider that the ideal control group for these studies would be a random sample of an age-and sexmatched population of older adults who are from the same area of residence and studied at the same time as cases. all included studies obtained upper respiratory tract specimens (described as nasopharyngeal secretions). assays might have specimen-specific sensitivities and specificities for detecting viruses, which could lead to heterogeneity in the estimation of virus-specific rates. the sensitivity of using nasopharyngeal washes for detecting any virus in adults was found to be higher than that for using nasopharyngeal swab specimens, which in turn was higher than that for using oropharyngeal swab specimens (84.9%, 73.3%, and 54.2%, respectively) [36] . the limited use (due to ethical concerns and feasibility) of invasive procedures to obtain lower respiratory tract specimens directly from the lung also influenced the diagnosis of viral infection in adults with ari [37] . pcr and serology-based diagnostic testing are more sensitive for detecting respiratory viruses than other methods, such as antigen detection and culture. high sensitivity is important for accurate assessment of etiological contribution, particularly in older adults who may have a lower nasopharyngeal viral load and an atypical clinical presentation [7] . moreover, despite being uncommon, detection of viral coinfection (range across studies, 1%-10%) may tend to overstate the contribution of individual respiratory viruses (although dual or multiple infections, in which both or several viruses have etiological importance, are possible). bacterial coinfections (range across studies, 15%-26%) were also reported. with improving diagnostic methods, multiple etiological agents are increasingly identified simultaneously in older adults with ari, making the individual contribution of each agent difficult to define. viruses can be detected in individuals with no respiratory symptoms. this is often seen in volunteer challenge studies and in some community surveys [38, 39] . the detection of viruses in control groups without respiratory symptoms might be due to a nascent infection or a persisting colonization from a previous infection [40] . these factors will tend to result in true associations being attenuated. the fact that molecular detection of viruses in older adults with ari is higher than the detection rate in controls without respiratory symptoms may not necessarily indicate causation. alternative explanations should be considered first before causality can be concluded. these include the respiratory viral infection acting as a so-called innocent bystander, without a causal role, and serving only as a predisposing risk factor for ari. similarly, the absence of a positive association (and afe) does not mean that a virus is not a cause of ari. moreover, without establishing the temporal sequence of exposure and outcome, determinations of causality are less secure. therefore, the association between viruses and ari and pneumonia should be interpreted carefully. in conclusion, this review provides clear evidence that is suggestive of the potentially causal role of rsv, flu, piv, hmpv, adv, rv, and cov in older adults with ari and presents the first estimate of the proportion of ari cases that can be attributed to virus exposure. etiological studies, which simply report rates of viral identification as causal, should make attempts to interpret findings in terms of the proportion of ari cases among older adults in whom a respiratory virus is identified that can be attributed to this viral exposure. the resceu investigators are as follows national institute for public health and the environment) penta foundation) jeroen aerssens, veronique wyffels, and matthias cleenewerck (janssen) cdc epic study team. communityacquired pneumonia requiring hospitalization among u.s. adults viral pneumonia respiratory syncytial virus infection in elderly and high-risk adults modelling estimates of the burden of respiratory syncytial virus infection in adults and the elderly in the united kingdom high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections rhinovirus outbreaks in longterm care facilities the diagnosis of viral respiratory disease in older adults aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis preferred reporting items for systematic reviews and meta-analyses: the prisma statement world health organization. integrated management of adolescent and adult illness (imai) the state of the world's children 2015: reimagine the future: innovation for every child what to add to nothing? use and avoidance of continuity corrections in meta-analysis of sparse data introduction to meta-analysis attributable risk percent in case-control studies a case-control study of acute respiratory tract infection in general practice patients in the netherlands respiratory viruses in adults with community-acquired pneumonia prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate microorganisms in respiratory tract of patients diagnosed with atypical pneumonia: results of a research based on the use of reverse transcription polymerase chain reaction (rt-pcr) dna microarray method and enzyme-linked immunosorbent assay viral respiratory tract infections in adult patients attending outpatient and emergency departments respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care respiratory virus is a real pathogen in immunocompetent community-acquired pneumonia: comparing to influenza like illness and volunteer controls incidence and characteristics of viral community-acquired pneumonia in adults a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection viral etiology of acute lower respiratory infection in adult inpatients detection on non-bacterium pathogen in 1232 cases of acute respiratory infection human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study accessed 29 drug candidates and model systems in respiratory syncytial virus antiviral drug discovery cdc epic study team. communityacquired pneumonia requiring hospitalization among u.s. children better tests, better care: improved diagnostics for infectious diseases respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia time lines of infection and disease in human influenza: a review of volunteer challenge studies flu watch group. comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections we thank joris menten from janssen for reviewing the manuscript.financial support. this work was supported by the innovative medicines initiative 2 joint undertaking (grant 116019), which in turn receives support from the european union's horizon 2020 research and innovation programme and efpia. is the elected president of the british society for immunology, which is an unpaid appointment but provides support for travel and accommodation at some meetings. all other authors report no potential conflicts. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord-320764-p4ydngag authors: breiman, robert f.; van beneden, chris a.; farnon, eileen c. title: surveillance for respiratory infections in lowand middle-income countries: experience from the centers for disease control and prevention's global disease detection international emerging infections program date: 2013-12-15 journal: j infect dis doi: 10.1093/infdis/jit462 sha: doc_id: 320764 cord_uid: p4ydngag nan in 2001 with its first international emerging infections program (ieip) established in bangkok, thailand, the centers for disease control and prevention (cdc) began building capacity in strategically located countries for infectious disease surveillance, diagnostics, epidemic detection and response, and collection of epidemiologic data to drive policy on prevention and control of priority infectious diseases. the vision of establishing programs that focus on emerging infectious disease detection and response evolved into what are now called global disease detection (gdd) regional centers. the gdd program was established in 2004 to provide support for the cdc's international programs and was expanded to include the ieip and other programs as part of the gdd regional centers when cdc's center for global health was established in 2010 [1] . the gdd program builds global capacity to identify and respond to emerging diseases, and to conduct applied public health research on disease prevention and control [2] . the gdd centers include 6 programs that support their host countries in building capacity to comply with the revised international health regulations (ihr 2005) . the 3 core programs are the international emerging infections program, the cornerstone of the gdd centers that serves as a platform to study emerging diseases and their prevention and control; the field epidemiology training program, which trains scientists in applied epidemiology and public health laboratory science; and the influenza program, which supports detection and response for seasonal and pandemic influenza. the remaining 3 gdd programs include the one health program, integrating animal and human health investigations of zoonotic diseases; the strengthening laboratory capacity program; and the riskcommunicationandemergencyresponseprogram, supporting health communication and helping countries establish infrastructure for emergency operations centers and systems. some gdd centers additionally have a refugee health program that works closely with ieip and other gdd programs. gdd centers work in partnership with ministries of health and the world health organization in their host countries, and often also work in nearby or adjacent countries. some gdd centers are embedded in ministries of health (eg, china) or their institutes (eg, kenya); others in universities (eg, guatemala), public-private research institutes (eg, bangladesh), or united states department of defense research units (eg, egypt). gdd centers have been established in countries where cdc had established ieip sites (thailand, kenya, guatemala, egypt, china, bangladesh), and new gdd centers and their embedded ieip programs have been subsequently established in kazakhstan and india, south africa, and georgia ( figure 1 ). six of the ieip programs in the regional centers (thailand, kenya, guatemala, egypt, china, and bangladesh) operate population-based infectious disease surveillance for pneumonia and acute respiratory infections. pneumonia and acute respiratory illness are well established as leading causes of child mortality worldwide, and the emergence of severe acute respiratory syndrome (sars) in 2003 and avian influenza a(h5n1) highlighted the need for continued detection and response for emerging respiratory pathogens. population-based surveillance provides the opportunity to define burden, risk factors, and transmission characteristics of new or emerging infectious diseases, as well as to assess effectiveness of strategies to reduce disease burden or prevent spread of pathogens causing diseases of significant public health importance. many ieips have also undertaken surveillance for additional syndromes, including acute febrile illness, acute diarrhea, acute jaundice, and acute infectious neurologic disease (meningitis, encephalitis, and acute flaccid paralysis), with testing for multiple etiologies. the methodologies and catchment population of the respiratory disease surveillance systems vary depending upon key local characteristics (table 1 ). in thailand, where healthcare utilization rates are high even in rural settings (sa kaeo and nahkhon phanom) [3, 4] , hospital-based surveillance was established to identify cases of moderate to severe illness. in contrast, in kenya, where healthcare utilization-even for cases of severe illness-is low both in urban [5] and rural [6] areas, a community-based system was established. this system consists of biweekly household visits during which information about illness is collected for all household members, and patients with serious illness are referred to the study clinic where more detailed clinical information and specimens are collected. because hospital-based systems are relatively inexpensive to operate [7] , they can cover large populations. in a community-based system with household visits, large population sizes would require resources beyond those available for conducting population-based surveillance. large hospitalbased systems provide more capacity to detect emerging pathogens and provide larger sample sizes for evaluating the effectiveness of interventions. rigorous community-based systems provide greater opportunities to evaluate transmission rates, determinants, and risk factors for common diseases, and sometimes yield incidence rates inclusive of more disease episodes; however, the smaller surveillance area limits the ability to fully define the spectrum of illness caused by particular pathogens. although subtle differences in the acute respiratory illnesses tracked in the ieip population-based surveillance exist, sites generally start by capturing acute respiratory infections (aris) as defined by evidence of acute infection (typically fever or abnormal white blood cell count) plus 1 or more signs or symptoms of surveillance for respiratory infections • jid 2013:208 (suppl 3) • s169 respiratory illness (table 2 ). most sites also track subsets of ari cases: influenza-like illness among outpatients; severe acute respiratory infection (sari) among hospitalized patients, and pneumonia, which may apply to outpatients or inpatients ( table 2 ). respiratory specimens from enrolled patients are collected for molecular diagnostic testing principally using nasopharyngeal and oropharyngeal swabs. sites test respiratory secretions by polymerase chain reaction (pcr) for a core ieip respiratory diagnostic panel of viral pathogens: respiratory syncytial virus (rsv), influenza a and b viruses, adenovirus, parainfluenza viruses 1-3, and human metapneumovirus. a detailed description of the gdd laboratory program, including methods used to identity rsv and other respiratory pathogens, is included in this journal supplement [8] . testing is performed by gdd laboratories (thailand, kenya, guatemala) or laboratories run by the gdd partner institution (china, bangladesh), or through a combination of gdd and partner laboratories (egypt) [8] . several sites use urine to routinely test for the presence of streptococcus pneumoniae (among older children/adults) and legionella pneumophila. more extensive testing has often been done for special studies of limited duration, such as testing of nasopharyngeal/oropharyngeal specimens for the presence of mycoplasma pneumoniae and chlamydia (chlamydophila) pneumoniae. blood cultures are collected from patients with pneumonia in several ieip sites ( table 2) in an attempt to identify bacteremic pneumonia. some sites also collect and store sera from febrile patients as part of acute febrile illness surveillance for emerging zoonotic diseases, and occasionally use sera from the subset of febrile patients meeting respiratory surveillance criteria for serologic studies of respiratory pathogens. in kenya, acute and convalescent sera have been tested by serology for influenza viruses, rsv, human metapneumovirus, adenovirus, and parainfluenza viruses in a special study to evaluate the additional diagnostic yield of serology for respiratory pathogens in addition to pcr [9] . in the event of an apparently newly introduced pathogen, such as the middle east respiratory syndrome coronavirus (mers-cov), these banked sera could be used to assess the extent of disease by evaluating the seroprevalence of antibodies or seroincidence of infection, as well as evaluating whether the pathogen was circulating previously without detection. in thailand, paired sera have been tested for m. pneumoniae, c. pneumoniae, legionella longbeachae, and coxiella burnetii; in bangladesh, paired sera have been tested for respiratory pathogens from patients with fever and/or ari and acute lower respiratory infection. respiratory specimens from matched healthy controls have been useful to determine usual rates of asymptomatic infection with respiratory viruses (especially adenoviruses and rhinoviruses) and colonization with s. pneumoniae and other bacteria for estimation of pathogen-attributable fractions of lower respiratory tract disease [10] [11] [12] . data on the incidence of the syndrome of pneumonia and sari have provided valuable information for considering the relative importance of specific etiologies, and for considering what fraction of pneumonia and associated poor health outcomes might be preventable through vaccines or other interventions. the gdd ieip sites have documented a high incidence of pneumonia and influenza-associated acute respiratory illness, especially among children [13] [14] [15] . among ieip sites with published data, a high incidence of rsv disease has been consistently demonstrated. for example, in hospital-based surveillance within known catchment populations in rural areas in thailand, rates of rsv-associated acute respiratory illness were highest among children aged <1 year (1067/100 000) and children aged 1-4 years (403/100 000), but were comparatively low but still substantial (42/100 000) among adults aged ≥65 years [16] . in community-based studies in rural kenya, the rate of rsv-associated acute respiratory illness was high in children aged <5 years (7100/100 000) with sari, and was 440/100 000 in persons aged >5 years with ari (including both inpatients and outpatients) [17, 10] . the ieip respiratory surveillance systems have also documented the incidence of acute respiratory illness due to other respiratory viruses and bacteria, including human metapneumovirus, parainfluenza viruses, and adenoviruses [13] . surveillance specimens from several sites have also been tested for additional viruses such as bocaviruses, coronaviruses, enteroviruses, parechoviruses, and rhinoviruses [12, 13] . the sites have also played a critical role in detection of and response to novel pathogens such as sars [18] and described one of the first known outbreaks of pandemic a(h1n1)2009 at a primary school in china [19] . some differences in surveillance methodology exist among the 6 gdd ieip sites that conduct respiratory surveillance. these differences are partially due to site-specific differences in local laboratory capacity, standards of clinical care, limitations in the use, quality, and interpretation of routine diagnostics (eg, blood cultures and chest radiographs), and healthcare-seeking behaviors of the population under surveillance. a universal challenge is the aseptic collection of blood for culture and adequate capacity for processing. because of the challenges of implementing blood culture, estimates of invasive bacterial infections, particularly bacteremic pneumococcal pneumonia, are uncertain. however, some sites, such as bangladesh and kenya, have successfully incorporated blood culture into their surveillance, enabling measurement of the proportion of invasive pneumococcal disease that may be prevented with use of newly developed conjugate vaccines [20] and documenting that blood culture-confirmed typhoid fever can be associated with significant respiratory manifestations [21] . investigators in the gdd regional center in thailand found that prior use of antibiotics (measured by serum antimicrobial drug levels) reduced the incidence estimates for pneumococcal bacteremia by 32% overall and 39% in children <5 years of age [22] . because specimens are archived from patients with acute respiratory illness in the gdd ieip programs with established respiratory surveillance sites, it is possible to rapidly characterize the incidence and epidemiology of newly identified pathogens (eg, pandemic a[h1n1]2009) [23] [24] [25] and to reexamine disease burden estimates when new, more sensitive and highly specific tests become available. thus, the novel mers-cov first identified in 2012 among patients in saudi arabia, qatar, and jordan [26] [27] [28] can be examined as a cause of respiratory illness by testing prospective as well as archived samples from ieip surveillance sites run by the gdd regional center in egypt and other gdd centers, surveillance sites run by other gdd programs (eg, influenza, one health, and refugee health), and affiliated surveillance networks (eg, the eastern mediterranean acute respiratory infections surveillance (emaris) sites established by the gdd regional center in egypt, the world health organization's eastern mediterranean regional office, and its member states). likewise, the sites are well poised to evaluate new diagnostic testing technologies, including multiple pathogen assays like the taqman array card (tac), which tests for a variety of pathogens with one specimen [29] . technologies like tac allow archived specimens collected over multiple years to be tested at one time, presenting the possibility to rapidly characterize a vast array of ari etiologies in multiple geographic locations with varied ecologic and demographic features. using well-characterized clinical, demographic, and geographic information, the ieip population-based surveillance sites are able to describe seasonality, risk factors, and spectrum of disease of newly identified pathogens. this journal supplement highlights the value of populationbased surveillance systems such as those initiated or catalyzed by gdd ieip sites. this multicountry network of populationbased surveillance sites provides a platform to test a variety of interventions in settings where ecology, economics, ethnicity, politics, predominant co-morbidities and co-infecting pathogens, and behavior vary, thereby providing a useful tool to design optimal intervention strategies targeted to each setting. gdd ieip surveillance data also provides a stimulus for more urgent development of novel vaccines and therapeutics for rsv and other diseases by demonstrating their burden and severity and are poised to assess the effectiveness, acceptance, and value of these tools compared with other prevention strategies. given the consistency in laboratory testing methods, the approach to surveillance of respiratory infections undertaken by gdd ieip sites enables comparison of the burden of rsv disease and its epidemiology among the 6 geographically and sociopolitically diverse countries. longitudinal surveillance data from these sites, combined with those from gdd influenza program sites, will add to the global understanding of rsv-associated mortality and risk factors for severe disease and death in low-and middleincome countries, result in better descriptions of rsv seasonality, and inform intervention strategies to mitigate the burden of rsv-associated disease. the ieip-run surveillance sites are well positioned to serve as platforms for future rsv vaccine efficacy or effectiveness studies in low-and middle-income countries, and to evaluate the utility and feasibility of rsv prophylaxis in these settings. ultimately, the evidence generated by studies from gdd ieip surveillance sites can inform countries' determinations of their public health priorities and generate the political will to implement effective prevention and control measures, through evidence-based policy change. notes a comparison of populationbased pneumonia surveillance and health-seeking behavior in two provinces in rural thailand putting surveillance data into context: the role of health care utilization surveys in understanding population burden of pneumonia in developing countries healthcare-use for major infectious disease syndromes in an informal settlement in nairobi healthcare-seeking behaviour for common infectious disease-related illnesses in rural kenya: a 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to seasonal influenza in young children: a systematic review and meta-analysis the burden of hospitalized lower respiratory tract infection due to respiratory syncytial virus in rural thailand viral and bacterial causes of severe acute respiratory illness among children aged less than 5 years in a high malaria prevalence area of western kenya control measures for severe acute respiratory syndrome (sars) in taiwan a primary school outbreak of pandemic 2009 influenza a (h1n1) in china invasive pneumococcal disease burden and implications for vaccine policy in urban bangladesh population-based incidence of typhoid fever in an urban informal settlement and a rural area in kenya: implications for typhoid vaccine use in africa antibiotic use in thailand: quantifying impact on blood culture yield and estimates of pneumococcal bacteremia incidence secondary household transmission of 2009 pandemic influenza a (h1n1) virus among an urban and rural population in kenya population-based surveillance for 2009 pandemic influenza a (h1n1) virus in guatemala incidence and epidemiology of hospitalized influenza cases in rural thailand during the influenza a (h1n1)pdm09 pandemic severe respiratory illness caused by a novel coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia novel coronavirus infection-update application of taqman lowdensity arrays for simultaneous detection of multiple respiratory pathogens the incidence of pneumonia in rural thailand acknowledgments. we appreciate the insightful comments of henry c. baggett financial support. this work was supported by the global disease detection program, centers for global health, and the influenza division, national centers for immunization and respiratory disease at the us centers for disease control and prevention.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-007013-tlvgyzft authors: chan, kok fei; carolan, louise a; korenkov, daniil; druce, julian; mccaw, james; reading, patrick c; barr, ian g; laurie, karen l title: investigating viral interference between influenza a virus and human respiratory syncytial virus in a ferret model of infection date: 2018-08-01 journal: j infect dis doi: 10.1093/infdis/jiy184 sha: doc_id: 7013 cord_uid: tlvgyzft epidemiological studies have observed that the seasonal peak incidence of influenza virus infection is sometimes separate from the peak incidence of human respiratory syncytial virus (hrsv) infection, with the peak incidence of hrsv infection delayed. this is proposed to be due to viral interference, whereby infection with one virus prevents or delays infection with a different virus. we investigated viral interference between hrsv and 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09) in the ferret model. infection with a(h1n1)pdm09 prevented subsequent infection with hrsv. infection with hrsv reduced morbidity attributed to infection with a(h1n1)pdm09 but not infection, even when an increased inoculum dose of hrsv was used. notably, infection with a(h1n1)pdm09 induced higher levels of proinflammatory cytokines, chemokines, and immune mediators in the ferret than hrsv. minimal cross-reactive serological responses or interferon γ–expressing cells were induced by either virus ≥14 days after infection. these data indicate that antigen-independent mechanisms may drive viral interference between unrelated respiratory viruses that can limit subsequent infection or disease. viral interference is a phenomenon whereby infection with one virus limits or delays infection with a second virus. it has been described in human epidemiological studies observing viral epidemic peaks [1] [2] [3] [4] , vaccine efficacy studies [5] , studies assessing virus infections in clinical samples [6] [7] [8] , animal studies [9] [10] [11] [12] [13] and in vitro infectivity studies [14] . viral interference has been observed between a range of viruses, including between arboviruses, such as yellow fever and dengue virus [15] ; between different respiratory viruses [9, 13, 16] ; and between influenza viruses of different types [10] and subtypes/lineages [10, 11] . at a population level, respiratory virus infections may display distinct epidemic peaks. observational studies from the netherlands, france, and hong kong showed that emergence of 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09) delayed infections with human respiratory syncytial virus (hrsv) [1, 3, 4] . influenza a virus infections also interrupted peak incidences of hrsv infections in japan during 2000-2002 [2] and in the netherlands during 2003-2012 [3] . negative associations between respiratory viruses have been reported when analyzing the proportion of coinfections with different respiratory viruses, using swab specimens from patients [6, 7, 17] . a(h1n1)pdm09 was least likely to be detected with any of the other respiratory viruses tested, including hrsv, in samples from all age groups [8, 18] . taken together, these data suggest that interference may occur between a(h1n1)pdm09 and hrsv. the ferret provides an ideal model of human influenza because animals can be directly infected with virus without adaptation and display similar disease symptoms to those in humans [19, 20] . historically, the ferret has also been used to study hrsv infection [21] [22] [23] , with recent studies assessing the pathogenesis, immunity, and transmission of hrsv [24, 25] . clinical symptoms are mild in ferrets infected with hrsv strains described to date [24, 25] . previously, we used the ferret model to demonstrate that viral interference can occur following infection with human influenza a and b viruses and will prevent, delay, or limit subsequent infection with an influenza virus of a different type, subtype, or lineage [10, 11] . notably, this effect depends on the virus combinations and the order and timing of sequential infections [10, 11, 26] . we have established complementary influenza viral dynamics models that explain these observations via the innate immune response [27] and cross-reactive adaptive immune responses [28] . ecological data suggest that infection with a(h1n1)pdm09 can prevent or delay infection with hrsv. using our ferret models of influenza and hrsv, we have systematically investigated this hypothesis. adult ferrets were housed at the peter doherty institute for infection and immunity bioresources facility. experiments were conducted with approval from the university of melbourne microbiology and immunology animal ethics committee, in accordance with the australian national health and medical research council code of practice for the care and use of animals for scientific purposes. all ferrets were seronegative for antibodies to currently circulating influenza viruses and hrsv (long and a2 strains) before use in experiments. a/tasmania/2004/2009 (a[h1n1]pdm09) virus was passaged allantoically in embryonated hen's eggs and stored at −80°c. the infectious influenza virus titer was measured by a 50% tissue culture infectious dose (tcid 50 ) assay [29] , read by hemagglutination with turkey red blood cells. hrsv long and a2 strains were passaged [24] . infectious hrsv titers were determined by plaque assay [24] . ferrets were infected intranasally with 10 3.5 tcid 50 a(h1n1) pdm09 in 500 µl and 10 5 plaque-forming units (pfu) of long hrsv or 10 6 pfu of long or a2 hrsv in 500 µl and monitored [24, 26] . ferrets were housed in pairs, by infection group. nasal wash specimens were collected and stored [24] . on the day of collection, viral rna was extracted from 140-µl nasal wash specimens for quantitative polymerase chain reaction (qpcr) analysis. blood samples were obtained from ferrets before primary virus infection and immediately before and 14 days after challenge, and serum was isolated. the proportional change in weight was calculated as the percentage difference from the weight on the day of challenge. four microliters of viral rna [24] was assayed by rt-qpcr with a(h1n1)pdm09 hemagglutinin-specific primers/probes from the cdc influenza virus rt-qpcr influenza a (h1/h3/h1pdm09) subtyping panel, obtained from the influenza reagent resource (available at: http://www.influenzareagentresource.org/) and hrsv n-specific primers/probes [24] . copy numbers for a(h1n1) pdm09 viral rna were calculated relative to plasmid phw2000-a/ tasmania/2004/2009 hemagglutinin; copy numbers for rsv rna were calculated relative to a hrsv rna standard [24] . mrna was isolated from nasal wash samples [30] . mrna expression of cytokines, chemokines, and housekeeping genes was quantified by qpcr [30, 31] . infectious hrsv in nasal wash samples was measured using the vs assay [24] . interferon γ (ifn-γ) enzyme-linked immunospot (elispot) assay ifn-γ-producing cells were detected by a ferret ifn-γ elispotplus assay (mabtech). single cell suspensions were prepared from ferret retropharyngeal lymph nodes [31] . a total of 5 × 10 4 lymph node cells were cultured with or without live influenza virus, hrsv, or 5 µg/ml concanavalin a (sigma) for 48 hours at 37 o c in 5% co 2 [11] . titers of antibodies to a/tasmania/2004/2009 were measured using hi assays [31, 32] . titers were expressed as the reciprocal of the highest dilution of serum for which hemagglutination was prevented. geometric mean titers (gmts) were calculated, with undetectable titers expressed as having a value of "5." seroconversion was defined as a titer of ≥40 at the end of the experiment and at least a 4-fold rise from baseline. titers of antibodies that neutralize hrsv long and a2 were measured using vs mn assays [24] . seroconversion was defined as a titer ≥160 at the end of the experiment and an increase of at least 4-fold from the baseline titer. antibodies that bind to the f glycoprotein of hrsv were detected by an elisa [24] . viral kinetics were assessed in viral rna from nasal wash specimens. for a(h1n1)pdm09, >10 6 copies of hemagglutinin/100 µl of nasal wash were positively correlated with replicating virus, based on the tcid 50 assay [10] and the level of infectious virus as measured by transmission in ferrets [33] . for hrsv, 10 3.8 copies of n/100 µl of nasal wash corresponded to a 50% chance of a sample being positive by the virospot assay, as determined using a probit regression model (supplementary figure 1) . accordingly, samples were considered to be infectious for hrsv when the amount of viral rna exceeded 10 3.8 copies/100 μl nasal wash and infectious for a(h1n1)pdm09 when viral rna exceeded 10 6 copies/100 µl of nasal wash for at least 1 measurement. clinical signs (ie, weight loss and fever) were assessed daily, and seroconversion was measured 14 days after challenge. statistical analysis was conducted using prism, version 6.0g, unless otherwise indicated and is described in the figure legends. ferrets were first infected with a(h1n1)pdm09 virus then challenged with hrsv 3, 7, or 11 days later, or vice versa ( figure 1a ). the intervals between inoculations spanned the times of peak titer and clearance of both virus infections [24, 30] and induction of humoral immunity ( figure 2a ). primary infection with a(h1n1)pdm09 prevented subsequent infection with hrsv in 3 of 4 ferrets when primary infection and challenge were separated by 3 days. shedding of hrsv was minimal in the single ferret infected, compared with control animals ( figure 1b and 1c) . no ferrets in this group seroconverted to hrsv (figure 2bi and 2bii ). primary infection with a(h1n1)pdm09 prevented infection with hrsv in 2 of 4 ferrets when infections were separated by 7 days ( figure 1d ). ferrets that did not shed virus did not seroconvert (figure 2bi and 2bii), while ferrets that shed virus seroconverted to hrsv (figure 2bi and 2bii). prior infection with a(h1n1)pdm09 did not prevent infection with hrsv 11 days later ( figure 1e ), with all ferrets showing a similar pattern of virus shedding ( figure 1b ) and antibody titers to control animals that received hrsv alone (figure 2bi and 2bii). the kinetics of hrsv shedding was examined in animals not protected from hrsv challenge. the peak of hrsv shedding was delayed in ferrets infected with a(h1n1)pdm09 followed by hrsv as compared to control animals infected with hrsv alone (median, 8 vs 6 days; p = .0091 by the mann-whitney test; figure 2ci ). there was no change in the duration of virus shedding ( figure 2cii ). clinical signs following hrsv challenge were minimal (supplementary figure 2) , consistent with our previous study [21] . all ferrets, except 1 control ferret infected with hrsv, maintained or gained weight (supplementary figure figure 2biv ). the median duration of a(h1n1)pdm09 shedding was increased in ferrets infected with hrsv followed by a(h1n1)pdm09 as compared to control animals infected with a(h1n1)pdm09 alone (8 vs 7 days; p = .0196 by the mann-whitney test; figure 2civ ). there was no change in the peak day of shedding (figure 2ciii ). prior infection with hrsv did reduce disease following infection with a(h1n1)pdm09. the mean maximum weight loss (±sd) among 8 control ferrets infected with a(h1n1)pdm09 was 10.6% ± 3.7% (supplementary figure 3a and 3d) . the mean maximum weight loss (±sd) for ferrets in all test groups (n = 12) was 4.1% ± 2.3% (supplementary figure 3b , 3c, and 3e). thus, prior infection with hrsv significantly reduced morbidity, as measured by weight loss, after challenge with a(h1n1) pdm09 (p = .0002 by the mann-whitney test). no fever was detected following a(h1n1)pdm09 infection (supplementary figure 3f -j). ferrets were infected (1) with an increased viral dose of the same hrsv strain, long, or (2) with an alternate hrsv strain, a2 (also at an increased viral dose), then challenged with a(h1n1) pdm09 3 days later. a2 is a laboratory-adapted strain that is shed at similar levels to long in ferrets and transmits between cohoused animals [24] . infection of ferrets with a 10-fold higher inoculum (ie, 10 6 pfu) of hrsv long led to a small increase in virus shedding on days 2-6 after infection, compared with animals infected with 10 5 pfu of hrsv long, although these differences were not significant (supplementary figure 4) . primary infection with 10 6 pfu hrsv long or a2 did not prevent infection with a(h1n1)pdm09 when infections were separated by 3 days (figure 4 ). all animals seroconverted to a(h1n1)pdm09 at similar levels ( figure 2bv and 2bvi). most ferrets lost weight after a(h1n1)pdm09 infection. the mean maximum weight loss (±sd) among 4 ferrets infected with a(h1n1)pdm09 alone was 7.1% ± 3.0%, whereas the mean maximum weight loss (±sd) for ferrets (n = 8) that received primary infection with hrsv prior to a(h1n1)pdm09 challenge was 4.9% ± 3.2% (p = .2828 by the mann-whitney test; supplementary figure 5a -c). there was no difference in fever (supplementary figure 5d -f) and no change to the kinetics of infection between animals that received a prior hrsv infection, compared with those that did not (data not shown). inflammation induced by viral infection may contribute to viral interference [10, 11] . we investigated the localized immune response following infection with hrsv or a(h1n1)pdm09. nasal wash specimens were collected early (day 2) and later (day 6/7) after infection, because the pattern of inflammatory mediators changes throughout h(h1n1)pdm09 [30] and hrsv [24] infections. expression of influenza virus matrix (m) mrna was highest on day 2 after infection, whereas expression of hrsv nucleoprotein (n) mrna was highest on day 6 ( figure 5a ). two days after infection, animals infected with a(h1n1)pdm09 had significantly higher levels of interferon β (ifn-β), granzyme b, ifn-γ, interleukin 6 (il-6), monocyte chemoattractant protein 1 (mcp-1), and tumor necrosis factor α (tnf-α) mrna as compared to animals infected with hrsv ( figure 5d, 5g-i, 5n , and 5o). expression of ifn-α and granzyme a mrna was increased but not significantly (ifn-α, p = .097; granzyme a, p = .18; figure 5e and 5f). on day 6/7 after infection, levels of granzyme closed circles and open circles indicate animals that did or did not, respectively, shed detectable challenge virus, as determined by quantitative reverse-transcription polymerase chain reaction analysis of viral rna from nasal wash (nw) samples. for statistical analysis, titers or fold changes were compared between test and control groups, using 1-way kruskal-wallis analysis of variance with the dunn multiple comparison test. *p < .05 and **p < .01. c, the kinetics of shedding was analyzed for all ferrets that shed challenge virus in figures 1 and 3 . data from ferrets obtained at the 3-day, 7-day, and 11-day intervals were pooled into the test group. the number of days from challenge inoculation to the peak level of challenge virus shedding (ci and ciii) and the number of days the challenge virus was shed (cii and civ) was determined for each ferret in the indicated groups. horizontal lines indicate median values. the number of days of virus shedding were compared between test and control groups, using the mann-whitney test. *p < .05 and **p < .01. a, granzyme b, ifn-γ, interleukin 17, and mcp-1 mrna were significantly higher in animals infected with a(h1n1)pdm09 as compared to those infected with hrsv ( figure 5f -h, 5m, and 5n). there was significant increase in expression of interleukin 8 (il-8) mrna 2 days after infection and of interleukin 1β, il-6, and il-8 mrnas 6/7 days after infection in ferrets infected with hrsv as compared to a(h1n1)pdm09 ( figure 5c, 5i, and 5j ). this suggests a localized inflammatory response was induced after hrsv infection, which coincided with the increase in hrsv virus replication ( figure 5a ). to directly compare the magnitude of expression of cytokines and chemokines induced by both virus infections, we assessed mrna expression on the day after infection at which the level of virus shedding was highest (ie, day 2 for a(h1n1) pdm09 and day 6 for hrsv). when assessed at these times, an equivalent fold change in mrna expression was observed for rsv n and influenza virus m ( figure 5a ). infection with a(h1n1)pdm09 induced significantly higher levels of ifn-β (p = .00021; figure 5d ), il-6 (p = .0088; figure 5i ), interleukin 12p40 (p = .0137; figure 5l ), mcp-1 (p = .0018; figure 5n ), and tnf-α (p = .00078; figure 5o ) mrna expression in ferrets, compared with hrsv. these data suggests there is increased inflammation in nasal tissues of animals infected with a(h1n1)pdm09 as compared to hrsv. there was no difference in expression of any cytokines or chemokines between ferrets infected with 10 5 or 10 6 pfu of hrsv long (data not shown). we have demonstrated that cross-reactive ifn-γ cellular responses can be detected between influenza b virus lineages and may contribute to viral interference [11] . thus, we assessed whether cellular immunity induced by infection with a(h1n1) pdm09 showed any cross-reactivity to hrsv. whereas retropharyngeal lymph node cells from a(h1n1)pdm09-infected ferrets were restimulated with a(h1n1)pdm09 ( figure 6b ), few cells produced ifn-γ when stimulated with hrsv ( figure 6a ). lymph node cells from hrsv-infected ferrets were restimulated with hrsv in vitro, although at much lower levels ( figure 6a ), and were not restimulated by a(h1n1)pdm09 ( figures 6b) . responses to concanavalin a were similar for all ferrets regardless of infection ( figure 6b ). moreover, there was limited serological cross-reactivity. animals infected with a(h1n1)pdm09 had high levels of influenza virus-specific neutralizing antibodies ( figure 6c ), yet minimal total serum or neutralizing antibodies to hrsv ( figure 6d and 6e) . similarly, infection with hrsv induced total serum and neutralizing antibodies to hrsv but few antibodies that were reactive with a(h1n1)pdm09 ( figure 6c-e) . discussion we have demonstrated that infection with a(h1n1)pdm09 can prevent infection and replication of hrsv in a ferret model of human disease for up to 7 days. infection with hrsv did not prevent subsequent infection with a(h1n1)pdm09; rather, animals were coinfected, albeit with reduced morbidity. infection with a(h1n1)pdm09 leads to increased levels of proinflammatory cytokines in the respiratory tract as compared to infection with hrsv. overall, these data support the ecological observation that viral interference induced by a(h1n1)pdm09 infection delayed infection with hrsv in the winter of 2009-2010. infection with a(h1n1)pdm09 induced higher expression of mcp-1, il-6, type i ifns, tnf-α, ifn-γ, and granzyme a/b mrnas as compared to hrsv infection. mcp-1 and tnf-α regulate the migration of macrophages/monocytes and natural killer (nk) cells into the respiratory tract. macrophages produce mcp-1, tnf-α, and il-6; thus, upregulation of these genes suggests an influx of macrophages and nk cells into the respiratory tissues [34] . nk cells produce ifn-γ, which activates macrophages and neutrophils and promotes t-cell proliferation and killing of virus-infected cells [34] . because cytotoxic t lymphocytes and nk cells also produce granzymes a/b, increased expression of ifn-γ and granzyme a/b mrnas on day 6 after infection suggests recruitment/activation of these cells to the site of infection. il-6 and type i ifns are produced by respiratory epithelial cells, monocytes/macrophages, and dendritic cells [34, 35] . il-6 is a proinflammatory cytokine, whereas type i ifns induce an antiviral state that may also limit replication and spread of hrsv [34, 35] it would be useful to explore the cellular infiltrate following a(h1n1)pdm09 and hrsv infections to gain further insight . ferrets were infected with 10 5 plaque-forming units (pfu) of hrsv strain long or 10 3.5 50% tissue culture infectious doses of a(h1n1)pdm09 (n = 4 ferrets/virus). nasal wash specimens were collected after challenge from ferrets on days 2 and 6 after infection, and mrna was assayed for the indicated genes, using quantitative polymerase chain reaction (qpcr) assays. for each graph, qpcr data are expressed as fold changes relative to values for nasal wash specimens from uninfected animals and normalized to atf4 and gapdh housekeeping genes. in panel a, expression of n is shown for hrsv-infected ferrets, and expression of m is shown for influenza virus-infected ferrets. for statistical analyses, inflammatory mediators were compared between (1) hrsv-infected and a(h1n1)pdm09-infected animals sampled on day 2 after infection, (2) hrsv-infected and a(h1n1)pdm09-infected animals sampled on day 6/7 after infection, and (3) a(h1n1)pdm09-infected animals sampled on day 2 and hrsv-infected animals sampled on day 6 after infection. fold changes were compared between viruses, using the mann-whitney u test. ifn, interferon; il-1, interleukin 1; il-6, interleukin 6; il-8, interleukin 8; il-10, interleukin 10; il-12p40, interleukin 12p40; il-17, interleukin 17; mcp-1, monocyte chemoattractant protein 1; tnf-α, tumor necrosis factor α. *p < .05, **p < .01, ***p < .001, and ****p < .0001. into potential differences in the level and cellular composition present in local inflammation. infection with a(h1n1) pdm09 induced a 7-fold higher cellular ifn-γ recall response as compared to infection with hrsv in our study. because there was no significant difference in ifn-γ responses to concanavalin a between the groups, this observation was not due to a difference in overall t-cell numbers but, instead, was due to an increase in the reactivation of a(h1n1)pdm09-specific cells. taken together, these data suggest that infection with a(h1n1)pdm09 induces a robust cytokine and chemokine response that strongly stimulates the adaptive and memory immune responses. conversely, infection with hrsv elicited a weaker and more limited cytokine and chemokine response that led to a reduced antigen-specific cellular response. however, it is possible that hrsv may not infect the ferret respiratory tract as efficiently as a(h1n1)pdm09 does, and this could result in reduced inflammatory responses. yet, infected animals seroconverted at titers consistent for sterilizing immunity, indicating a productive infection (data not shown) [24] . furthermore, increasing the inoculum of hrsv did not significantly affect the pattern or amount of virus shedding nor the expression of inflammatory mediators, suggesting that the hrsv level was already maximal in this ferret model. notably, increased expression of inflammatory mediators following infection with influenza virus as compared to hrsv has been observed in studies assessing human clinical samples and in vitro airway epithelial cell cultures [36] [37] [38] [39] . what is the mechanism of viral interference induced by a(h1n1)pdm09? the increased antiviral state and inflammation observed after a(h1n1)pdm09 infection has the potential to prevent subsequent infection or delay shedding of hrsv, as was observed here. both viruses predominantly infect ciliated airway epithelial cells, and we have shown that a(h1n1)pdm09 and hrsv long replicated in the upper and lower respiratory tracts of ferrets [24, 30] . infection with a(h1n1)pdm09 can also prevent infection with an influenza b/yamagata virus [10] . there are minimal shared epitopes between influenza a and b viruses [40] , and we showed that minimal cross-reactive ifn-γ-producing cells were induced between hrsv and influenza virus. these data suggest that short-lived mechanisms drive this effect, as no effect was detected in ferrets after one week or, as shown by others, in mice, when infections were separated by 35 days [41] . the timing of interference indicate that interactions between different viruses may also be important. it is possible that different mechanisms act on different virus combinations. gene expression analysis of early markers of the immune response (cona; b) . the number of interferon γ (ifn-γ)-producing cells was determined by an enzyme-linked immunospot (elispot) assay. c-e, sera were tested for antibodies to a(h1n1)pdm09, by a hemagglutination inhibition (hi) assay (c); for total serum antibody binding to hrsv f protein, by an enzyme-linked immunosorbent assay (elisa; d); and for neutralizing serum antibody to hrsv long or a2, by a virospot microneutralization (mn) assay (e). data were obtained from 2 ferrets per group. of respiratory epithelium infected with the virus strains used in these studies may provide further insight. it is notable that infection with hrsv reduced morbidity induced by a(h1n1)pdm09 infection. although virus loads were not decreased in nasal wash specimens, virus shedding may be reduced in the lower respiratory tract, limiting clinical disease. il-8 mrna expression was elevated in nasal wash samples of ferrets infected with hrsv as compared to a(h1n1) pdm09. increased il-8 expression has been associated with milder disease in ferrets infected with pathogenic influenza virus strains, potentially mediated by rapid recruitment of neutrophils, which assist in clearing virus [42] . analysis of the lung influenza virus loads in animals that have been infected with hrsv prior to challenge with a(h1n1)pdm09 would be of interest. epidemiological data reported in france described a 3-4week delay in the peak incidence of hrsv infections following the emergence of the a(h1n1)pdm09, compared with previous years [1] . similarly, a delay of 2-4 weeks of the expected peak of hrsv was reported following an early influenza a season in the netherlands. [3] . these population-level observations of viral interference arise from the interplay between (1) immunodynamics (ie, host-level viral interference), (2) heterogeneity between hosts (ie, differences in immunity to virus strains between individuals), and (3) transmission dynamics (ie, within or between different age groups) [43] . for influenza, these processes have been investigated in some detail. others have demonstrated that a short period (ie, days rather than weeks) of viral interference at the host level may result in substantial separation between epidemic waves at the population level [43] . our results provide the first hostlevel immunodynamic evidence in support of these processes driving the epidemiological interactions observed previously in europe [1, 2] . our study has limitations. we used a circulating strain of a(h1n1)pdm09 from early 2009 and laboratory strains of hrsv, long and a2. the long and a2 strains induce consistent infections and disease in ferrets, with 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parainfluenza virus infections plasticity and virus specifcity of airway epithelial cell immune response during respiratory virus infection interferon in nasal secretions from infants with viral respiratory tract infections cross-reactive human b cell and t cell epitopes between influenza a and b viruses influenza virus lung infection protects from respiratory syncytial virus-induced immunopathology severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction does homologous reinfection drive multiple-wave influenza outbreaks? accounting for immunodynamics in epidemiological models supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.notes key: cord-341667-ayl71jpc authors: van reeth, kristien; nauwynck, hans; pensaert, maurice title: bronchoalveolar interferon-α, tumor necrosis factor-α, interleukin-1, and inflammation during acute influenza in pigs: a possible model for humans? date: 1998-04-17 journal: j infect dis doi: 10.1086/517398 sha: doc_id: 341667 cord_uid: ayl71jpc biologically active interferon-α, tumor necrosis factor-α (tnf-α), and interleukin-1 (il-1) were detected in bronchoalveolar lavage (bal) fluids of 3-week-old cesarian-derived colostrum-deprived pigs inoculated with h1n1 influenza virus. cytokine titers and lung virus titers were significantly higher 18–24 h after inoculation than at 48–72 h after inoculation in all 4 litters of pigs examined. all three cytokines were positively correlated with a 3to 4-fold increase in bal cell numbers (p < .036) and with a drastic neutrophil infiltration (24%–77% of bal cells vs. 0–1.5% in controls) (p < .001). in addition, cytokine production coincided with the onset of general and respiratory symptoms of influenza and with the development of a necrotizing bronchopneumonia. this study is the first demonstration of tnf-α and il-1 in bal fluids of a natural influenza virus host. it documents that pigs may be a highly valuable experimental model in human influenza virus pneumonia. biologically active interferon-a, tumor necrosis factor-a (tnf-a), and interleukin-1 (il-1) were detected in bronchoalveolar lavage (bal) fluids of 3-week-old cesarian-derived colostrum-deprived pigs inoculated with h1n1 influenza virus. cytokine titers and lung virus titers were significantly higher 18 -24 h after inoculation than at 48 -72 h after inoculation in all 4 litters of pigs examined. all three cytokines were positively correlated with a 3-to 4-fold increase in bal cell numbers (p õ .036) and with a drastic neutrophil infiltration (24% -77% of bal cells vs. 0 -1.5% in controls) (p õ .001). in addition, cytokine production coincided with the onset of general and respiratory symptoms of influenza and with the development of a necrotizing bronchopneumonia. this study is the first demonstration of tnf-a and il-1 in bal fluids of a natural influenza virus host. it documents that pigs may be a highly valuable experimental model in human influenza virus pneumonia. swine influenza (si) is a highly important respiratory disease onset of fever, anorexia, tachypnea, dyspnea, and coughing. of pigs. the causative viruses are type a influenza viruses of considerable economic losses result from growth retardation h1n1 and h3n2 subtype, which are antigenically related to or weight loss. experimental viral infections of pigs have docuhuman influenza viruses. typical outbreaks involve an abrupt mented massive viral replication in lung epithelial cells, accompanied by polymorphonuclear leukocyte infiltration and epithelial degeneration [1] . however, the exact mechanisms by which si virus produces lung pathology and disease have not been studied. phragmatic lung were collected for standard histopathology, influorexia, and weight loss, particularly if produced at higher levels enza virus titrations, and fluorescent antibody stainings [7] . the [5] . evidence for a role of tnf-a and il-1 in influenza virus right lung was lavaged with 60 ml of pbs. bronchoalveolar lavage pathogenesis and disease is growing [4, 6] . (bal) cells were separated by centrifugation at 400 g and counted, the pathogenesis of human influenza has been studied aland cytospin preparations were stained with diffquik (baxter, most exclusively in volunteers and in small laboratory animals. here we studied the production of ifn-a, tnf-a, and il-1 fluid samples in 96-well microtiter plates. ifn-a activity was dein the lungs of pigs and its relation with disease or inflammation termined in a cytopathic effect reduction assay by use of mdbk cells and vesicular stomatitis virus [8] . was defined as the reciprocal of the dilution producing 50% inhibition of cytopathic effect. ifn-a specificity was demonstrated by neutralization of samples with rabbit antiserum against recombinant porcine ifn-a (gift from c. la bonnardière, jouy en josas, france). tnf-a was assayed as cytotoxic activity in pk(15) sub-experimental design, virologic examinations, and lung inflamclone 15 cells (gift from g. bertoni, bern, switzerland) in the matory parameters. four litters (18 total) of 3-week-old cesarianpresence of actinomycin d [9] . the plates were stained with crystal derived colostrum-deprived (cdcd) pigs were used. they were violet and read spectrophotometrically. the number of units of inoculated intratracheally with 10 7.0 eid 50 of influenza virus tnf-a per milliliter was defined as the reciprocal of the dilution (h1n1 a/sw/belgium/1/83), third passage in embryonated eggs. producing 50% cytotoxicity. tnf-a specificity was established control pigs were left either uninoculated or inoculated with pbs by neutralization with rabbit anti-human tnf-a (innogenetics, or with sterile allantoic fluid ( in all groups. maximum il-1 titers were 535, 245, 520, and 150 u/ml in the 4 respective groups. only in group 1 pigs was il-1 also found at 72 h after inoculation. levels of the three cytokines were significantly higher 18 -24 h after inoculation than at 48 -72 h after inoculation (p õ clinical responses, influenza virus titers, bal cell numbers, percentage of neutrophils, and cytokine titers of individual pigs .016 for all three cytokines). significant correlations were noted between production of all three cytokines and bal cell num-are summarized in table 1. clinical symptoms. control pigs remained healthy. in in-bers (p õ .036 for each) and percentage of neutrophils (p õ .001 for each). cytokine production coincided with the onset fluenza virus -inoculated pigs, lethargy, shivering, anorexia, tachypnea, and labored abdominal respiration developed be-of clinical disease and development of bronchopneumonia. tween 18 and 24 h after inoculation. recovery started between 48 and 72 h after inoculation. pigs in group 1 were most discussion severely affected. influenza virus replication. control pigs tested negative for at the start of this study, it was unknown whether the lungs of 3-week-old cdcd pigs are fully capable of tnf-a and virus. virus titers and immunofluorescence scores were similar in the 4 influenza virus -inoculated groups (p ú .577). titers il-1 production. thus far, tnf-a and il-1 have only been demonstrated in the lungs of pigs 6 -8 weeks old and older, in apical and diaphragmatic lung lobes were significantly higher 18 -24 h after inoculation than at 48 -72 h after inoculation (p either conventional [11] or gnotobiotic [12] . in our study, the use of cdcd pigs was required, since we regularly detect õ .016 and .008, respectively). fluorescence was evident in all sections examined. eighteen and 24 h after inoculation, tnf-a and/or il-1 in bal fluids from conventional pigs in the absence of experimental viral infections. the age of 3 weeks bronchi/bronchioli and alveoli had, respectively, 90% and 30% of their epithelial cells fluorescing. by 48 -72 h, more of the was selected for practical reasons. because sanitary status and age may influence cytokine production, we performed a prelim-alveolar tissue became involved. lung inflammatory changes. control pigs did not have inary experiment in 3-week-old cdcd pigs. two pigs were inoculated intratracheally with 17 mg of escherichia coli o111: macroscopic or microscopic lung pathology. bal cell numbers were between 35 and 60 1 10 6 . fewer than 1.5% of cells were b4 lipopolysaccharide, a known potent tnf-a and il-1 inducer. bal fluids collected 6 and 12 h after inoculation re-neutrophils; ú95% had macrophage morphology. after influenza virus inoculation, gross lung lesions appeared vealed tnf titers of 184 and 128 u/ml and il-1 titers of 564 and 596 u/ml in the respective bioassays. consequently, 3-between 48 and 72 h after inoculation. at that time, ç85%, 18%, 18%, and 8% of lung tissue was affected in groups 1, 2, week-old cdcd pigs were found suitable for further influenza virus -cytokine studies. 3, and 4, respectively. on histopathology, bronchi/bronchioli and, to a lesser degree, alveoli showed epithelial necrosis and to our knowledge, this is the first demonstration of influenza virus -induced tnf-a and il-1 in bal fluids of a natural virus massive neutrophil infiltration at 18 -24 h after inoculation. forty-eight and 72 h after inoculation, bronchioli and alveoli host. interestingly, influenza virus was an equally effective inducer of tnf-a and il-1 as was e. coli endotoxin. tnf-a were filled with exudate containing necrotic debris and macrophages and only few neutrophils. histologic changes were most and il-1 have remarkably overlapping and synergistic effects, several of which are consistent with clinicopathologic manifes-dramatic in group 1. bal cell numbers were between 112 and 160 1 10 6 at 18 -24 h after inoculation and consisted of a tations of influenza [5] . our findings in pigs are in agreement with previous reports in mice [3, 4] and further substantiate the maximum of 56% -77% neutrophils in groups 1, 2, and 3. in / 9d43$$ap48 03-04-98 19:03:33 jinfa uc: j infect group 1 pigs, which had consistently higher values of tnf than the other groups, showed most severe disease and lesions effect of intratracheal challenge of fattening pigs previously immunised with an inactivated influenza h1n1 vaccine group 4 pigs, with barely detectable tnf levels, also had lower interferon production by leukocytes interferon-a was detected at extremely high titers in some infiltrating the lungs of mice during primary influenza virus infection. pigs. as for tnf-a and il-1, ifn-a production was tightly production of interleukin 1 and tumour necrosis factor activities in and peak ifn production coincided with the appearance of bronchoalveolar washings following infection of mice by influenza viillness. these findings support the idea that interferons contribrus peper rl, van campen h. tumor necrosis factor as a mediator of inflam-ifn-a is intrinsically pyrogenic [13] and can mediate neutromation in influenza a viral pneumonia tumour necrosis factor and interleukin 1: cytokines with vitro that ifn-a may enhance the neutrophil respiratory burst multiple overlapping biological activities kluger a in pig bal fluids, ifn could considerably add to the pyro-mj. thermal and behavioral effects of lipopolysaccharide and influenza genic and inflammatory effects of tnf-a and il-1. in interleukin-1b -deficient mice porcine respiratory coronavirus -mediated interference against influenza virus replication in the respiratory tract the other groups. most striking was the virtual lack of tnf of feeder pigs high interferon titer in newborn pig intestine and previous groups cannot be attributed to differences in viral during experimentally induced viral enteritis improved bioassay for the belonged to a genetically different line. it would be worthwhile detection of porcine tumor necrosis factor using a homologous cell line: pk(15) mur-although influenza viruses in humans primarily infect the taugh mp. inflammatory cytokine expression in swine experimentally upper respiratory tract, influenza pneumonia yearly causes high infected with actinobacillus pleuropneumoniae increased levels of tumor necrosis factor elderly. experimental research thus remains of high priority, and interleukin 1 in bronchoalveolar lavage fluids from pigs infected and commonly used animal models have some limitations ferrets, for example, upper respiratory tract infection predomi tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of innounced [15]. mice and guinea pigs, on the other hand, are terleukin 1 interferon-a enhances neutrophil respito them. also, mice show a fall in body temperature instead of ratory burst responses to stimulation with influenza a virus and fmlp. fever and, with more virulent strains, the infection is invariably lessons for human influenza from pathogenicity studies with ferrets hypothesis that tnf-a and il-1 contribute to clinicopathologic effects of influenza. first of all, both cytokines were positively we thank r. ducatelle for help with histology and d. j. shaw correlated (r ú .879) with neutrophil recruitment to the lungs, for statistics. and peak cytokine production coincided with the onset of clinical disease. furthermore, there was a clear association between individual tnf levels on the one hand and the extent of neutrophil infiltration and severity of lung pathology on the other. key: cord-320445-pdvkyzci authors: fry, alicia m.; lu, xiaoyan; chittaganpitch, malinee; peret, teresa; fischer, julie; dowell, scott f.; anderson, larry j.; erdman, dean; olsen, sonja j. title: human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand date: 2007-04-01 journal: j infect dis doi: 10.1086/512163 sha: doc_id: 320445 cord_uid: pdvkyzci background. we detected human bocavirus (hbov) infection in 4.5% of hospitalized patients with pneumonia in rural thailand. however, the role of hbov as a pathogen is unclear. methods. we compared hbov infection in patients with pneumonia with that in asymptomatic control patients enrolled between 1 september 2004 and 31 august 2005 in the same hospitals in thailand.we examined outpatients with influenza-like illness for hbov infection and tested for 13 additional respiratory viruses. epidemiologic and clinical characteristics of hbov infection are described. results. hbov infection was detected in 20 (3.9%) of 512 outpatients and 3 (1%) of 280 control patients. coinfection with other viruses was detected in 83% of patients with pneumonia and in 90% of outpatients. compared with control patients, hbov infection was significantly associated with pneumonia requiring hospitalization (adjusted odds ratio, 3.56 [95% confidence interval, 1.06–11.91]; p = .04). eighty-three percent of hbov infections were detected in patients with pneumonia who were <5 years old. more patients with pneumonia associated with hbov—respiratory syncytial virus (rsv) or human parainfluenza virus (hpiv) coinfections had wheezing than patients with rsv and hpiv infections alone (9 [53%] of 17 vs. 32 [23%] of 138]; p = .01). conclusions. hbov infection was epidemiologically associated with pneumonia among young children in rural thailand, but infection and illness may be dependent on coinfection with other viruses. human bocavirus (hbov) is a recently identified parvovirus that was detected in respiratory secretions from children with lower respiratory tract infections (lrtis). first reported in sweden [1] in september 2005, it is only the second member of the family parvoviridae to be potentially associated with human disease and is the first to be linked with respiratory illness. recently, additional studies identified hbov from banked respi-ratory specimens from children with lrti [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . together, these studies reported a prevalence of hbov infection of 1.5%-11.3%, with most hbov detections occurring in young children. coinfection with other viruses was examined in several studies and ranged from 17% to 67% [1, [5] [6] [7] [8] [9] [10] of specimens. however, the viruses tested and viral diagnostic assays varied between studies. although several studies detected hbov in respiratory specimens from patients presenting with lrti, they neither assessed whether hbov infection was epidemiologically associated with respiratory illness by comparison with control groups that controlled for age and month of infection nor estimated populationbased rates of disease in different age groups. we sought to determine whether hbov infection was associated with severe respiratory illness by comparing hbov infection among hospitalized patients with pneumonia with infection among persons without respiratory symptoms. using recently developed real-time polymerase chain reaction (pcr) assays for hbov [12] , we examined hospitalized patients of all ages with pneumonia enrolled through active population-based surveillance in rural thailand between 1 september 2004 and 31 august 2005 for hbov infection, to generate estimates of incidence and disease burden. we also tested a sample of patients with influenza-like illness and persons without fever or respiratory illness during the preceding 3 days who attended outpatient departments of the same hospitals during the study period. in addition, we used sensitive pcr assays to test for 13 other respiratory viral pathogens. the association of hbov infection with pneumonia requiring hospitalization and with viral coinfections and the clinical and epidemiologic characteristics of hbov infections during the study period are described. we enrolled hospitalized patients with pneumonia identified through active population-based surveillance in sa kaeo province, [13] . in general, a patient was enrolled in the study if he or she had at least 1 sign of acute infection (fever, chills, temperature !35.5њc, abnormal white blood cell [wbc] count or differential [111,000 or !3000 cells/ ml]) plus signs or symptoms of lower respiratory tract disease (abnormal breath sounds, tachypnea, cough, sputum production, or dyspnea) and the physician ordered a chest radiograph (cxr) within 48 h of admission. each enrolled patient provided a nasopharyngeal (np) swab, acute and convalescent serum, urine, and clinical and demographic information. in addition, a sample of outpatients with acute influenza-like illness was recruited from the outpatient departments of 5 hospitals in sa kaeo province [14] . influenza-like illness was defined by the world health organization definition (fever within the preceding 3 days and cough or sore throat in the absence of other diagnoses). each outpatient participant provided a np swab and demographic information. the key to the present study was the collection of respiratory samples from control patients (i.e., patients from the same province who did not have a fever and did not report a fever, cough, or sore throat within the preceding 3 days), which began in august 2004. the control patients were collected from the outpatient clinics described above. we sought to enroll equal numbers of control patients from each age group (persons р2, 3-5, 6-15, 16-54, and у55 years old) during each month of the year and an equal number of control patients each month (median number per month, 25; range, 10-30). each control patients provided a np swab and demographic information. nasopharyngeal swabs were collected and shipped to the thailand national institute of health (thai nih) and then to the cdc as described elsewhere [12] . acute and convalescent serum specimens were also collected. np specimens were inoculated into mdck and hep-2 cells and tested for respiratory syncytial virus (rsv), human parainfluenza viruses (hpivs), influenza viruses, and adenovirus by immunofluorescent staining at thai nih. respiratory specimens and select serum specimens were tested for hbov by real-time pcr assays that targeted the hbov ns1 and np-1 genes [12] . we defined an hbov infection as a positive pcr test for both the ns1 and np-1 targets or a positive test for a single target that was confirmed with a second extraction from a new, previously unopened sample aliquot. semiquantitative estimates of virus load in clinical samples expressed as genome equivalents (ges) per milliliter of viral transport media were determined by comparison of sample cycle threshold value with those obtained from standard curves prepared from serial 10-fold dilutions of hbov recombinant plasmids for each real-time pcr target [12] . the respiratory specimens were also tested for rsv; hpiv 1, 2, and 3; adenovirus; influenza viruses a and b; human metapneumovirus (hmpv); and rhinoviruses using pcr methods described elsewhere [15, 16] and for human coronaviruses (hcov) 229e, oc43, hku1, and nl63 using in-house realtime pcr assays. a specimen was considered to have a positive result if either culture or pcr testing was positive. diagnostic testing for bacterial pathogens during the study period was limited to urine pneumococcal surface antigen assay among patients у18 years old who had pneumonia. we compared hbov infection in hospitalized patients with pneumonia and control patients using the x 2 test and a multivariable unconditional logistic regression model controlling for age group (р2, 3-5, 6-15, 16-54, and у55 years old) and month. the proportion of patients with pneumonia in each age group was 24%, 9%, 9%, 25%, and 34%, respectively; the proportion of control patients was 18%, 11%, 22%, 25%, and 23%, respectively. our analysis was limited to 1 september 2004-31 august 2005. we assessed risk factors for hbov infection among hospitalized patients with pneumonia using an unconditional logistic regression model that included all potential confounders and variables in the model that were significant at the univariate level at . all 2-way interactions p ! .05 were evaluated. to estimate the minimum age group-specific incidence of hbov-associated pneumonia requiring hospitalization, we divided the number of hbov infections by the appropriate age-group population estimate from the 2000 national thai census of sa kaeo province. each pneumonia admission was counted individually, including readmissions у14 days since a previous discharge. readmissions within р13 days were considered to be 1 admission. clinical characteristics that were obtained from patients with pneumonia associated with hbov infection with rsv, hpiv 1-3, or rhinovirus infection, but no coinfection with any of the other viruses, were compared with patients with hbov-rsv and hbov-hpiv coinfection or with hbov-rhinovirus coinfection. rsv-and hpiv-associated pneumonias were combined to increase the numbers available for comparison. variables were compared using the x 2 test for dichotomous variables or the wilcoxon signed-rank test for continuous variables. two-tailed was considered to be p ! .05 statistically significant. patients with missing values for clinical characteristics were excluded from the comparison of clinical characteristics, and patients with triple virus infections were excluded from clinical comparisons and logistic regression models. all analysis was performed by use of sas software (version 9.1; sas institute). all participants were informed of the study objectives, and written consent was obtained. the study protocol was reviewed and approved by the ethics review boards of the cdc and thai moph. during 1 september 2004-31 august 2005, 3960 patients with signs and symptoms of clinical pneumonia were admitted to a sa kaeo province hospital, and 2288 (58%) of these patients had a cxr. we enrolled 1171 (51%) of the hospitalized patients with pneumonia who had a cxr; 3 patients did not have a sufficient amount of specimen remaining to test for hbov and were excluded from the analysis. among patients with pneumonia, 51 were readmissions. in addition, we enrolled a sample of 512 outpatients with upper respiratory tract illness and 280 control patients without fever or respiratory illness. one outpatient did not have enough specimen to test for hbov and was excluded. np swab specimens from all participants were tested for hbov (table 1) . as reported elsewhere [12] , hbov infection was detected in 4.5% of patients with pneumonia. most infections were detected among patients with pneumonia who were !5 years old; 13% were among infants !1 year old, and 70% were among children 1-4 years old. few hbov infections were found among young or middle-aged adults; 4% were among persons у65 years old. by contrast, only 1% of control patients had hbov infection. hbov infection was detected in 20 (3.9%) of 512 outpatients with influenza-like illness; 70% were among children !5 years old. among hbovpositive specimens, the estimated virus load ranged from 600 to ge/ml. as noted elsewhere [12] , most of the pos10 4 ϫ 10 itive hospitalized patients and all of the control patients had moderate to low virus loads (! ge/ml). six patients 4 5 ϫ 10 with pneumonia and 3 outpatients had high levels of hbov in their respiratory samples (у10 7 ge/ml). among patients with pneumonia, hbov was the third most common viral infection detected among children !5 years old, after rhinovirus and rsv, and it accounted for 12% of all pneumonia requiring hospitalization in this age group. the unadjusted estimated annual incidence of hbov-associated hospitalized pneumonia, including all hbov infections, in sa kaeo province during the study year was 123 cases/100,000 population for infants 0-11 months old, 129 cases/100,000 population for children 1-4 years old, 3 cases/100,000 population for persons 5-19 years old, 1 case/100,000 population for persons 20-49 years old, 2 cases/100,000 population for persons 50-64 years old, and 9 cases/100,000 population for persons 165 years old. overall, the unadjusted estimated incidence of hospitalized pneumonia in sa kaeo province with hbov detected was 12 cases/100,000 population. among the 53 hbov-infected patients with pneumonia, 44 we compared the detection of hbov in respiratory specimens between hospitalized patients with pneumonia and control patients (table 3) . the risk of hbov infection was 4 times higher among patients with pneumonia than among control patients and was statistically significant even after we controlled for age group and month. however, if we compared hbov infections in patients with pneumonia who did not have coinfection with other viruses and control patients, the detection of hbov was not significantly greater in patients with pneumonia, compared with control patients. we also compared outpatients with influenza-like illness with control patients. after controlling for age group and month, the risk of hbov detection in outpatients with influenza-like illness was slightly higher than that in control patients, but the association was not significant (adjusted odds ratio [aor], 1.58 [95% confidence interval {ci}, 0.34-7.31]; ). p p .55 during the study period, hbov infections in patients with pneumonia and outpatients with influenza-like illness occurred throughout the year. however, 34 (47%) of 73 hbov infections occurred during february and march (figure 1); 6 (55%) of 11 hbov infections with no coinfection occurred during february and march. the seasonality of hbov infections with viral coinfections appeared to coincide with the seasonality of rsv and rhinovirus. hbov-rhinovirus coinfections occurred throughout the year, similar to rhinovirus infections (data not shown). however, 11 (50%) of hbov-rhinovirus coinfections occurred during february and march. also, 8 (62%) of hbov-rsv coinfections occurred in june and july, the months when 55% of rsv infections occurred (data not shown). the remaining hbov-rsv coinfections occurred during february and march. we looked at independent risk factors for hbov infection among hospitalized patients with pneumonia. because coinfections with rhinoviruses, hpiv3, and rsv were common with hbov and coinfection occurred between these viruses, we included these viral infections, as well as age group and month, in our model. age was the greatest risk factor for hbov infection, with younger children, a few clinical characteristics differed when we compared patients with pneumonia associated with hbov infection with patients with rsv and hpiv infections (combined) and with patients with hbov-rsv/hpiv coinfection (table 4) . more patients with pneumonia associated with hbov-rsv/hpiv coinfections had wheezing than patients with rsv and hpiv infections. hbov-rsv/hpiv coinfections also were more likely to cause elevated wbc counts (111,000 cells/ml) at admission. individually, results were similar for rsv but not hpiv ( ). none of the patients with p p .03 pneumonia associated with hbov-rhinovirus infections were !1 year old. nine (6%) patients with rhinovirus infection were !1 year old. there was no difference in fever or elevated wbc counts between patients with rhinovirus infection and those with hbov-rhinovirus coinfection. finally, we tested serum collected from some hospitalized patients with pneumonia for hbov dna by real-time pcr. initially, we screened serum from 5 patients with the highest levels of hbov dna (110 7 ge/ml) in their respiratory specimens. hbov dna was detected in the acute-phase serum specimen from 4 (80%) of 5 patients and in the convalescentphase serum specimen from 2 (40%) patients. the virus loads were substantially lower than those in the corresponding respiratory specimens. viral coinfections were detected in 3 (60%) of 5 patients. for comparison, we examined, in a blinded fashion, acute serum from 6 patients with mid-to low levels of hbov dna ( ge/ml) and 5 patients with 4 5 2 ϫ 10 -4 ϫ 10 no detectable hbov dna in their respiratory specimens. hbov dna was not detected in any of these specimens. our results suggest that hbov infection is epidemiologically associated with pneumonia requiring hospitalization in rural thailand. because hbov infection was uncommon among our sample of persons without fever or signs and symptoms of respiratory illness, it seems unlikely that hbov infection in patients with pneumonia was a coincidental event and not linked to their illness. however, the role that hbov infection plays in the disease process is confounded by the high proportion of coinfections with other respiratory viruses. we tested table 4 for a greater number of viruses than has been reported in other published studies, and we found a much higher proportion of viral coinfections-among children !5 years old, the proportion of hbov infections with viral coinfections was 91%. in addition, the burden of hbov-associated pneumonia was substantial. our population-based estimates of the incidence of hbov-associated pneumonia place it among such viruses as rsv and influenza, which cause significant morbidity among young children. finally, our findings suggest that hbov infection, or coinfection, may influence a patient's clinical presentation. the apparent dependency of hbov infection and illness on coinfection with other respiratory viruses is intriguing. parvoviruses are among the most dependent on host cellular functions for replication, and they only multiply in cells that are in the process of replicating their own dna [17] . human parvovirus b19, an erythrovirus, only replicates during the dna synthesis phase of the cell cycle. adeno-associated viruses in the genus dependovirus depend on coinfection with adenovirus or herpesviruses that induce dna synthesis in the host cell to facilitate their own replication. however, the other members of the genus bocavirus-bovine bocavirus and canine minute virus-do not require a helper virus, and most of the hbov coinfecting viruses that we identified do not induce dna replication in the host cell. one possibility is that coviral-induced cellular damage results in high levels of cellular division and differentiation, creating conditions permissive for hbov replication. a similar mechanism has been shown for polyomavirus infection in mice [18] . in addition, respiratory viral infections may suppress immune system functions to make the host more susceptible to infection with hbov. further studies are needed to understand the interaction between hbov and other viral infections. interestingly, patients with pneumonia associated with hbov-rsv/hpiv coinfection and hbov-rhinovirus coinfection appeared to have more wheezing recorded at admission than patients with rsv, hpiv, or rhinovirus infection. children with viral respiratory infections often present with wheezing [19] . although we did not have a large sample of patients with only hbov infection, our results raise the possibility that hbov coinfection may play an important role in the clinical presentation of wheezing among children. also, several studies have documented an association with respiratory viral infections and exacerbations of asthma in adults and children [20] [21] [22] . confirmation of our results with other patient cohorts and obtaining additional information on underlying illness, such as a history of asthma, will be important to more fully understand the role of hbov infection and human disease. the presence of nucleic acid in acute-phase serum specimens from patients with pneumonia with high titers of hbov in their respiratory specimens suggests that hbov viremia may have occurred in these patients, although virus concentrations were consistently higher in respiratory secretions than in serum collected on the same day. we did not find evidence of viremia in samples from patients with low hbov respiratory titers or those infected with other respiratory viruses. therefore, this phenomenon may not be common. interestingly, hbov nucleic acid was present in convalescent serum specimens from 2 of the patients with hbov positive acute serum. persistent viremia of another parvovirus causing human illness, parvovirus b19, has been described [23] . one study also reported finding hbov nucleic acid in respiratory tissues and one bowel-tissue specimen from autopsy specimens from 9 children [24] . therefore, it is possible that this recently described virus does cause viremia and possibly even illness other than that of the respiratory tract. persistent hbov viremia could have implications for blood-or organ-donation programs. tissue-culture techniques for growing hbov that could establish hbov viability in serum specimens have not yet been described. our study has some strengths and limitations with bearing on its interpretation. in contrast to most studies to date, which reported hbov in a convenience sample of patients submitting respiratory specimens, the present study was built on an ongoing, prospective, population-based, surveillance system, and it included a control group without respiratory disease, which allowed us to estimate incidence and burden of disease and to determine epidemiologically whether hbov infection was associated with hospitalized pneumonia. however, the number and rate of cases of hbov-associated pneumonia were likely underestimated. only 58% of patients admitted to a sa kaeo hospital during the study period with signs and symptoms of clinical pneumonia got a cxr, and only 51% of these enrolled in the etiology study did so. we found a high proportion of hbov infections that had coinfection with other viruses. however, although we tested for more viruses than other studies, we may have missed additional viral infections not detected by pcr or tissue culture. the pcr primers for viral diagnostics were based on published sequences. therefore, viral variants that are not recognized by our primers or identified by our culture techniques will have been missed. during the study year, diagnostic testing for bacterial pathogens was limited to urine pneumococcal surface antigen assay among patients with pneumonia who were 118 years old. no adults had hbov-streptococcus pneumoniae coinfection. however, we do not know whether hbov-bacterial coinfections occurred among children or whether hbov-atypical bacterial coinfections occurred. to our knowledge, we present the first study that clearly demonstrates an association between hbov infection and pneumonia requiring hospitalization and likely with less severe respiratory illness that is managed in outpatient clinics. however, understanding the interaction between hbov and other coinfecting viruses will be crucial for establishing the role of this newly identified virus in human disease. in addition, hbov coinfection may influence the clinical symptoms that the patient displays. additional studies to better define hbov infection, including interactions with other viruses, determining whether hbov causes illness other than respiratory illness, and whether hbov infection can persist after acute illness will be critical to determine the burden of hbov infection. cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection evidence of human coronavirus hku1 and human bocavirus in australian children detection of human bocavirus in japanese children with lower respiratory tract infections human bocavirus in children frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus in french children the association of newly identified respiratory viruses with lower respiratory tract infections in korean children human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus real-time pcr assays for detection of bocavirus in human specimens the incidence of pneumonia in rural thailand the cost of influenza in thailand human metapneumovirus infections in young and elderly adults ) • 1045 transcription-pcr assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness parvoviridae: the viruses and their replication adult mouse kidneys become permissive to acute polyomavirus infection and reactivate persistent infections in response to cellular damage and regeneration the tucson children's respiratory study: 2. lower respiratory tract illness in the first year of life the incidence of respiratory tract infection in adults requiring hospitalization for asthma respiratory viruses and exacerbations of asthma in adults community study of role of viral infections in exacerbations of asthma in 9-11 year old children persistent parvovirus b19 infection without the development of chronic anemia in hiv-infected and -uninfected children: the women and infants transmission study high prevalence of human bocavirus in pediatric autopsy respiratory specimens we thank brian holloway and karen mccaustland (centers for disease control and prevention biotechnology core facility branch), for oligonucleotide synthesis and expert technical advice; and george gallucci and pongpun sawatwong, for technical assistance. key: cord-287210-sars5dmi authors: woo, patrick c. y.; lau, susanna k. p.; tsoi, hoi-wah; huang, yi; poon, rosana w. s.; chu, chung-ming; lee, rodney a.; luk, wei-kwang; wong, gilman k. m.; wong, beatrice h. l.; cheng, vincent c. c.; tang, bone s. f.; wu, alan k. l.; yung, raymond w. h.; chen, honglin; guan, yi; chan, kwok-hung; yuen, kwok-yung title: clinical and molecular epidemiological features of coronavirus hku1–associated community-acquired pneumonia date: 2005-12-01 journal: j infect dis doi: 10.1086/497151 sha: doc_id: 287210 cord_uid: sars5dmi backgroundrecently, we described the discovery of a novel group 2 coronavirus, coronavirus hku1 (cov-hku1), from a patient with pneumonia. however, the clinical and molecular epidemiological features of cov-hku1–associated pneumonia are unknown methodsprospectively collected (during a 12-month period) nasopharyngeal aspirates (npas) from patients with community-acquired pneumonia from 4 hospitals were subjected to reverse-transcription polymerase chain reaction, for detection of cov-hku1. the epidemiological, clinical, and laboratory characteristics of patients with cov-hku1–associated pneumonia were analyzed. the pol spike (s), and nucleocapsid (n) genes were also sequenced resultsnpas from 10 (2.4%) of 418 patients with community-acquired pneumonia were found to be positive for cov-hku1. all 10 cases occurred in spring and winter. nine of these patients were adults, and 4 had underlying diseases of the respiratory tract. in the 6 patients from whom serum samples were available, all had a 4-fold change in immunoglobulin (ig) g titer and/or presence of igm against cov-hku1. the 2 patients who died had significantly lower hemoglobin levels, monocyte counts, albumin levels, and oxygen saturation levels on admission and had more-extensive involvement visible on chest radiographs. sequence analysis of the pol s, and n genes revealed 2 genotypes of cov-hku1 conclusionscov-hku1 accounts for 2.4% of community-acquired pneumonia, with 2 genotypes in the study population. without performance of diagnostic tests, the illness was clinically indistinguishable from other community-acquired pneumonia illnesses tify novel agents. of the 3 novel agents identifie in the last 3 years-including human metapneumovirus [3] , severe acute respiratory syndrome (sars) coronavirus (sars-cov) [4] , and human coronavirus nl63 (hcov-nl63) [5, 6 ]-2 are coronaviruses. on the basis of serological and phylogenetic characterization, coronaviruses were divided into 3 distinct groups: human coronavirus 229e (hcov-229e) and hcov-nl63 are group 1 coronaviruses, and human coronavirus oc43 (hcov-oc43) is a group 2 coronavirus [7] . hcov-229e and hcov-oc43 account for 5%-30% of human respiratory tract infections [8] , whereas, in a few recent studies, hcov-nl63 was found to be present in 2%-3.6% of respiratory specimens [9] [10] [11] . in 2002 and 2003, the epidemic caused by sars-cov affected 18000 people, with 750 deaths [4, [12] [13] [14] [15] [16] [17] [18] . table 1 . primers used for amplification and sequencing of the pol, spike (s), and nucleocapsid (n) genes. forward primer reverse primer pol lpw1465 5 -gttcaagtgtcgctgttca-3 lpw1822 5 -ctatcattatcacaatccacag-3 lpw1467 5 -gggtatgaagtatcatccta-3 lpw1825 5 -gataatcccaacccataagaac-3 lpw1826 5 -catcttataaaggatgttgac-3 lpw1829 5 -acaaacaacacatgcacctacac-3 s lpw1830 5 -ttgcctattattttacaaggt-3 lpw1864 5 -acactacctataactatagtac-3 lpw1832 5 -tatgttaataawactttgtatagtg-3 lpw1866 5 -tacaattgacaagaactagaag-3 lpw1836 5 -gatttgcarttgggcagttctgg-3 lpw1868 5 -ccattagaatcatacaaaagat-3 lpw1840 5 -ggtatttttaaagaagtttctgc-3 lpw1870 5 -agcttcaacaaaaccwacatctg-3 lpw1844 5 -taggtycacamtgyggttcttc-3 lpw1872 5 -amccttgyttaggtgcaatacct-3 lpw1848 5 -ttaaaactgtyttagtaagtcc-3 lpw1874 5 -tagtaaaactagttayaacacc-3 n lpw1887 5 -tagtggtatggatactgccttgt-3 lpw1890 5 -gctttaacatttcagmattacca-3 lpw1891 5 -cagtgttttggtaaaagaggacc-3 lpw1892 5 -taccacctagtgtcgaattagg-3 recently, we have described the discovery of a novel group 2 coronavirus, coronavirus hku1 (cov-hku1), from a patient with pneumonia [19] . complete genome sequencing and phylogenetic analysis revealed that cov-hku1 is a distinct group 2 coronavirus, with g + c content of 32%, the lowest among all known coronaviruses. although preliminary screening also identifie an additional patient with pneumonia with cov-hku1 in her nasopharyngeal aspirate (npa), the epidemiological and clinical features of cov-hku1-associated pneumonia remain to be determined. in the present study, using prospectively collected npas, we examined the prevalence of cov-hku1 in patients with community-acquired pneumonia during a 12-month period. the clinical, laboratory, and radiological characteristics of patients with cov-hku1-associated pneumonia were described. the molecular epidemiological profil of cov-hku1 was also analyzed. all prospectively collected npas from patients with community-acquired pneumonia that were sent to the clinical microbiology laboratories of 4 hospitals in hong kong during a 12-month period (22 march 2003 [the beginning of the sars epidemic in hong kong] to 21 march 2004) for detection of sars-cov and were found to be negative for sars-cov rna, by reverse-transcription polymerase chain reaction (rt-pcr) [20] , were included in the study. community-acquired pneumonia is de[16] . to determine possible risk factors associated with cov-hku1-associated pneumonia, 2 age-and sex-matched controls per patient with cov-hku1-associated pneumonia were randomly selected from patients with community-acquired pneumonia whose npas were found to be negative for cov-hku1. controls were within 5 years in age (older or younger) of the corresponding patients with cov-hku1-associated pneumonia and were admitted within 15 days before or after admission of the corresponding patients with cov-hku1-associated pneumonia. the hospital records, laboratory results, and chest radiographs of the controls were examined by 2 infectious disease physicians. rna extraction. viral rna was extracted from npas using the qiaamp viral rna mini kit (qiagen) within 10 h of receipt of specimens. the eluted rna (template for rt-pcr) was stored immediately at ϫ70њc until use. a 453-bp fragment of the pol gene of cov-hku1 was amplifie by rt-pcr using cov-hku1-specifi primers (lpw1926 [5 -aaaggatgttg-acaaccctgtt-3 ] and lpw1927 [5 -atcatcatactaaa-atgcttaca-3 ]) designed by multiple alignment of the nucleotide sequences of the pol genes of cov-hku1 [19] and those of other coronaviruses. rt was performed using the su-perscript ii kit (invitrogen). the pcr mixture (50 ml) contained cdna, pcr buffer, 200 mmol/l each dntp, and 1.0 u of taq polymerase (boehringer). the mixtures were amplifie in 40 cycles of 94њc for 1 min, 48њc for 1 min, and 72њc for 1 min and a fina extension at 72њc for 10 min. to ensure the high specificit of the cov-hku1-specifi primers, rna extracted from 200 npas positive for influenz a and b viruses, parainfluenz viruses 1-3, rsv, or adenovirus antigens and rna from 12 npas positive for hcov-229e, hcov-oc43, hcov-nl63, or sars-cov rna (3 samples/coronavirus) were also subjected to rt-pcr using the 2 cov-hku1-specifi primers. the amplifie products were detected by agarose gel electrophoresis, as described elsewhere [19] . both strands of the pcr products were sequenced twice by use of an abi prism 3700 dna analyzer (applied biosystems), using the 2 pcr primers. the sequences of the pcr products were compared with the sequences of the pol genes of cov-hku1 [19] and those of other coronaviruses in the genbank database. elisa using recombinant nucleocapsid (n) protein of cov-hku1. the elisa-based igg and igm antibody tests were performed in accordance with our published protocol [19] . each sample was tested in duplicate, and the mean absorbance for each serum sample was calculated. the complete pol, s, and n genes of cov-hku1 from npas from 9 of the 10 patients from whom adequate amounts of rna were available were amplifie and sequenced using the strategy described in our previous study [19] and the primers listed in table 1. the nucleotide and deduced amino acid sequences of the pol, s, and n genes were compared with those of cov-hku1 [19] and other group 2 coronaviruses. phylogenetic tree construction was performed using the pileup method with growtree (genetics computer group). statistical analysis. a comparison of characteristics was made between patients with cov-hku1-associated pneumonia and those with non-cov-hku1-associated pneumonia and between patients who died of and those who survived cov-hku1-associated pneumonia. fisher's exact test was used for categorical variables, and the mann-whitney u test was used for continuous variables. was regarded as statistically p ! .05 significant clinical and laboratory characteristics. during the 12-month period, npas from 418 patients (men:women, 198:220; mean ‫ע‬ sd age, years) in 4 hospitals with community-ac-49 ‫ע‬ 26 quired pneumonia were found to be negative for sars-cov rna by rt-pcr. a 453-bp fragment of the pol gene of cov-hku1 was amplifie and sequenced for 10 patients (2.4%). sequence analysis revealed 0%-2% nucleotide differences between the sequences of the fragments and the sequence of the pol gene from the epidemiological, clinical, and radiological characteristics of the 10 patients with cov-hku1-associated community-acquired pneumonia are summarized in table 2. no epidemiological linkage was identifie among the 10 cases. all cases occurred during either spring or winter (january-may). the median age of these patients was 71.5 years (range, 13-96 years). seven were men, and 3 were women. eight had underlying diseases, of whom 4 had underlying diseases of the respiratory tract. four had recently traveled to southern china. five were smokers. clinically, the illness was not distinguishable from other community-acquired pneumonia illnesses. fever, productive cough, and dyspnea were common symptoms at presentation. upper respiratory tract symptoms were present in only 2 patients (patients 1 and 5). one patient (patient 7) had loose-stool diarrhea. oxygen saturation levels on room air on admission were !95% in 2 patients. airspace shadows were observed in the right lungs of 6 patients and in the left lungs of 6 patients. the upper, middle, and lower zones were affected in 2, 4, and 9 patients, respectively. all patients, except patient (table 3) . none of the 10 patients in this control group from whom serum samples were available had a 4-fold increase in igg titer or the presence of igm against cov-hku1. two of the 10 patients died of cov-hku1-associated pneumonia. the firs patient (patient 2) was a 66-year-old man who had presented with dyspnea for 1 day. he had type 2 diabetes mellitus, old myocardial infarction, and gastric lymphoma with total gastrectomy in 2002 and was treated with chemotherapy. he had severe lymphopenia ( lymphocytes/l) and an 9 0.2 ϫ 10 oxygen saturation level of only 83% on admission. chest radiographic examination revealed patchy airspace shadows in both lungs, with predominant involvement of the lower zones (figu e 2a). he died 11 days after admission. the other patient (patient 10) was a 72-year-old man who had presented with fever and productive cough for 1 week. he had type 2 diabetes mellitus, cerebrovascular accident, and prostatic carcinoma with bone metastasis complicated by spinal cord compression; laminectomy and luque instrumentation were performed. he had lymphopenia ( lymphocytes/l), thrombocytopenia 9 0.9 ϫ 10 ( thrombocytes/l), deranged liver and renal function 9 33 ϫ 10 test results, and an oxygen saturation level of only 88% on admission. chest radiographic examination revealed extensive airspace shadows in both lungs, with the middle zones more severely involved (figu e 2b). he died 5 days after admission. the clinical, laboratory, and radiological characteristics of patients who survived and those who died of cov-hku1-associated community-acquired pneumonia were compared (table 4). patients who died had lower hemoglobin concentrations (p p .04), monocyte counts (p p .04), serum albumin levels (p p .04), and oxygen saturation levels on admission (p p .03) and had bilateral involvement ( ) and more zones involved p p .003 ( ) on chest radiographs. p p .01 the complete pol, s, and n genes of cov-hku1 from npas from 9 of the 10 patients from whom adequate amounts of rna were available were amplifie and sequenced. the phylogenetic trees and nonsynonymous mutations and the corresponding amino acid changes are shown in figu e 3. for the s gene, there were 317 and 306 nt positions with synonymous and nonsynonymous mutations, respectively (figu e 3b); 72% and 28% of the nonsynonymous mutations were in the putative s1 and s2, respectively (figu e 4). for the n gene, there were 42 and 53 nt positions with synonymous and nonsynonymous mutations, respectively (figu e 3c). the nucleotide sequences of 7 of the 9 s or n genes showed similar sequences (genotype a), and those of the other 2 genes also showed similar sequences (genotype b) (figu e 3b and 3c). for the cov-hku1 specimen from patient 1, 2 peaks (t and c) were consistently observed at nt 1300 of the n gene, suggesting the presence of a quasi species (figu e 3c). for the pol gene, there were 95 and 13 nt positions with synonymous and nonsynonymous mutations, respectively (figu e 3a). the nucleotide sequences of the pol genes in the 7 cov-hku1 specimens of genotype a were also clustered together (figu e 3a). interestingly, the 7 cov-hku1 specimens of genotype a were from 7 patients with underlying diseases, and the 2 specimens of genotype b were from the 2 patients without underlying diseases (table 2) . furthermore, multiple alignments of the nucleotide sequences of the pol genes from the 9 cov-hku1 specimens and those from the hcov-oc43, hcov-229e, hcov-nl63, and sars-cov supported the notion that the primers used in the present study were specifi for cov-hku1. cov-hku1, a novel group 2 coronavirus, is associated with community-acquired pneumonia. since the sars epidemic in 2003, we have started to prospectively collect npas from patients with community-acquired pneumonia and store the extracted rna, so that, when a novel virus is discovered, the epidemiological and, hence, the clinical, laboratory, and radiological features of the disease can be studied in a timely manner. in january 2004, we discovered a novel coronavirus, cov-hku1, from a patient with community-acquired pneumonia [19] . the rna extracted from prospectively collected npas was immediately retrieved, and the presence of cov-hku1 was investigated. ten of the 418 npas were positive for cov-hku1, giving an incidence of 2.4%. the presence of cov-hku1 in these specimens was genuine-that is, it was not due to contamination-since amplificatio and sequencing of multiple genes (pol, s, and n) of cov-hku1 indicated the presence of cov-hku1 with different nucleotide sequences from the different patients. moreover, the clinical significanc of cov-hku1 was confi med by the presence of a specifi antibody response in all 6 patients from whom serum samples were available. the antibody response in these patients was unlikely to be the result of cross-reaction with other known human coronaviruses, since none of the 20 serum samples obtained from patients with hcov-oc43 infections or sars-cov pneumonia had positive results in the recombinant n elisa (authors' unpublished data). further studies on detection of cov-hku1 in npas from healthy individuals will determine whether asymptomatic shedding of cov-hku1 occurs. similar to hcov-229e, hcov-oc43, and hcov-nl63, cov-hku1 is a human coronavirus that is endemic in humans. similar to other human coronavirus infections, cases of cov-hku1-associated pneumonia occurred during winter and spring. most patients with cov-hku1-associated pneumonia were old (80% were 165 years old) and had major underlying diseases, especially those of the respiratory and cardiovascular systems. to study the phylogeny and relationships among the 10 cov-hku1 specimens, we sequenced the pol, s, and n genes from the 9 cov-hku1 specimens that contained adequate amounts of rna. combined with the data on partial sequencing of the pol genes from the 10 cov-hku1 specimens (figu e 1), results showed that, unlike the epidemiological profil of sars-cov, the 10 cov-hku1 specimens were not clonal, and the topology of the phylogenetic trees did not follow the pattern of a clonal outbreak (figu e 3). interestingly, the phylogenetic trees constructed using the sequences of both the s and n genes showed that cov-hku1 of genotype b was detected in npas from with 2 patients without underlying diseases, but cov-hku1 of genotype a was detected in npas from patients with underlying diseases (figu e 3b and 3c). sequencing of more cov-hku1 specimens may reveal the presence of genotypes or clades of cov-hku1 with differential virulence. compared with sars-cov pneumonia, cov-hku1-associated pneumonia is a monophasic disease, and most patients had relatively mild symptoms that were localized to the respiratory tract and were, therefore, hospitalized only briefl . sars-cov pneumonia is often described as a biphasic disease, with the firs phase due to cell lysis as a result of viral replication and the second phase due to immunopathological damage [4, 12] . on the other hand, all 10 patients with cov-hku1-associated pneumonia showed the pattern of a monophasic disease. although dyspnea was present in one-half of the patients with cov-hku1-associated pneumonia at presentation, compared with only ∼20% of patients with sars-cov pneumonia at presentation [4] , patients with cov-hku1-associated pneumonia often recovered quickly, but patients with sars-cov pneumonia deteriorated after 7-10 days [4, 12] . for the 8 patients who recovered, the median duration of hospitalization was only 5.5 days. this rapid recovery of patients with cov-hku1-associated pneumonia could be related to the rapid control of the virus by the immune system. this is in line with the results of our previous study, which showed that the index patient with cov-hku1-associated pneumonia had his peak viral load at around day 3 after the onset of illness [19] . moreover, only 1 of the patients had extrapulmonary symptoms, and all available extrapulmonary specimens (stool, urine, and serum) were negative, by rt-pcr, for cov-hku1 (authors' unpublished data). on the other hand, for sars-cov pneumonia, patients usually had their peak viral loads 7-10 days after onset of illness [12] . furthermore, the virus can be readily detected in extrapulmonary specimens, in which the viral loads correlated with the manifestations in the corresponding systems [22] . these finding imply that the virus was not well controlled by the immune system during the initial phase of the illness. despite the relatively mild course of disease in most patients, cov-hku1-associated pneumonia is associated with mortality in a minority of patients who have lower hemoglobin concentrations, monocyte counts, serum albumin levels, and oxygen saturation levels on admission and more-extensive involvement on chest radiographs. as in most cases of pneumonia, extensive involvement in the lungs will result in poor gaseous exchange and, hence, hypoxia and eventually death. the lower hemoglobin concentrations, monocyte counts, and serum albumin levels could represent poorer premorbid states and narrower margins to figh against infections. both patients who died had underlying diabetes mellitus, malignancy (gastric lymphoma in one and prostate carcinoma in the other), and cardiovascular disease (old myocardial infarction in one and cerebrovascular accident in the other). prospective study of aetiology and outcome of adult lower-respiratory-tract infections in the community etiology of community-acquired pneumonia: impact of age, comorbidity, and severity a newly discovered human pneumovirus isolated from young children with respiratory tract disease coronavirus as a possible cause of severe acute respiratory syndrome identificatio of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans the molecular biology of coronaviruses seroepidemiologic studies of coronavirus infection in adults and children human coronavirus nl63 infection in canada new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia detection of human coronavirus nl63 in young children with bronchiolitis clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china molecular epidemiology of the novel coronavirus causes severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia isolation and characterization of viruses related to the sars coronavirus from animals in southern china viral replication in the nasopharynx is associated with diarrhea in patients with severe acute respiratory syndrome characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients viral loads in clinical specimens and sars manifestations key: cord-308979-qhlvd2mt authors: sumino, kaharu c.; agapov, eugene; pierce, richard a.; trulock, elbert p.; pfeifer, john d.; ritter, jon h.; gaudreault-keener, monique; storch, gregory a.; holtzman, michael j. title: detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment date: 2005-09-15 journal: j infect dis doi: 10.1086/432728 sha: doc_id: 308979 cord_uid: qhlvd2mt backgroundinfections with common respiratory tract viruses can cause high mortality, especially in immunocompromised hosts, but the impact of human metapneumovirus (hmpv) in this setting was previously unknown methodswe evaluated consecutive bronchoalveolar lavage and bronchial wash fluid samples from 688 patients—72% were immunocompromised and were predominantly lung transplant recipients—for hmpv by use of quantitative real-time polymerase chain reaction (pcr), and positive results were correlated with clinical outcome and results of viral cultures, in situ hybridization, and lung histopathological assessment resultssix cases of hmpv infection were identified, and they had a similar frequency and occurred in a similar age range as other paramyxoviral infections. four of 6 infections occurred in immunocompromised patients. infection was confirmed by in situ hybridization for the viral nucleocapsid gene. histopathological assessment of lung tissue samples showed acute and organizing injury, and smudge cell formation was distinct from findings in infections with other paramyxoviruses. each patient with high titers of hmpv exhibited a complicated clinical course requiring prolonged hospitalization conclusionsour results provide in situ evidence of hmpv infection in humans and suggest that hmpv is a cause of clinically severe lower respiratory tract infection that can be detected during bronchoscopy by use of real-time pcr and routine histopathological assessment of infection [4] . despite these aggressive approaches, an infectious agent cannot be identified in 20%-65% of patients [3] [4] [5] . missed diagnoses may be due to the poor sensitivity of the diagnostic tests as well as the occurrence of emergent pathogens. moreover, even when a pathogen is found, it may be difficult to be certain that the isolated organism is the cause of the illness. improvements in the diagnosis of viral infection may be achieved when innovative approaches are used to discover new viruses. in this regard, human metapneumovirus (hmpv) was newly discovered by random polymerase chain reaction (pcr) amplification of viral rna that was isolated from children with respiratory tract illnesses [6] . after its initial isolation in the netherlands in 2001, hmpv was identified in lower respiratory tract infections in children worldwide [7] [8] [9] [10] . analysis of stored nasopharyngeal swab samples indicated that hmpv may account for a significant portion of respiratory tract infections with previously unknown etiology [6, 10, 11] . the consequences of hmpv infection range from acute upper respiratory tract infection to severe bronchiolitis 17 (2) autoimmune disease 12 (2) none 47 (7) uncertain 46 (7) or pneumonitis, and the pattern of illness is similar to that found in respiratory syncytial virus (rsv) infection [8, 9, 11] . illness associated with hmpv develops in a broad age range, but young children, the elderly, and immunocompromised hosts appear to be at greatest risk for severe illness [8, 9, 11] . in addition, hmpv has been isolated in 51%-55% of patients with severe acute respiratory syndrome (sars), but its contribution to that illness remains uncertain [12, 13] . indeed, to our knowledge, neither a pathological characterization of hmpv infection nor a correlation with clinical findings has been reported in humans. moreover, the prevalence and other characteristics of hmpv infection in the clinical setting of patients undergoing bronchoscopy for respiratory tract infections still need to be defined. in the present study, we therefore developed and applied quantitative real-time pcr to screen for hmpv in bronchial wash (bw) and/or bal fluid samples. positive results afforded us the opportunity to further define the clinical and pathological features of illness in patients infected with hmpv and to identify a subset of patients with severe illness. a total of 806 sequential samples were collected from patients who underwent bronchoscopy from 15 december 2002 to 1 august 2003. samples obtained within 10 days from the same patient were excluded from analysis, so that a total of 688 fluid samples (576 bal and 112 bw) from 688 patients were analyzed. all samples that were screened for viruses were routinely submitted to the st. louis children's hospital clinical virology laboratory, which serves washington university medical center and acts as a referral laboratory for the st. louis metropolitan area. bronchoscopy was performed at st. louis children's hospital and barnes-jewish hospital at washington university medical center as well as at affiliated community hospitals. the sample period encompassed the peak winter-spring period for detection of hmpv in nasopharyngeal swab samples [14] . the mean age of the patients was 38.6 years; 26% of the patients were !18 years old, 62% were 18-65 years old, and 12% were 165 years old. eighty-six percent of the patients had underlying conditions, most commonly in the setting of lung transplantation (table 1) . predominantly, indications for bronchoscopy were unexplained respiratory tract symptoms or abnormalities found on chest radiographs (table 2) . the cohort also included 210 patients without acute illness who underwent routine bronchoscopy for surveillance after lung transplantation or follow-up bronchoscopy after treatment for transplant rejection. virus detection. for the detection of several non-hmpv viruses (influenza virus, parainfluenza virus, adenovirus, rsv, herpes simplex virus, and rhinovirus), samples were examined by direct immunofluorescence assay (difa) and for cytopathic effect (cpe) in cell culture, as described elsewhere [15] . cy-tomegalovirus (cmv) was detected by shell-vial culture [15] . testing for hmpv on samples stored at ϫ70њc was performed after institutional review board approval. for each sample, a 140-ml aliquot was used for the isolation of rna by use of the qiaamp viral rna kit (qiagen). a 5-ml aliquot of this solution was used for the detection of virus by use of quantitative real-time pcr with the taqman one-step rt-pcr kit, in accordance with the manufacturer's protocol (pe biosystems). primers and probes were derived from a conserved fragment of the nucleocapsid (n) gene from the nl00-1 sequence of hmpv (genbank accession no. af371337). direct and reverse primers amplified nt 993-1064 of the n gene. this system specifically detects hmpv genotype a, which was predominant in the study region during the study period; genotype a accounted for 75% of all hmpv-positive samples (e.a., k.c.s., and m.j.h., unpublished data). a synthetic rna standard for hmpv was generated from a portion of the n gene (nt 40-1172) and was cloned into the pgem plasmid (promega). rna was transcribed in vitro using the t7 mega script kit (ambion). the dna template was eliminated by trizol extraction (invitrogen), dnase treatment, rneasy mini kit preparation (qiagen), and final phenol extraction. the concentration of the rna standard was determined by measurement of the optical density and was used to calculate the copy numbers of hmpv rna. the pcr amplification cycles were as follows: initial heating at 48њc for 30 min and 95њc for 10 min, followed by 40 cycles of 95њc for 15 s and 60њc for 1 min. these conditions allowed for the detection of as few as 5 copies of the rna standard and linear amplification of the standard from 10 to 10 6 molecules. for all hmpv-positive samples, rna isolation and hmpv real-time pcr were repeated, and hmpv-negative samples were excluded from further analysis. in addition, all samples that were found to be positive for hmpv by real-time pcr were tested by a second real-time pcr, which detected genomic rna for a different gene than that used for the initial detection. the primers and probe of this second real-time pcr were designed to amplify a fragment (nt 7155-7241) of hmpv stl43-84, which was isolated in our laboratory from a nasopharyngeal swab sample [14] . all samples were also routinely processed for bacterial and fungal culture and were selectively processed for atypical pathogens (pneumocystis, legionella, and mycoplasma species). in situ hybridization. detection of hmpv was performed using in situ hybridization, because no sensitive and reproducible anti-hmpv antibody was yet available. cdna encoding the hmpv n gene (nt 40-1172; genbank accession no. af371337) was inserted into the pgem-3zf(+) plasmid (promega) and was transcribed into viral genomic rna using the t7 promoter. the antisense rna probe template was linearized by sspi digestion, and the probe was labeled with 35 s using an rna labeling kit (promega). the transcribed rna was purified by phenol extraction and ethanol precipitation and was used as a probe to detect hmpv n gene mrna by in situ hybridization, as described elsewhere [16] . because a sense control probe will also detect hmpv genomic rna, rhesus monkey kidney llc-mk2 cells inoculated with or without hmpv were used as positive and negative controls, respectively, for lung biopsy samples. the fidelity of the probe was also tested on llc-mk2 cells that were cultured in a-mem with bovine serum albumin (1 mg/ml) and trypsin (1 mg/ml) and then inoculated with or without hmpv stl43-84. before in situ hybridization, cells were checked for syncytial formation and for reactivity with a convalescent-phase serum sample from an hmpv-infected patient (patient 6). for these experiments, cultured cells were blocked with 2% gelatin, were incubated with the convalescent-phase serum sample (1:100 dilution) for 1 h and then with fluorescein isothiocyanate-conjugated anti-human igg (1:400 dilution; jackson laboratory), and finally were subjected to immunofluorescence photomicrography. histopathological assessment and clinical outcome. the histopathological appearance of the lung tissue samples and the clinical outcome were reviewed for each patient with detectable hmpv, in accordance with a protocol that was approved by the institutional review board of washington university school of medicine. lung biopsy samples were available for study in 5 of 6 patients and were subjected to hematoxylin-eosin staining and immunostaining for adenovirus, cmv, and herpes simplex virus as described elsewhere [17] . in addition, pathologists examined lung tissue samples from 4 additional patients whose bal fluid samples were positive for rsv, rhinovirus, or parainfluenza virus. pathologists reviewed all lung tissue samples in a blinded manner. clinical characteristics and outcomes were determined by a retrospective review of medical records. hmpv was detected by real-time pcr in samples from 6 patients (designated patients 1-6) at a rate that was similar to that found for other common respiratory tract viruses detected by difa and cpe (table 3). as was expected for an immunocompromised population, cmv was also detected at a high frequency in the samples (table 3) . the detection rate for influenza virus was low, probably because a rapid screening test was available and the need for proceeding to bronchoscopy was decreased. none of the 6 patients with hmpv were coinfected with other viruses, and no hmpv was detected in the 210 asymptomatic patients who underwent routine bronchoscopy for surveillance after lung transplantation or follow-up after treatment for transplant rejection. all occurrences of hmpv infection were clustered in late winter/spring, when hmpv was prevalent in the area. hmpv was the sole organism isolated from bal or bw fluid samples in 4 patients (patients 2, 3, 5, and 6). in 2 patients, we compared viral copy numbers in bw fluid samples with those in bal fluid samples and found that the bw fluid samples contained higher viral copy numbers than the bal fluid samples. for all patients with severe lower respiratory tract illness (patients 1, 2, 3, 5, and 6), the results of routine bacterial and fungal cultures were negative for significant pathogens. in addition, the results of the difa for pneumocystis species (patients 1, 2, 5, and 6), pcr for mycoplasma (patient 1), and culture or urine antigen testing for legionella species (patients 1, 2, and 5) were negative. clinical course of hmpv infection. all patients presented with nonspecific acute respiratory tract symptoms that included fever, cough, dyspnea, and wheezing. four of 6 illnesses occurred in immunocompromised patients (table 4) . patients 2 and 6 presented with acute respiratory failure that required mechanical ventilation. chest radiographs of these patients showed diffuse infiltrates that were suggestive of acute lung injury (figure 1). patients 1, 5, and 6 required prolonged hospitalization because of their acute respiratory tract illness. patient 3 presented with exacerbation of chronic obstructive pulmonary disease and required mechanical ventilation. patient 4, who had the mildest illness, coincidentally developed mild upper respiratory tract symptoms at the time of routine surveillance bronchoscopy after lung transplantation. patients with severe illness due to hmpv infection (patients 1, 2, 5, and 6) tended to have higher levels of hmpv in bal and bw fluid samples than did patients with mild illness, but the small number of patients was insufficient to establish a significant correlation. additional clinical samples were available from patients 1 and 6. for patient 1, a nasopharyngeal swab sample was negative for hmpv at 1 week after the bal fluid sample was obtained. for patient 6, tracheal aspirate and nasopharyngeal swab samples showed a low hmpv titer of 50 and 1032 copies, respectively, at 15 days after bronchoscopy. each of these findings was compatible with partial or complete clearance of the virus as well as greater sensitivity of bal and bw fluid samples for viral detection. in situ detection of hmpv infection. infection with hmpv was confirmed using immunofluorescence microscopy and in situ hybridization of samples. here, we took advantage of an hmpv isolate (designated stl43-84) that we had obtained from a nasopharyngeal swab sample and had characterized by nucleotide sequencing [14] . this isolate was used to inoculate cultured llc-mk2 cells that exhibited cpe and syncytial formation that are typical in paramyxoviral infection (figure 2). this system was then used as a substrate for testing an available convalescentphase serum sample that was obtained from a patient in the present cohort. thus, hmpv-infected llc-mk2 cells, but not control cells, showed immunofluorescent staining with the convalescent-phase serum sample from patient 6, thereby indicating the presence of anti-hmpv antibody in this patient's serum (figure 2) . in addition, we were able to demonstrate that hmpvinfected llc-mk2 cells, but not control cells, gave a positive signal by in situ hybridization with a 35 s-labeled antisense probe for hmpv n gene mrna (figure 2), thereby validating this approach for further analysis of lung biopsy samples. using the same antisense probe for hmpv, we next found a positive signal by in situ hybridization for hmpv mrna in the open lung biopsy sample from patient 1 (figure 3). the signal seemed (on the basis of morphological appearance) to localize predominantly to type ii alveolar epithelial cells and to have a heterogeneous and patchy distribution (figure 3). a lower but still significant signal by in situ hybridization was also found in bronchial epithelial cells (figure 3). signals for hmpv mrna were not detected in the few smudge cells found in the samples that were subjected to in situ hybridization. the transbronchial biopsy samples that were available for study from patients 2, 5, and 6 did not show any focal signal by in situ hybridization for hmpv. histopathological assessment of hmpv infection. lung tissue samples were available from 5 of 6 patients with hmpv infection (all but patient 3). lung tissue samples from 3 patients-from an open lung biopsy (patient 1) and 2 transbronchial biopsies (patients 2 and 5)-showed similar findings. the major finding in each case was acute and organizing lung injury; this included areas of what resembled diffuse alveolar damage with hyaline membrane formation and foci of bronchiolitis-obliterans/organizing pneumonialike reaction. each sample included enlarged type ii pneumocytes with smudged hyperchromatic nuclei that resembled the smudge cells found in adenovirus infection ( figure 4) . however, immunostaining for adenovirus, cmv, and herpes simplex virus was negative in all lung biopsy samples. a transbronchial biopsy sample from patient 4 showed changes after lung transplantation without evidence of lower respiratory tract infection. the small transbronchial biopsy sample from patient 6 showed only nonspecific acute and chronic inflammation. smudge cells were not detected in the samples from the 4 patients with lower respiratory tract infection who had rsv, rhinovirus, or parainfluenza virus detected in bal fluid samples. cultured rhesus monkey kidney llc-mk2 cells were inoculated with either hmpv (+ hmpv) or a control (ϫ hmpv) and then were incubated at 37؇c for 5 days. cells were then subjected to phase-contrast microscopy (first row) at 5 days after inoculation, immunofluorescence microscopy with patient convalescent-phase serum (1:100 dilution) and fluorescein isothiocyanate-conjugated goat anti-human igg (second row) at 2 days after inoculation, and in situ hybridization (ish) with 35 s-labeled antisense probe for viral nucleocapsid gene mrna under brightfield (third row) and darkfield microscopy (fourth row) at 2 days after inoculation. scale bars, 20 mm. ab, antibody since the discovery of hmpv, several studies have confirmed that the virus can be detected (generally by pcr) in clinical samples (generally nasopharyngeal swab samples) from children and adults with acute respiratory tract illness. its occasional detection in asymptomatic patients has also been reported [9] , which raises the question of whether there is sufficient specificity in this diagnostic approach. in the present study, we add several critical pieces to the diagnostic and clinical matrix for hmpv infection: (1) we detected hmpv at a frequency similar to that of other respiratory tract viruses in patients who underwent bronchoscopy for suspected respiratory tract infection; (2) we quantitatively detected hmpv by real-time pcr in bronchoscopy samples from patients with severe respiratory tract illness but not in those without symptoms or signs of respiratory tract infection; (3) we used a viral culture system to develop a specific antisense probe for hmpv and then demonstrated hmpv mrna in the lung tissue samples; and (4) we found organizing and acute lung injury and the prominent formation of smudge cells in the lung tissue samples of these patients, thereby suggesting that this pattern may be characteristic of hmpv infection. taken together, the detection of high copy numbers of hmpv by real-time pcr and of smudge cell formation by transbronchial biopsy can be of significant diagnostic value in defining the etiology of severe respiratory tract illness. the pattern of the present histological findings for hmpv infection is somewhat unusual. we found that, in severe illness, hmpv is capable of causing acute pneumonia and lung injury in a pattern of diffuse alveolar damage with hyaline membrane formation that is similar to the lung injury found in other types of viral respiratory tract infections. the other member of the subfamily pneumovirinae, rsv, which causes clinical disease that is similar to hmpv infection, can also cause acute lung injury with desquamated reactive pneumocytes, and this leads to accumulation of cellular debris within the alveoli and small airways. the presence of multinucleated giant cells (syncytial cells) with eosinophilic cytoplasmic inclusions are strongly suggestive of rsv infection [18, 19] , and we showed that hmpv can also cause syncytial cell formation in cell culture in vitro. by contrast, in vivo, hmpv infection appears to cause the formation of easily distinguishable smudge cells that have not been found in other paramyxoviral infections [20] . these smudge cells closely resemble the enlarged pneumocytes with darkly stained homogeneous inclusion bodies that are found in respiratory tract infection with adenovirus (a virus that causes a pattern of respiratory tract illness that is indistinguishable from that caused by hmpv). the smudge cells found in adenovirus infection have been shown to contain the virus by use of electron microscopy, in situ hybridization, and immunohistochemistry in autopsy series [21] [22] [23] . in contrast, we were able to detect hmpv mrna in the alveolar and airway epithelial cells but not in the smudge cells. this difference could be due to sampling error, because hmpv rna was detected in only a patchy distribution even in the open lung biopsy sample. it is also possible, however, that smudge cells could be reactive in hmpv infection. the present findings are also distinct from those found in experimental hmpv infection of nonhuman primates. in that situation, hmpv replication occurs mainly in ciliated epithelial cells and rarely in type i pneumocytes, and histological assessment shows that erosive and inflammatory changes are confined to the conducting airways [24] . whether these differences reflect species specificity, disease severity, host immune status, or other factors requires further study. similarly, although the results of studies of monkeys support a primary role for a new coronavirus in sars, the present findings again raise the possibility that hmpv contributes to moresevere respiratory tract illness, including sars [12, 13] . although the pattern of acute lung injury with smudge cell formation appears distinctive for hmpv infection, at least in the immunocompromised host, additional samples will be needed to more fully determine the diagnostic value of this pattern. in general, the detection of virus in bal fluid samples is not sufficient (without concomitant histopathological or clinical presentation) to establish a diagnosis of infection. nonetheless, this approach has been used to achieve prompt and accurate diagnoses in patients with possible respiratory tract infection. the highest diagnostic yield and impact of bal fluid sample analysis on therapeutic decisions has been found in immunocompromised hosts, and the combination of bal with transbronchial biopsy provides an additional diagnostic yield [25] . under these circumstances, detection of common respiratory tract viruses (influenza virus, parainfluenza virus, rsv, rhinovirus, and ad-enovirus) is similar to that in the present study (∼3% of cases by use of conventional detection methods) [1, 26] . these respiratory tract viruses have been recently recognized as an important cause of lower respiratory tract infection in immunocompromised patients, and their detection may alter the clinical course of the illness [1, 27, 28] . among these viruses, rsv in particular has been associated with high mortality (26%-100%), especially in bone marrow transplant recipients [27, 29, 30] . in our study population, which was weighted toward immunocompromised patients, hmpv (detected by pcr) was found as frequently as other common respiratory tract viruses (detected by difa and culture). although the relative insensitivity of standard lab tests may underestimate the occurrence of other viruses, this shortcoming should not detract from the importance of using the detection of hmpv to improve patient care. in particular, the detection of hmpv in bal fluid samples in a proper clinical setting may prevent these high-risk patients from undergoing additional diagnostic procedures and treatments and thereby avoid their attendant morbidity and costs. whether there is an additional benefit conferred by instituting antiviral treatment still needs to be determined. for rsv, combination treatment with ribavirin and anti-rsv antibody may improve the clinical outcome in immunocompromised patients, but no treatment has yet been tested in vivo for hmpv infection [29, 31, 32] . the results of the present study provide a further rationale for the development of anti-hmpv antibody and other possible therapies to intervene in what appears to be a severe respiratory tract illness. although we were able to identify only 6 patients with hmpv infection, the consistent clinical features and characteristic pattern of histopathological changes in these patients strongly indicate a clinical significance. in summary, hmpv is a significant cause of lower respiratory tract infection and is associated with morbidity in the immunocompromised host. we recommend that rapid assessment by pcr for hmpv in bal fluid samples be included in the evaluation of these patients. a positive result (combined with the absence of other respiratory tract viruses, such as adenovirus) can be correlated with histopathological findings of smudge cells in the background of acute and organizing lung injury to suggest the diagnosis of lower respiratory tract infection due to hmpv. conventional respiratory viruses recovered from immunocompromised patients: clinical considerations respiratory tract viral infections in bone marrow transplant patients respiratory viruses and severe lower respiratory tract complications in hospitalized patients role of flexible bronchoscopy in immunocompromised patients with lung infiltrates role of bronchoalveolar lavage in immunocompromised patients with pneumonia treated with a broad spectrum antibiotic and antifungal regimen a newly discovered human pneumovirus isolated from young children with respiratory tract disease ruuskanen o. metapneumovirus and acute wheezing in children virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups human metapneumovirus infections in young and elderly adults human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus detection in patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada diversity of human metapneumovirus infection in a us population (abstract w7-6) essentials of diagnostic virology retinoic acid attenuates o 2 -induced inhibition of lung septation viral induction of a chronic asthma phenotype and genetic segregation from the acute response demonstration of respiratory syncytial virus in an autopsy series fatal pneumonia in an allogeneic bone marrow transplant recipient spencer's pathology of the lung adenovirus pneumonia in lung transplant recipients expression of early and late adenoviral proteins in fatal adenovirus bronchopneumonia fatal neonatal pneumonia caused by adenovirus type 35 experimental human metapneumovirus infection of cynomolgus macaques (macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract pulmonary infiltrates in non-hiv immunocompromised patients: a diagnostic approach using non-invasive and bronchoscopic procedures recovery of viruses other than cytomegalovirus from bronchoalveolar lavage fluid paramyxovirus infection in lung transplant recipients community respiratory virus infections following lung transplantation combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients respiratory syncytial virusassociated infections of adult recipients of solid organ transplants respiratory syncytial virus upper respiratory tract illnesses in adult blood and marrow transplant recipients: combination therapy with aerosolized ribavirin and intravenous immunoglobulin comparison of inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro key: cord-335375-n6q70o35 authors: chan, paul k. s.; lim, pak-leong; liu, esther y. m.; cheung, jo l. k.; leung, danny t. m.; sung, joseph j. y. title: antibody avidity maturation during severe acute respiratory syndrome–associated coronavirus infection date: 2005-07-01 journal: j infect dis doi: 10.1086/430615 sha: doc_id: 335375 cord_uid: n6q70o35 the maturation of virus-specific immunoglobulin g avidity during severe acute respiratory syndrome–associated coronavirus infection was examined. the avidity indices were low (mean ± sd, 30.8% ± 11.6%) among serum samples collected ⩽50 days after fever onset, intermediate (mean ± sd, 52.1% ± 14.1%) among samples collected between days 51 and 90, and high (mean ± sd, 78.1% ± 8.0%) among samples collected after day 90. avidity indices of 40% and 55% could be considered as cutoff values for determination of recent (⩽50 days) and past (>65 days) infection, respectively. measurement of antibody avidity can be used to differentiate primary infection from reexposure and to assess humoral responses to candidate vaccines 2003 and january 2004) suggests that the clinical presentation of disease and the transmission behavior of the reemerged sars-cov strain can be different from what was known before [4] . when a sars outbreak occurs again, it is mandatory that a serological survey be conducted, to define the epidemiological character of the outbreak. since these outbreaks may happen in places where a proportion of the population was exposed to the virus during a previous outbreak of sars, a reliable method for differentiating between recent infection and past exposure is vital if a meaningful interpretation is to result from such investigations [4] . the avidity (functional affinity) of an antibody is a measure of the overall strength of interaction between antibody and antigen. the avidity of virus-specific igg antibody is low during primary viral infection and increases with time [6] [7] [8] . however, exceptions to this rule have been observed for some viruses [9, 10] . here, we report the maturation pattern of anti-sars-cov nucleocapsid protein-specific igg antibody (hereafter, "anti-sars-cov igg antibody") avidity over the course of a 10-month period after primary infection and discuss the potential applications of our findings. patients, materials, and methods. sixty-one patients with sars were recruited into the present study; they ranged in age from 21 to 81 years (mean ‫ע‬ sd, 37.0 ‫ע‬ 14.9 years), and 54.1% were female. these patients presented with acute-onset fever that progressed to pneumonia, which was otherwise unexplained. nine patients (14.8%) required intensive care, all of whom eventually recovered. all 61 patients fulfilled the us centers for disease control and prevention criteria for sars [11] and had serological evidence of sars-cov infection, as determined by the anti-sars-cov igg antibody immunofluorescence assay described elsewhere [12] . forty-one patients (67.2%) seroconverted, and 20 (32.8%) developed a у4-fold increase in antibody titer. a total of 90 serum samples were available from the 61 patients, of whom 26 provided 11 sample for the study. anti-sars-cov igg antibody avidity was measured by a 2step approach. the first step was to assess the concentrations of anti-sars-cov igg antibody in samples, so that samples that required further dilution for testing during the second step could be identified. on the basis of our serial-dilution experiments, samples with ods of 12.5 needed to be further diluted to provide a linear range for measurement of avidity during the second step. concentrations of anti-sars-cov igg antibody were measured by a recombinant nucleocapsid proteinbased enzyme immunoassay, elisars (iggene), in accordance with the manufacturer's instructions. briefly, samples were diluted to a concentration of 1:50 by mixing 22 ml of serum with 1.1 ml of sample diluent. an 100-ml aliquot of the prediluted serum was added to an antigen-coated well. after incubation at room temperature (∼25њc) for 30 min, the wells were washed 3 times with the washing buffer provided. antihuman igg antibodies conjugated with horseradish peroxidase were added and were incubated at room temperature for 15 min. after a second washing step, 3,3 ,5,5 -tetramethylbenzidine was added as a substrate for color development. optical density was measured at 450 nm. the second step was also based on the elisars assay but included a urea-elution procedure. briefly, samples were further diluted, if necessary, according to the results obtained during the first step. the neat or prediluted serum samples were mixed with sample diluent as described above. the sample mixtures were added in duplicate to 2 antigen-coated wells. after the first incubation step, 300 ml of urea was added to one of the wells, whereas the same volume of washing buffer was added to the other well, which served as a reference. on the basis of our initial optimization experiments using 5 early and 5 late samples (collected !20 and 1250 days after fever onset, respectively), a soaking step at room temperature for 10 min with 4 mol/l urea diluted in washing buffer was found to be most suitable and, thus, was used in the present study. the ureasoaking step was followed by washing 3 times with the washing buffer provided. the subsequent conjugate-addition and colordevelopment steps were conducted in accordance with the manufacturer's protocol. the antibody avidity index was calculated as od urea /od reference and is expressed as a percentage. samples collected р50 days after fever onset were also tested for anti-sars-cov igm antibody, so that igm antibody detection and igg antibody avidity measurement could be compared with respect to demonstrating a recent infection. anti-sars-cov igm antibody was also detected by the elisars assay. briefly, samples were treated by use of a rheumatoid factor removal kit (chemicon) and then mixed with sample diluent to a final concentration of 1:50. a 100-ml aliquot of the diluted sample was added to an antigen-coated well and subjected to the incubation, wash, and color-development steps described above, except that anti-human igm antibody was used as the conjugate. anti-sars-cov igg antibody titers for paired serum samfigure 2 . changes in severe acute respiratory syndrome-associated coronavirus-specific igg antibody avidity in paired serum samples ples were measured by an in-house indirect immunofluorescence assay that has been described elsewhere [12] . this was done so that the value of using changes in igg antibody titers and that in antibody avidity could be compared with respect to demonstrating a recent infection. for the 90 serum samples, the optical-density values obtained during the first step ranged from 0.639 to 3.509 (mean ‫ע‬ sd, 2.057 ‫ע‬ 0.862); 31 samples had an od of 12.5 and, thus, required further dilution for the second step. of these, 19 required further dilution of 1:2, and 12 required further dilution of 1:5, to achieve a reference od of р2.5 before they were subjected to the second step for measurement of antibody avidity. figure 1 shows the pattern of maturation of anti-sars-cov igg antibody avidity after infection. the avidity indices were low (mean ‫ע‬ sd, 30.8% ‫ע‬ 11.6%; range, 7.4%-51.8%) among the 45 samples collected р50 days after fever onset and increased to intermediate levels (mean ‫ע‬ sd, 52.1% ‫ע‬ 14.1%; range, 25.9%-73.1%) among the 11 samples collected between days 51 and 90. the avidity indices were high (mean ‫ע‬ sd, 78.1% ‫ע‬ 8.0%; range, 61.0%-94.7%) among the 34 samples collected after day 90. of the 45 samples collected р50 days after fever onset, only 18 (40.0%) were positive for anti-sars-cov igm antibody, as determined by the elisars assay. for the 18 igm-positive samples, the avidity indices ranged from 13.3% to 46.8%, with a median of 32.3% and an interquartile range of 23.1%-37.6%. for the 27 igm-negative samples, the avidity indices ranged from 7.4% to 51.8%, with a median of 31.9% and an interquartile range of 21.8%-40.8%. there was no significant difference in avidity level between these 2 groups of samples (p p .746, mann-whitney u test). all together, 1 sample was available from 35 patients, 2 samples were available from 23 patients, and 3 samples were available from 3 patients. the results for the 26 patients with at least 2 samples were further analyzed (the third samples from the 3 patients with 3 samples were not considered). their first samples were collected between days 17 and 54 (mean ‫ע‬ sd, 32.3 ‫ע‬ 9.6 days) after fever onset, and the time span between collection of the first and second samples ranged from 18 to 253 days (mean ‫ע‬ sd, 128.8 ‫ע‬ 73.6 days). of the 26 paired samples, only 6 (23.1%) showed a significant (у4-fold) increase in anti-sars-cov igg antibody titer (as determined by an in-house indirect immunofluorescence assay) from the first to the second sample, a result that could be regarded as evidence of recent infection. when the antibody avidity indices for the 26 paired samples were analyzed, they all showed an increase in avidity index with time. the changes in avidity levels for the paired samples are shown by collection time interval in figure 2. discussion. our data show that anti-sars-cov igg antibody avidity is low during primary infection and increases with time in a unidirectional manner. on the basis of this phenomenon, measurement of antibody avidity can be used to resolve certain difficulties that may be encountered in assessment of sars-cov infection. first, it can be used to differentiate between primary infection and reexposure. although it was not possible to include patients who had been reexposed in the present study, on the basis of experience with other viral infections that have a similar pattern of antibody avidity maturation [13] , it is reasonable to infer that patients reexposed to sars-cov will mount a humoral memory immune response that includes the production of antibodies with high avidity within a short period of time. second, the presence of antibodies with low avidity could provide alternative evidence for demonstrating a primary infection when the igm assay result is in doubt. this is important, given that viral serological testing based solely on the determination of the presence of igm can lead to false conclusions, because igm responses last for only a very short period of time and could be missed if serum samples are collected too early or too late [14] . on the other hand, igm can persist for months or even years after primary infection and reappear during secondary infection [15] . when interpreting our igm results, one should be aware that the igm assay used in the present study was based on the indirect enzyme immunoassay format, the sensitivity of which might be inferior to that of the igm capture format, and that our omission of the igg antibody removal step might have decreased the assay's sensitivity. nevertheless, our igm assay results for the 45 samples collected р50 days after fever onset support the view that low antibody avidity could be a valuable alternative marker for defining primary infection, in particular when serum sample availability is limited in terms of collection time points. our data show that all 36 samples with an avidity index of !40% were collected before day 50, whereas all 39 samples with an avidity index of 155% were collected after day 65. avidity indices between 40% and 55% could be considered to represent "the maturation zone," in which the correlation between avidity and time since infection is less strong. third, our comparison of the avidity indices for the 26 paired samples indicated that this approach could provide a helpful alternative to the use of increasing antibody concentration as serological evidence of recent infection. this is particularly important if convalescent samples are collected when antibody concentrations are no longer increasing, as was the case for most of our paired samples. other possible applications of measurement of antibody avidity to sars-cov infection include use of the technique to assess humoral responses to vaccine candidates and to discriminate between primary and secondary vaccine failures. world health organization. summary of probable sars cases with onset of illness from 1 laboratory-acquired severe acute respiratory syndrome severe acute respiratory syndrome (sars) in taiwan, china update 4: review of probable and laboratory-confirmed sars cases in southern china investigation into china's recent sars outbreak yields important lessons for global public health changes in antibody avidity after virus infections: detection by an immunosorbent assay in which a mild protein-denaturing agent is employed avidity of igg in serodiagnosis of infectious diseases comparative evaluation of the use of immunoblots and of igg avidity assays as confirmatory tests for the diagnosis of acute ebv infections differential maturation of avidity of igg antibodies to gp41, p24 and p17 following infection with hiv-1 the relative functional affinity of specific anti-core igg in different categories of hepatitis b virus infection severe acute respiratory syndrome immunofluorescence assay for serologic diagnosis of sars differentiation of primary from nonprimary genital herpes infections by a herpes simplex virus-specific immunoglobulin g avidity assay hepatitis a virus infection among the hemophilia population at the bonn hemophilia center serological diagnosis of tick-borne encephalitis by determination of antibodies of the igm class key: cord-321006-rxuq3ux8 authors: chang, luan-yin; chiang, bor-luen; kao, chuan-liang; wu, mei-hwan; chen, pei-jer; berkhout, ben; yang, hui-ching; huang, li-min title: lack of association between infection with a novel human coronavirus (hcov), hcov-nh, and kawasaki disease in taiwan date: 2006-01-15 journal: j infect dis doi: 10.1086/498875 sha: doc_id: 321006 cord_uid: rxuq3ux8 we investigated whether infection with a novel human coronavirus (hcov), called “new haven coronavirus” (hcov-nh)—which is similar to and likely represents the same species as another novel hcov, hcov-nl63—is associated with kawasaki disease (kd) in taiwan. fifty-three patients with kd were enrolled in our study. serum, peripheral-blood mononuclear cells, nasopharyngeal aspirates, throat swabs, and rectal swabs from these patients were assayed for hcov-nl63 by real-time reverse-transcriptase (rt) polymerase chain reaction (pcr), and the throat swabs, nasopharyngeal aspirates, and rectal swabs were also assayed for hcov-nh by rt-pcr. all pcr results were negative for both hcov-nl63 and hcov-nh; therefore, we did not find any association between hcov-nh infection and kd in taiwan of which is the development of life-threatening coronary arterial abnormalities. since the implementation of national health insurance (nhi) in taiwan in 1995, 195% of taiwan's population has been entitled to use the same health care system, and a database has been built that includes data on hospitalization and outpatient care. using the nhi data, we have found that there is a seasonal clustering of kd, usually in the summer, and that the annual incidence in taiwan is 50-70 cases/100,000 children !5 years old [2] , which is much higher than the annual incidence in western countries and is the second highest annual incidence in the world, just after japan's [3] [4] [5] [6] [7] [8] . therefore, kd can be considered one of the most important childhood diseases in taiwan, and it should be thoroughly investigated. the evidence that suggests that the etiology of kd can be traced to an infectious agent includes temporal clustering and marked seasonality, geographic clustering, a strong association between the frequency of kd and infectious-disease surveillance [2, 9] , and age distribution (with the highest incidence among children 6 months to 2 years old, who have little maternal antibody and are, in general, the most susceptible to infection) [2] . various infectious agents have been implicated as potential causes of kd, as have certain immunological agents and toxins, including bacterial toxin-mediated superantigens, allergens such as anionic detergents and house dust mites, and some chemicals. to date, no link between any of these individual agents and kd has been irrefutably established. recently, infection with a novel human coronavirus (hcov), called "new haven coronavirus" (hcov-nh) [10] -which is similar to and likely represents the same species as another novel hcov, hcov-nl63 [11] -was reported to be associated with kd [12] . shortly thereafter, 2 groups of researchers reported that they did not find any association between hcov-nh infection and kd [13, 14] . therefore, controversy exists as to whether there is an association between hcov-nh infection and kd, and further studies are needed to investigate whether the virus plays a role in the etiology of kd. thus, we evaluated whether hcov-nh infection is associated with kd in taiwan. patients, materials, and methods. the institutional review board of national taiwan university hospital approved the collection of clinical specimens and data for the present study. between february 2004 and april 2005, patients with kd were enrolled at national taiwan university hospital and taiwan adventist hospital in the city of taipei, at far eastern memorial hospital in taipei county, and at min-sheng hospital in tao-yuan county. we enrolled patients with kd who had at least 5 of the following 6 manifestations: fever for 15 days, skin rash, neck lymphadenopathy, nonpurulent bulbar conjunctivitis, red lip with fissure and/or strawberry tongue, and palm/sole erythema and induration followed by desquamation. we also enrolled patients with incomplete kd, defined as occurring in patients who had !5 of the above manifestations but who had a coronary arterial abnormality. a coronary arterial abnormality was defined as a lumen diameter (inner border to inner border) of у3 mm in patients р5 years old and of у4 mm in patients 15 years old. after informed consent was obtained from the parents of the patients, serum, peripheral-blood mononuclear cells (pbmcs), throat swabs, nasopharyngeal aspirates, and rectal swabs were obtained during hospitalization (acute illness) and at convalescence (∼2 weeks after hospital discharge). the acute-illness specimens were assayed for viral isolation by bacterial culture with hep-2, mk-2, rd, hel, and vero cells and for specific pathogens by polymerase chain reaction (pcr). a questionnaire was also administered, by which contact history and clinical symptoms were ascertained. for all patients, 2-dimensional echocardiography was performed during hospitalization and again ∼8 weeks after hospital discharge. laboratory data and data on responses to intravenous immunoglobulin (ivig) and coronary arterial abnormalities were also collected. serum, pbmcs, nasopharyngeal aspirates, throat swabs, and rectal swabs were processed for rna extraction, which was performed using an rna isolation kit in accordance with the manufacturer's instructions (qiaamp viral rna mini kit; qiagen). for detection of hcov-nl63 (genbank accession number ay567487), the forward primer (5 -ctagttcttctggt-acttccactcc-3 ; nt 26746-26770), the reverse primer (5 -tctggtaggaacacgcttccaa-3 ; nt 26858-26837), and the taqman probe (5 -taagcctctttctcaacccagggc-3 ; nt 26783-26806) were designed for detection of the n gene by real-time reverse-transcriptase (rt) pcr (lightcycler; roche). a 255-bp product covering this region was generated by rt-pcr with the forward primer 5 -gataaccagtcgaagtca-cctagttc-3 (nt 26727-26752), the reverse primer 5 -atta-ggaatcaattcagcaagctgtg-3 (nt 26981-26956), and the rna template derived from the hcov-nl63 strain (gift from l. van der hoek, university of amsterdam). the rt-pcr product was cloned into a ta cloning vector (yt&a vector; yeastern biotech), to generate the construct. the in vitro-transcribed rna was purified and quantified to determine the copy number and was subsequently used as the positive template for real-time rt-pcr. an aliquot (2.6 ml) of rna isolated from the clinical specimens, the positive template (10, 100, 1000, and 10,000 copies of the in vitro-transcribed rna), and the negative control (distilled water) were subjected to real-time rt-pcr. the real-time rt-pcr amplification program was as follows: 20 min at 60њc, for reverse transcription; 2 min at 95њc, to denature the cdna; 45 cycles of 5 s at 95њc and 10 s at 60њc, for amplification; and 30 s at 40њc, for cooling. the sensitivity of this real-time rt-pcr was р10 copies of in vitrotranscribed rna, and the linear regression coefficient of the standard dilution series was 0.99 to 1. to investigate possible inhibitors in the clinical specimens, a spike assay with 100 copies of the in vitro-transcribed rna was performed on randomly selected hcov-nl63-negative specimens, including 20 rectal swabs, 10 throat swabs, and 8 serum specimens. the mean copy number of the spiked clinical specimens was 78 copies/100 copies of the in vitro-transcribed rna (sd, 22 copies; se, 5.1 copies) and did not differ significantly from the standard value of 67 copies/100 copies of the in vitro-transcribed rna ( ). thus, the pcr conditions for the clin-p p .08 ical and standard specimens were comparable, and no significant pcr inhibitors were found in our clinical specimens. this assay has been successfully used to identify circulation of hcov-nl63 in taiwan within the period of the present study. we screened 219 clinical respiratory tract specimens that had been collected between may and october 2004 and identified 5 children (2.3%) infected with hcov-nl63 (authors' unpublished data). following the method of esper et al. [10, 12] , we screened for hcov-nh by rt-pcr with primers specific for the hcov-nh replicase 1a gene (forward primer, 5 -gcgctatgagggtggt-tgtaac-3 ; reverse primer, 5 -cgcgcagttaaaagtccag-aattaac-3 [underscoring indicates g/c clamps]). each set of rt-pcr amplifications included appropriate positive (hcov-nl63 rna) and negative (distilled water) controls. pcr products were analyzed by agarose gel electrophoresis. results. a total of 53 taiwanese patients with kd were enrolled. clinical information for these patients is shown in table 1, and the numbers of specimens obtained for analysis are shown in table 2. fifty-two patients (98%) fulfilled the criteria for kd (28 fulfilled all 6 of the criteria, and 24 fulfilled 5 of the criteria), and only 1 patient had incomplete kd (4 criteria plus left coronary arterial dilatation of 3.9 mm in diameter). the mean ‫ע‬ sd age of the patients was 2.30 ‫ע‬ 1.93 years, and 27 (51%) were male. all of the patients had fever; the mean ‫ע‬ sd duration of fever was 8.2 ‫ע‬ 2.8 days, and the mean ‫ע‬ sd peak body temperature was 39.7њc ‫ע‬ 0.6њc. of the patients, 96% had skin rash, 40% had erythema and induration over the bacille calmette-guérin scar, 96% had nonpurulent bulbar conjunctivitis, 92% had red lip with fissure and/or strawberry tongue, 74% had neck lymphadenopathy, 85% had palm/sole erythema and induration, and 100% had desquamation (96% had periungual desquamation, and 40% had perianal desquamation). in addition to the above typical symptoms of kd, 75% had cough, 72% had rhinorrhea, and 72% had diarrhea. all of the blood cultures were negative, no group a streptococcus was identified in throat swabs, and no significant urine culture was obtained. echocardiography showed that 4 (8%) of the 53 patients had a coronary arterial abnormality during the acute stage of kd: the first with a left coronary arterial diameter of 4.4 mm and a right coronary arterial diameter of 4.2, the second with a left anterior descending coronary arterial diameter of 4.2 mm, the third with a left coronary arterial diameter of 3.9 mm, and the fourth with a left coronary arterial diameter of 3.3 mm. all 53 patients received a second echocardiography ∼8 weeks after hospital discharge, and it was found that 2 (the first and the fourth) of the 4 patients who had had a coronary arterial abnormality during the acute stage still had a coronary arterial abnormality: the first with a right coronary arterial diameter of 3.7 mm and a normal left coronary arterial diameter (2.2 mm), and the fourth with a left coronary arterial diameter of 3.3 mm and a right coronary arterial diameter of 2.7 mm. fiftyone patients (96%) received ivig treatment (2 g/kg), and 2 did not (because their fevers subsided spontaneously). two patients needed re-treatment with ivig. all patients received low-dose aspirin (3-5 mg/kg/day) for 8-10 weeks or until their coronary arterial aneurysms subsided. the mean ‫ע‬ sd interval between specimen collection and disease onset was 6.1 ‫ע‬ 1.9 days, and the range of the interval was 3-11 days (median, 6 days). specimens were obtained within 10 days of onset of illness for 51 patients (96%). serum, pbmcs, throat swabs, nasopharyngeal aspirates, and rectal swabs were processed for real-time rt-pcr detection of hcov-nl63, and the throat swabs, nasopharyngeal aspirates, and rectal swabs were also processed for rt-pcr detection of hcov-nh. the sample sizes for the tested specimens are shown in table 2. all pcr results were negative for both hcov-nl63 and hcov-nh. discussion. the novel hcovs identified in new haven, connecticut (hcov-nh), and the netherlands (hcov-nl63) are similar and likely represent the same species [10, 11] . a study by esper et al. suggested an association between hcov-nh infection and kd [12] , a finding that required confirmation in broader epidemiologic settings, using data from other locations, other years, and other populations, as well as other detection methods [15] . we tested for hcov-nh and hcov-nl63 in another location, taiwan, but failed to confirm the association in 53 patients with kd. ebihara et al. studied 19 patients with kd in japan [13] , and belay et al. studied 10 patients with kd in san diego, california [14] ; neither group found an association between hcov-nh infection and kd, either. further study is warranted to determine whether this new hcov is associated with kd. identification of the infectious etiology of kd has proved to be very difficult. since kd was first reported in 1967, 130 years have passed. despite plenty of hard work by many scientists and physicians worldwide, the causative pathogen has remained unknown. although esper et al. reported that hcov-nh infection is associated with kd [12] , we did not find such a correlation. there are several possibilities with respect to the etiology of kd. the disease might be caused by 1 pathogen, or it might be caused by the interaction of 11 pathogen. furthermore, different pathogens might be responsible for kd in different places or in different seasons. because recent new evidence has failed to further support the association between hcov-nh infection and kd, further investigations are required to discover the real causative pathogen of kd and whether it differs in different countries and in different time periods. kawasaki acute febrile mucocutaneous syndrome with lymphoid involvement with specific desquamation of the fingers and toes in children epidemiological features of kawasaki disease in taiwan from epidemiologic pictures of kawasaki disease in japan: from the nationwide incidence survey in 1991 and 1992 kawasaki syndrome hospitalizations in the united states kawasaki syndrome in hawaii incidence of henoch-schonlein purpura, kawasaki disease, and rare vasculitides in children of different ethnic origins kawasaki disease in australia, 1993-95 kawasaki disease in sweden: incidence and clinical features seasonality and temporal clustering of kawasaki syndrome evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children identification of a new human coronavirus association between a novel human coronavirus and kawasaki disease lack of association between new haven coronavirus and kawasaki disease kawasaki disease and human coronavirus coronaviruses in the limelight we thank dr. lia van der hoek, for critical reading of the manuscript. key: cord-327501-8s6dvanf authors: schwaiger, julia; karbiener, michael; aberham, claudia; farcet, maria r; kreil, thomas r title: no sars-cov-2 neutralization by intravenous immunoglobulins produced from plasma collected before the 2020 pandemic date: 2020-09-17 journal: j infect dis doi: 10.1093/infdis/jiaa593 sha: doc_id: 327501 cord_uid: 8s6dvanf the 2020 sars-cov-2 pandemic is caused by a zoonotic coronavirus transmitted to humans, similar to earlier events. whether the other, seasonally circulating coronaviruses induce cross-reactive, potentially even cross-neutralizing, antibodies to the new species in humans is unclear. the question is particularly relevant for people with immune deficiencies, as their health depends on treatment with immunoglobulin preparations that need to contain neutralizing antibodies against the pathogens in their environment. testing 54 intravenous immunoglobulin preparations, produced from plasma collected in europe and the united states, confirmed highly potent neutralization of a seasonal coronavirus; however, no cross-neutralization of the new sars-cov-2 was seen. intravenous immunoglobulins (ivig) are produced from thousands of pooled plasma donations, and thus contain a wide variety of antibodies the donors have generated against infectious disease agents. ivig can therefore protect people with immune deficiencies against circulating bacterial and viral infections. when a new pathogen emerges, antibodies to the new agent only become detectable in ivig preparations after a certain proportion of plasma donors have contracted the infection and successfully recovered from it. an even higher number of convalescent plasma donors is needed to result in neutralizing antibody (nab) levels high enough for the resulting ivig to afford protection against the new infectious agent. after the arrival of west nile virus (wnv) in the united states, for example, it took several years before the prevalence of wnv nabs reached approximately 0.5% in the plasma donor community, at which point they became detectable in ivig lots produced from plasma donated in the united states. thereafter, a significant proportion of ivig lots even reached in vivo protective levels [1] . severe acute respiratory syndrome coronavirus-2 (sars-cov-2) spread around the globe at unprecedented speed, infecting millions of people within the first 6 months of circulation. the virus belongs to the coronaviridae family of viruses, which contains several species of importance for human health. the human coronaviruses (hcovs) 229e, nl63, oc43, and hku1 are in circulation as seasonal respiratory viruses that mostly cause self-limiting and mild infections but which can also lead to pneumonia and bronchiolitis [2] [3] [4] , whereas the middle east respiratory syndrome coronavirus (mers-cov) caused a prolonged outbreak mainly limited to the arabian peninsula, and the sars-cov and sars-cov-2 viruses have established global transmissions chains-the latter 3 associated with significant human mortality. due to the widespread and long-term circulation of hcovs, and the pooling of plasma from thousands of donors for every lot, ivigs contain significant levels of hcov nab levels, as was shown for example for hcov-nl63 [5] . whether these hcov antibodies cross-react with, or even neutralize, the related sars-cov-2 has not been fully elucidated. to date, antibody binding assays have shown some cross-reactivity between different hcovs and sars-cov-2; however, functional and therefore clinically more relevant virus neutralization assays have shown no or only very low levels of cross-reactive antibodies [6] [7] [8] . the question is of significant clinical relevance, as sars-cov-2 cross-neutralizing antibodies in ivigs, if they were present, might afford some protection to people with immune deficiencies and may even represent a treatment option for coronavirus disease 2019 (covid-19) patients. the current study tested a representative number of ivig lots for nabs against sars-cov-2 and the longer-circulating hcov-229e, to establish clarity about cross-neutralization of the pandemic virus by antibodies induced by earlier circulating seasonal coronaviruses. in addition, results from the ongoing monitoring of the plasma donor community for the development of sars-cov-2 antibodies are presented. a total of 54 ivig lots fractionated from plasma collected prior to the circulation of sars-cov-2 were analyzed. the ivig lots were manufactured from plasma either donated by plasmapheresis (source) or recovered from whole-blood donations (recovered), in the united states (gammagard liquid; baxter healthcare corp.; n = 30) or central europe, that is austria, germany, and czech republic (kiovig; baxter ag; n = 24). these 54 ivig lots were tested in 2 independent experiments for nabs (1 european lot was in a single assay due to volume constraints). two prepandemic plasma donations were collected in april and may 2019 by biolife austria. human plasma pool samples were generated by the combination of 6 individual donations obtained in the same week at austrian plasma donation centers (biolife). the pools were assembled from donations collected in week 13 (last week of march; n = 40), week 14 (n = 80), week 15 (n = 80), week 16 (n = 80), week 20 (n = 100), week 24 (n = 80), and week 28 (first week of july; n = 100) of 2020, that is during the sars-cov-2 pandemic. cumulative incidence of covid-19 was calculated from data provided by the austrian federal ministry for social affairs, health, care and consumer protection (www. sozialministerium.at) and statistics austria (www.statistik.at). sars-cov-2 nab titers were determined in ivig and human plasma samples that were used undiluted, or prediluted with cell culture medium 1:4, 1:5, 1:10, or 1:20 depending on sample amount available, and then serially diluted in 2-fold steps. equal volumes of sample dilutions were mixed with virus stock at 10 3.0 tissue culture infectious doses 50% per milliliter (tcid 50 / ml) sars-cov-2 (strain bavpat1/2020, kindly provided by c. drosten and v. corman, charité berlin, germany) and incubated for 150 minutes ± 15 minutes, before titration on vero cells (cat. no. 84113001, european collection of authenticated cell cultures, porton down, salisbury, uk) in 8-fold replicates per dilution. the virus-induced cytopathic effect was determined after 5-7 days of incubation. the reciprocal sample dilution resulting in 50% virus neutralization (nt 50 ) was determined using the spearman-kärber formula, and the calculated neutralization titer for 50% of the wells reported as 1:x. the detection limits were as follows: <1:0.8 for undiluted, <1:3.1 for 1:4 prediluted, <1:3.9 for 1:5 prediluted, <1:7.7 for 1:10 prediluted, and <1:15.4 for 1:20 prediluted ivig. the neutralization assay (µnt) included several validity criteria, that is confirmatory titration of input virus infectivity, cell viability, and neutralization testing of an internal reference standard, all of which had to comply with defined ranges. the neutralization assay for hcov-229e antibodies is essentially identical to the sars-cov-2, where samples were used undiluted or prediluted, then serially diluted in 2-fold steps and mixed 1:2 with 10 3.0 tcid 50 /ml hcov-229e (cat. no. vr-740, american type culture collection [atcc], rockville, md), incubated, and titrated on mrc-5 cells (cat. no. ccl-171, atcc, rockville, md). the virus-induced cytopathic effect was determined after 7-9 days of incubation. graphical illustration and statistical analysis (paired t tests) were done using graphpad prism v8.1.1 software. the validity, specificity, and reliability of the sars-cov-2 neutralization assay used in the current study were demonstrated by the analysis of 100 convalescent plasma donations from polymerase chain reaction (pcr)-confirmed sars-cov-2 cases [9] and was the basis for the correlation of function against diverse binding assays [10] . as controls, 2 plasma samples collected before the emergence of sars-cov-2 did not contain sars-cov-2 nabs, as expected, but neutralized hcov-229e with nt 50 values of 1:43 and 1:26, respectively. sars-cov-2 nab titers were below the limit of detection for all 54 ivig lots tested, irrespective of geographic origin of the plasma (europe vs united states) and plasma collection modality (recovered vs source) ( figure 1a) . in contrast, hcov-229e nab titers between 1:43 and 1:196 (mean = 1:98) were measured for the 54 ivig lots tested ( figure 1b) . ivig lots produced from recovered plasma contained significantly higher levels of nab to hcov-229e as compared to ivig lots produced from source plasma, independent of the geographic origin. a significant, although quantitatively minor, difference was also found between ivig lots manufactured from source plasma collected in either the united states or europe. to evaluate the potential development of sars-cov-2 antibodies in the plasma donor community, samples of plasma pools of 6 donations each were tested. the use of pools enabled testing of a high amount of plasma donations for sars-cov-2 nabs with the biosafety level-3 functional assay. as the mean sars-cov-2 µnt 50 of plasma donations is rather high (approximately 1:230 [9] ) even the nabs of only 1 positive sample within a pool are detectable in this assay. testing a total of 560 plasma pools of 6 donations each, in total reflective of 3360 plasma donations, from week 13 (ie, last week of march) until week 28 (ie, first week of july) 2020 revealed that most of these pools had sars-cov-2 µnt titers below the limit of detection ( table 1 ). the first pool with detectable nabs to sars-cov-2 was collected in week 14. further positive pools were found in weeks 15, 16, 24, and 28. up to 7% of the tested pools showed nabs to sars-cov-2, which indicates that up to 1.17% of the plasma donors were positive for sars-cov-2 nabs at a cumulative incidence of covid-19 in austria of 0.21% (table 1) . our analysis of ivig lots revealed high nab titers to hcov-229e ( figure 1b) , which confirmed the presence of functionally intact nabs in these lots. it is interesting to note that in ivig lots produced from recovered plasma significantly higher titers to hcov-229e were found compared to ivig lots produced from source plasma, independent of plasma origin. recovered plasma is usually obtained from older donors [11] . a trend towards increasing probability of hcov-229e infections with age has been detected in the scottish [3] and us [4] population. furthermore, higher hcov-229e-specific nab titers were found in older study participants (60-85 years) compared to younger ones (21-40 years) [12] . thus, a higher infection rate with hcov-229e in older people and consequently the generation of neutralizing hcov-229e-specific antibodies in the older plasma donors would explain the higher hcov-229e titers found in ivig lots produced from recovered plasma. low levels of cross-neutralizing abs have earlier been demonstrated between specific pairs of coronaviruses, specifically between sars-cov-2 sera and sars-cov [6] and sars-cov sera and mers [13] . in clear contrast, testing the same lots of ivig that were shown to contain high hcov-229e nab titers with a highly specific sars-cov-2 µnt did not detect sars-cov-2-specific nabs. these results are entirely consistent with the absence of sars-cov-2 antibodies in the plasma used for production of these ivig lots, as this plasma was donated well before the start of the sars-cov-2 pandemic. the current study, as well as an earlier study that tested 21 ivig lots (9 gamunex c, 10 gammagard liquid, 2 other) with a sars-cov-2-specific enzyme-linked immunosorbent assay (elisa; receptor-binding domain or spike protein) that was shown to correlate well with a neutralization test [14] revealed the absence of cross-reactive antibodies against sars-cov-2 in ivig lots produced from prepandemic plasma. currently available ivigs can therefore not be expected to afford protection from sars-cov-2 infection. nevertheless, several clinical trials currently investigate ivig at high dosage in the treatment of covid-19 patients (clinicaltrials.gov), with the expected benefit attributable to the anti-inflammatory and immunomodulatory capacity of ivig, rather than an antiviral effect. with increasing numbers of human infections, including in the plasma donor community, it is interesting to follow the development of antibodies against sars-cov-2 in plasma donations and, after the several months production cycle time between plasma donation and ivig lot release, also in ivig lots. a longitudinal study on this topic is currently in progress. in this context it is noteworthy that the mean nab titers induced by wnv [1] and sars-cov-2 [9] infection are of similar magnitude. after the emergence of wnv in the united states, nab titers became detectable in ivigs after approximately 0.5% of the population had contracted and recovered from the infection. in austria, testing of plasma pool samples indicated that up to 1.17% of plasma donors were positive for sars-cov-2 nabs. based on the current number of reported sars-cov-2 infections in the united states (approximately 4.3 million per 30 july 2020; www.cdc.gov), and an estimated rate of >40% asymptomatic infections [15] , more than 7.2 million people in the united states could have been infected already, that is 2.2% of the approximately 330 million population. based on these facts, the detection of sars-cov-2 nabs in ivig lots produced from us plasma, the major source for fractionation, is expected within the next few months. another, more immediately available possibility for the treatment of covid-19 is production of a hyper-ivig from the plasma of covid-19 convalescent donors (covig-19), and developmental work is currently under way through a large alliance of plasma stakeholders (https://www.covig-19plasmaalliance.org). west nile virus infection in plasma of blood and plasma donors, united states update on human rhinovirus and coronavirus infections epidemiology of seasonal coronaviruses: establishing the context for the emergence of coronavirus disease 2019 human coronavirus circulation in the united states inhibition of human coronavirus nl63 infection at early stages of the replication cycle lack of crossneutralization by sars patient sera towards sars-cov-2 a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease. medrxiv severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients characterization of 100 sequential sars-cov-2 convalescent plasma donations quantification of sars-cov-2 antibodies with eight commercially available immunoassays measles virus neutralizing antibodies in immunoglobulin lots produced from plasma collected in europe or the united states antibodies to coronaviruses are higher in older compared with younger adults and binding antibodies are more sensitive than neutralizing antibodies in identifying coronavirus-associated illnesses cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests a serological assay to detect sars-cov-2 seroconversion in humans prevalence of asymptomatic sars-cov-2 infection: a narrative review key: cord-313000-as507p4t authors: dare, ryan k.; fry, alicia m.; chittaganpitch, malinee; sawanpanyalert, pathom; olsen, sonja j.; erdman, dean d. title: human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays date: 2007-11-01 journal: j infect dis doi: 10.1086/521308 sha: doc_id: 313000 cord_uid: as507p4t background. we sought to determine whether infections with human coronaviruses (hcovs) 229e, oc43, hku1, and nl63 are associated with pneumonia and to define the epidemiology of hcov infection in rural thailand. methods. we developed a real-time reverse-transcription polymerase chain reaction (rt-pcr) assay panel for the recognized hcov types and compared hcov infections in patients hospitalized with pneumonia, outpatients with influenza-like illness, and asymptomatic control patients between september 2003 and august 2005. results. during study year 1, 43 (5.9%) of 734 patients with pneumonia had hcov infections; 72.1% of the infections were with oc43. during study year 2, when control patients were available, 21 (1.8%) of 1156 patients with pneumonia, 12 (2.3%) of 513 outpatients, and 6 (2.1%) of 281 control patients had hcov infections. compared with infection in control patients, infection with any hcov type or with all types combined was not associated with pneumonia (adjusted odds ratio for all hcov types, 0.67 [95% confidence interval, 0.26–1.75]; p= .40 ). hcov infections were detected throughout both study years; 93.6% of oc43 infections in the first year occurred from january through march. conclusions. hcov infections were infrequently detected in rural thailand by use of sensitive real-time rtpcr assays. we found no association between hcov infection and illness. however, we noted year-to-year variation in the prevalence of hcov strains, which likely influenced our results. establish hcov etiology, including (1) use of sensitive, wellvalidated assays that detect and distinguish all 4 currently recognized hcov types; (2) comprehensive testing for other respiratory pathogens that may be present in mixed infections with hcovs and, thus, may be responsible for the disease; and (3) comparison of hcov infections between asymptomatic control patients and symptomatic patients, controlling for age and month of illness. in the present study, we developed a sensitive and specific real-time rt-pcr assay panel for all currently recognized hcovs (229e, oc43, nl63, and hku1) that complements our previously described severe acute respiratory syndrome (sars)-cov-specific real-time rt-pcr assay [28] . we used this assay panel to test specimens from patients hospitalized with pneumonia, outpatients with influenza-like illness (ili), and asymptomatic control patients from a rural thai province. we describe the epidemiology of hcov infection in rural thailand and compare infections in control patients versus patients with pneumonia. viral nucleic acid. hcov rna used for development and validation of the real-time rt-pcr panel was obtained from several sources (see acknowledgments). hcov prototype strains 229e (atcc vr-740) and oc43 (atcc vr-1558) were available from centers for disease control and prevention (cdc) archives, whereas the nl63 prototype strain was donated. thirty-one available hcov-positive clinical specimens (16 oc43, 4 229e, 10 hku1 and 1 nl63) were previously identified by rt-pcr and probe hybridization [29] or by rt-pcr and sequencing [30, 31] . other respiratory viruses were available for specificity testing from cdc archives, including laboratory strains of influenza virus a and b, respiratory syncytial virus (rsv), human parainfluenza viruses (hpiv)-1-4, human metapneumovirus (hmpv), adenovirus, rhinovirus, human bocavirus (hbov), and sars-cov. rna transcripts. primer pairs containing t7 and sp6 rna polymerase promoter sequences were used to amplify regions bracketing the real-time rt-pcr signatures for each hcov type. a 488-bp fragment of the oc43 nucleoprotein (n) gene was generated using the forward primer 5 -sp6-gagctcaacccaagcaaactg-3 and the reverse primer 5 -t7-tgtgcgcgaagtagatctgg-3 ; 476 bp of the 229e n gene was generated using the forward primer 5 -t7-atgg-ctacagtcaaatgggctg-3 and the reverse primer 5 -sp6-ccgcgactctgcgacct-3 ; 401 bp of the nl63 n gene was generated using the forward primer 5 -t7-atcagt-gctaacctcactatcaaagaat-3 and the reverse primer 5 -sp6-cttctcgtagcacttcaagacaaca-3 ; and 440 bp of the hku1 replicase 1b gene was generated using the pre-viously published primers [23] 5 -sp6-ggttgggattatcct-aaatgtga-3 (forward) and 5 -t7-ccatcatcactcaaaa-catcata-3 (reverse). amplification products were gel purified using the qiaquick gel extraction kit (qiagen), and rna transcripts were prepared using the megashortscript high yield transcription kit (ambion). rna transcripts were purified using the megaclear kit (ambion) and were quantified by uv light spectroscopy. for limit-of-detection tests, serial dilutions of the quantified rna transcripts were prepared in a diluent containing yeast trna (50 ng/ml; ambion). primers and probes. multiple primer and probe sequences for hcov-hku1 and hcov-nl63 were designed to identify type-specific conserved regions of the viral replicase 1b and n genes identified from alignments of sequences available in genbank (ay884001, dq190472, and dq206693-9 for hku1 [31, 32] and nc_005831, ay563107-8, and ay758276-82 for nl63 [13, 14] ), using primer express (version 3.0; applied biosystems) and beacon designer (version 5.0; premier biosoft international) software (table 1) . primer and probe sequences for hcov-oc43 and hcov-229e have been described elsewhere [26] . all probes were labeled at the 5 end with fam and quenched at the 3 end or internally with black hole quencher-1 (biosearch technologies). sequences of different representative hcovs were aligned to check for potential crossreactivity of the real-time assays ( [14, 15, [33] [34] [35] . real-time rt-pcr assays. the real-time rt-pcr panel consisted of 4 differential hcov monoplex assays run under identical amplification conditions. reaction mixtures were prepared using the iscript one-step rt-pcr kit for probes (bio-rad). twenty-five-microliter reactions contained 2ϫ reaction buffer, 50ϫ iscript reverse transcriptase, primers, probe, nuclease-free water, and nucleic acid. amplification was performed on an icycler iq real time detection system (bio-rad) with the following cycling conditions: 48њc for 10 min (1 cycle); 95њc for 5 min (1 cycle); and 95њc for 15 s followed by 55њc for 1 min (45 cycles). each specimen was also tested for the human ribonuclease p gene to measure nucleic acid integrity [28] . study subjects and specimens. specimens were collected from patients of all ages enrolled in an enhanced active surveillance program for pneumonia requiring hospitalization in sa kaeo province, thailand [36] , over a 2-year period from 1 september 2003 to 31 august 2005; we previously tested these specimens for hbov infections [37, 38] . in general, a patient was enrolled in the study if he or she had at least 1 sign of acute infection (e.g., fever, chills, and/or abnormal white blood cell count), symptoms of lower respiratory tract disease (e.g., abnormal breath sounds, tachypnea, and/or cough), and the physician ordered a chest radiograph (cxr) within 48 h of admission. final cxr findings were not available for the present analysis. a nasopharyngeal swab, acute and convalescent serum, urine, and clinical and demographic information were collected from each patient with pneumonia. a sample of outpatients with ili, defined as fever within the previous 3 days and cough or sore throat in the absence of other diagnoses, were recruited from the outpatient departments of 5 hospitals in sa kaeo province [39] . only outpatient specimens from the second year of the study (1 september 2004 to 31 august 2005) were tested for hcov infection. a nasopharyngeal swab and demographic information was collected from each outpatient participant. in addition, during the second study year, we enrolled a sample of control patients from the same outpatient clinics; these were patients with other health-related complaints but without a fever and with no history of fever, cough, sore throat, or diarrhea within the previous 3 days. we sought to enroll equal numbers of control patients and patients hospitalized with pneumonia from each age group (р2 years, 3-5 years, 6-15 years, 16-54 years, and у55 years) during each month of the year. a nasopharyngeal swab and demographic information was collected from each control patient. nasopharyngeal swabs were collected in viral transport medium, and aliquots were processed and transported as described elsewhere [37, 38] . the swabs were tested for rsv, hpivs, influenza virus a and b, and adenovirus by culture at the thailand national institute of health [38] , and total nucleic acid extracts were tested for influenza virus a and b, rsv, hpiv-1-3, hmpv, adenovirus, rhinovirus, and hbov (year 2 only) by pcr at the cdc [37, [40] [41] [42] . aliquots of the extracts were kept at ϫ70њc for hcov testing. we did not test for sars-cov infection. acute and convalescent serum samples were tested for rsv, hpiv-1-3, adenovirus, and hmpv by inhouse eias and for influenza viruses by hemagglutination-inhibition assays (year 1 only). a specimen was considered to be positive if either culture or pcr results were positive or if there was a 4-fold or greater increase in antibody titer between acute and convalescent serum samples. all participants were informed of the study objectives, and written consent was obtained. the study protocol was reviewed and approved by the internal review boards of the cdc and the thailand ministry of public health. statistical methods. admissions for pneumonia were counted individually, including readmissions у14 days after a previous discharge; readmissions occurring within 13 days were considered to be 1 admission. we compared hcov infections between patients hospitalized with pneumonia and control patients during the study year using the x 2 test and a multivariable unconditional logistic regression model controlling for age group and month of illness. the clinical characteristics of patients with hcov infection, without coinfections, were compared to patients with infection with other common viruses. variables were compared with the x 2 test for dichotomous variables. two-tailed was considered to indicate statistical p ! .05 significance. analyses were performed using sas software (version 9.1; sas institute). different primer and probe combinations were evaluated for the nl63 and hku1 assays, and those with the most efficient amplification were selected for further studies. all real-time assays were internally specific for the other hcovs, and no amplification was obtained with other respiratory viral pathogens-including influenza viruses a and b, rsv, hpiv-1-4, hmpv, adenovirus, rhinovirus, hbov, and sars-covor with human dna. all assays were successfully validated against 31 clinical specimens previously shown to be positive for hcovs by alternate methods; 3 specimens originally reported to be positive for oc43 by rt-pcr [30] were identified as being hku1 by the real-time assay and were confirmed by sequencing. of the outpatients with ili, 12 (2.3%) had hcov infections (table 3) . nl63 was the most common type detected among the outpatients (75.0% of all hcovs). we detected a similar proportion of hcov infections among asymptomatic control patients; 6 (2.1%) of the 280 control patients had 1 of the hcov types detected. among the control patients, hcov infections were detected in all age groups except for children !1 year of age. we compared hcov infections between patients hospitalized with pneumonia and control patients for study year 2. neither all hcov infections combined nor infections with individual hcov types were associated with pneumonia requiring hospitalization, even after controlling for age and month of illness (for all hcov infections, adjusted odds ratio [ . fewer nl63 infections were detected p p .79 among patients with pneumonia than among control patients, and the association between nl63 and pneumonia appears to be protective. however, the numbers are small, and this result should be interpreted with caution. cohort, age group tested, no. hku1 nl63 229e oc43 all hcov of all 82 patients with hcov infections, 30.5% were coinfected with other respiratory viruses, including almost half of those who were infected with nl63 (table 4) . among the patients with pneumonia, 17 (26.6%) of the 64 who were infected with hcov had a viral coinfection. among the outpatients and control patients with hcov infections, 7 (58.3%) of 12 and 1 (16.7%) of 6, respectively, were coinfected with another virus. hcov infections were detected in sa kaeo province throughout the 2-year study (figure 1). oc43 was the most frequently detected hcov type, representing 36 (43.4%) of all 83 hcov infections. during study year 1, oc43 was present in 31 (72.1%) of the 43 hcov-infected patients; these infections clustered in january-march 2004 and made up 93.6% of the oc43 infections that year. the following year, 8 (53.3%) of all 15 hku1 infections were detected during the same 3 months and only 1 oc43 infection (2.7%) was detected, indicating a shift in circulation of the predominant hcov type. nl63 infections appeared to peak in september 2004. 229e was detected at low levels throughout the study. we compared the clinical characteristics of oc43-infected patients with pneumonia to those of patients with pneumonia who were infected with influenza virus or rsv (2 common infections) and limited our analysis to study year 1, when the prevalence of oc43 infections was highest. fifty percent of the 24 patients with oc43 infections had fever, 91.7% had cough, 41.7% had tachypnea, 22.7% received oxygen therapy, and 8.3% required mechanical ventilation; no statistically significant differences were noted between patients infected with oc43 and those infected with influenza virus or rsv (data not shown). a few differences were noted-3 (12.5%) of 24 patients with oc43 infections had rhinitis versus 0 of 47 patients with influenza virus infections ( ), and only 3 persons (12.5%) p p .04 with oc43 infections had wheezing on admission versus 9 (47.4%) of 19 persons with rsv infections ( ). patients p p .01 with hcov infections other than with oc43 also had similar proportions of fever (63.2%), cough (89.5%), and tachypnea (21.1%). in the present study, we developed a sensitive and specific realtime rt-pcr assay panel for the 4 hcov types (229e, oc43, nl63 and hku1) and used this assay panel to assess the disease burden and epidemiology of hcov infection in a rural province of thailand over a 2-year period. infections were infrequently detected among hospitalized patients with pneumonia and among outpatients with ili. during study year 2, the asymptomatic control patients and the ill patients had similar proportions of hcov infections. oc43 infections were detected in significantly higher numbers during the first study year. however, we did not have a control group for comparison during that year. thus, in rural thailand from 1 september 2003 to 31 august 2005, infection with hcovs occurred infrequently. we were unable to show an epidemiologic association between any of the hcov types and pneumonia requiring hospitalization. the real-time rt-pcr assays for the 4 hcov types were combined into a single test panel using identical amplification conditions. the assay panel was shown to be sensitive and specific with defined targets and was validated by use of human clinical specimens that were independently confirmed to be positive for the different virus types. samples previously determined to be oc43 positive by use of conventional methods were found to be hku1 positive and oc43 negative by use of this panel, exemplifying the panel's specificity in relation to other hcov diagnostic tools. although real-time assays have been described for some hcov types [26, 43] , most studies failed to report analytical and clinical data by which assay performance could be evaluated. although our assay panel could detect and discriminate among the 4 known hcov types, it could have failed to detect or improperly classify unrecognized hcovs. our study represents the first comprehensive analysis of hcov infection in a tropical rural population in southeast asia. despite sensitive assays, we found the prevalence of infection with hcovs to be very low during the study period. similar to our findings, a recent study from bangkok, thailand, conducted during 2002-2003 found 229e and oc43 infections in 4.9% of young children with acute respiratory tract illness; however, the majority were 229e infections rather than oc43 infections [44] . the proportion of hcov types we detected is similar to those of other international studies reporting hcovs from both upper and lower acute respiratory tract illness by use of pcr assays [12-16, 18, 20-23, 31, 45] . in the present study, we detected very few nl63 infections. moreover, a larger proportion of the patients with detected nl63 infections were coinfected with other viruses, compared with patients infected with other hcov types. additional years of surveillance will be necessary to clearly establish the prevalence and circulation patterns of hcovs in thailand. we did not find an epidemiologic association between hcov infection and pneumonia. however, correlation between hcov and severe pneumonia during the first study year could not be assessed because of the absence of a control group, and the low number of hcov infections during the second study year made it difficult to accurately assess disease correlation. given the year-to-year variation in the prevalence of hcov infections, only a multiyear study may be able to definitively assess whether an association between hcov infection and severe illness exists. only a few studies have tested asymptomatic control subjects for hcov infection, and most of these studies have tested only for oc43 and 229e. using serologic assays, mcintosh et al. [46] detected oc43 and 229e infection in 34 (8%) of 417 children hospitalized with acute lower respiratory tract disease and in 1 (8%) of 13 control children. nokso-koivisto et al. [47] failed to find 229e or oc43 by pcr among hospitalized children aged 1 month to 16 years with or without a history of respiratory tract illness. in contrast, van elden et al. [26] found significantly more 229e and oc43 by pcr in patients with respiratory tract illness than in bone-marrow transplant recipients and healthy volunteers without respiratory tract illness. however, they did not control for age and month of illness/admission, and it is unclear how frequently oc43 and 229e were detected among patients with pneumonia versus those with the common cold. thus, although numerous studies have tentatively linked 229e and oc43 infections to severe respiratory tract illness over many years [48] , no study controlling for age and month of illness has demonstrated an epidemiologic association between infection with these hcovs and any illness other than the common cold. also, no study has demonstrated an epidemiologic association between nl63 and hku1 infection and severe respiratory tract illness in relation to asymptomatic control subjects. two studies did find nl63 and hku1 in bronchoalveolar lavage specimens from patients with severe illness [49, 50] . in temperate regions, hcov infections occur most frequently in late winter and early spring [7, 8, 17, 20] . however, there are few reports describing the seasonal circulation patterns of these viruses in the tropics [18, 23] . hcov infections were detected year-round in the present study. however, during study year 1, oc43 infections appeared to cluster from january through march. in contrast, influenza virus infections clustered from june through october in sa kaeo province [39] . there was a statistically significant difference in the number of oc43 infections between year 1 and year 2, suggesting year-to-year variation in strain circulation. previous multiyear studies [6, [8] [9] [10] [11] in temperate regions have made similar observations. our study has some limitations. first, the number of hcov infections detected among patients with pneumonia was likely underestimated. only 55%-59% of patients admitted to a sa kaeo province hospital with signs and symptoms of clinical pneumonia had a cxr taken, and only 37%-51% of these were enrolled in the study; children and severely ill patients were less likely to enroll. second, adults were underrepresented in the outpatient sample. third, we found a high proportion of patients infected with hcov to be coinfected with other viruses, especially among those infected with nl63, making it difficult to determine the responsible etiology; however, we tested for more viruses than have other studies [26] . finally, we cannot exclude the possibility that some control patients may have been shedding hcov following an earlier symptomatic infection. using sensitive pcr assays, van elden et al. [26] reported detecting hcov 14 days after illness. future studies that include control subjects should account for this possibility. well-planned studies are needed to evaluate whether newly described viruses are epidemiologically associated with severe respiratory tract illness. we sought to determine whether infections with new and previously recognized hcov types were associated with pneumonia requiring hospitalization in thailand. we found very low numbers of hcov infections and failed to find an association with severe respiratory tract disease. however, because year-to-year variation in overall prevalence and type of circulating hcov can occur, a multiyear study with appropriate control subjects will be necessary to adequately assess whether there is an association between hcov infection and severe respiratory disease. a new virus isolated from the human respiratory tract growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease 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suggests a relatively recent zoonotic coronavirus transmission event the incidence of pneumonia in rural thailand real-time pcr assays for detection of bocavirus in human specimens human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand the cost of influenza in thailand genescan reverse transcription-pcr assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness human metapneumovirus infections in young and elderly adults rhinovirus-associated hospitalizations in young children clinical disease in children associated with newly described coronavirus subtypes human coronavirus infection among children with acute lower respiratory tract infection in thailand human coronavirus nl63 infection in canada coronavirus infection in acute lower respiratory tract disease of infants human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms coronavirus infection in acute lower respiratory tract disease of infants a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection human respiratory coronavirus hku1 versus other coronavirus infections in italian hospitalised patients we thank the following people who provided hcov rna for assay validation: dr. lia van der hoek (university of amsterdam, amsterdam, the netherlands), dr. nancy bellei (university of sao paulo, sao paulo, brazil), dr. atul humar (mount sinai hospital, toronto, canada), and dr. ian mackay (royal children's hospital, queensland, australia). we also thank george gallucci, brian holloway, xiaoyan lu, and karen mccaustland (cdc, atlanta, georgia) for technical assistance; pongpun sawatwong (thailand ministry of public health-us cdc collaboration) for laboratory assistance; and the surveillance officers and research nurses in sa kaeo province, thailand. key: cord-317232-qk72i0gv authors: gierer, stefanie; müller, marcel a.; heurich, adeline; ritz, daniel; springstein, benjamin l.; karsten, christina b.; schendzielorz, alexander; gnirß, kerstin; drosten, christian; pöhlmann, stefan title: inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity date: 2015-03-15 journal: j infect dis doi: 10.1093/infdis/jiu407 sha: doc_id: 317232 cord_uid: qk72i0gv middle east respiratory syndrome coronavirus (mers-cov) infection is associated with a high case-fatality rate, and the potential pandemic spread of the virus is a public health concern. the spike protein of mers-cov (mers-s) facilitates viral entry into host cells, which depends on activation of mers-s by cellular proteases. proteolytic activation of mers-s during viral uptake into target cells has been demonstrated. however, it is unclear whether mers-s is also cleaved during s protein synthesis in infected cells and whether cleavage is required for mers-cov infectivity. here, we show that mers-s is processed by proprotein convertases in mers-s–transfected and mers-cov–infected cells and that several rxxr motifs located at the border between the surface and transmembrane subunit of mers-s are required for efficient proteolysis. however, blockade of proprotein convertases did not impact mers-s–dependent transduction of target cells expressing high amounts of the viral receptor, dpp4, and did not modulate mers-cov infectivity. these results show that mers-s is a substrate for proprotein convertases and demonstrate that processing by these enzymes is dispensable for s protein activation. efforts to inhibit mers-cov infection by targeting host cell proteases should therefore focus on enzymes that process mers-s during viral uptake into target cells. the emergence and subsequent pandemic spread of the severe acute respiratory syndrome (sars) coronavirus in 2002-2003 caused almost 800 deaths and wreaked enormous economic havoc [1, 2] . the virus was transmitted from bats, potentially via intermediate hosts, to humans, demonstrating that the zoonotic transmission of novel coronaviruses from animal reservoirs to humans can pose a significant threat to public health [3, 4] . a similar outbreak scenario unfolded in 2012, when a novel coronavirus, initially named human coronavirus emc and now termed middle east respiratory syndrome coronavirus (mers-cov), was detected in a patient from jordan hospitalized with a severe and ultimately fatal pneumonia [5] . subsequently, the virus spread within the middle east and, through travel activity, occasionally to europe, africa, asia, and north america [6] [7] [8] [9] . the outbreak has, as of 4 july 2014, entailed 827 laboratory-confirmed infections and 287 deaths [10] , and adaptation of the virus to moreefficient human-to-human spread is a public health concern. therefore, it is imperative to identify and explore novel targets for antiviral therapy. the viral surface protein spike (s), a type i transmembrane protein synthesized in the constitutive secretory pathway of infected cells, mediates coronavirus entry into target cells [11, 12] . for this, the mers-cov spike protein (mers-s) binds to its receptor dipeptidyl-peptidase-4 (dpp4, cd26) on the surface of target cells [13] and drives fusion of the viral envelope with a target cell membrane, which allows delivery of viral proteins and rna into the host cell cytoplasm, the site of viral replication. however, mers-s and other coronavirus s proteins are synthesized as inactive precursors in infected cells and only acquire the ability to drive membrane fusion upon processing into the surface unit (s1) and the transmembrane unit (s2) by host cell proteases [14, 15] . the activity of the responsible proteases is essential for viral infectivity, which makes these enzymes potential targets for antiviral intervention. it has been demonstrated that the cysteine protease cathepsin l [14, 16, 17] and the type ii transmembrane serine protease tmprss2 [14, 16, 17] can activate mers-s during viral binding and uptake into target cells. however, activation of viral glycoproteins, including activation of the s protein of certain strains of the coronavirus infectious bronchitis virus (ibv) [18] , may also proceed in the constitutive secretory pathway of infected cells and is often accomplished by furin and other proprotein convertases [19] [20] [21] [22] . whether mers-s is also cleaved during the passage of the secretory pathway and whether these cleavage events contribute to s protein activation is at present unknown. here, we show that proprotein convertases process mers-s in transfected and infected cells, and we demonstrate that the integrity of several rxxr motifs located at the border of the s1 and s2 subunit is required for s protein processing. treatment of mers-cov-infected cells with a proprotein convertase inhibitor (pci) abrogated s protein cleavage but did not alter viral infectivity, indicating that s protein processing in infected cells is dispensable for mers-s activation. expression plasmids encoding mers-s [14] , vesicular stomatitis virus glycoprotein (vsv-g) [23] , zaire ebolavirus glycoprotein (ebov-gp) [23] , lassa virus glycoprotein (lasv-gpc) [23] , dpp4 [13] , and tmprss2 [24] have been described previously. plasmids pgal4-vp16 [25] , pgal5-luc [25] , pnl4-3-luc-r − e[26] , and p96zm651gag-opt [27] , as well as plasmids encoding mers-s [14] and ebov-gp [28] with a c-terminal v5 tag have also been described. a constructs expressing lasv-gpc and mers-s potential cleavage site mutants (pcm) 1 (626-axxa-629), pcm2 (691-axxa-694), pcm3 (748-axxa-751), and pcm4 (884-axxa-887) with a c-terminal v5 tag were generated by polymerase chain reaction (pcr)based mutagenesis. the integrity of all pcr-amplified sequences was confirmed by automated sequence analysis. 293t cells were propagated in dulbecco's modified eagle's medium (dmem; life technologies) supplemented with 10% fetal bovine serum (fbs; pan-biotech), penicillin, and streptomycin. caco-2 cells were cultured in dmem-glutamax medium (invitrogen) supplemented with 10% fbs, penicillin, and streptomycin. vero b4 cells were cultured in dmem supplemented with 5% fbs, 1% l-glutamine, 1% sodium pyruvate, 1% nonessential amino acids (all from life technologies), and antibiotics as stated above. all cell lines were grown in humidified atmosphere at 37°c and 5% co 2. for the detection of mers-s expression in transfected cells, 293t cells underwent calcium phosphate transfection with the respective plasmids encoding s proteins with a c-terminal v5 antigenic tag. for immunoblotting, the lysates were separated by sodium dodecyl sulfate gel electrophoresis and transferred onto nitrocellulose membranes (hartenstein). mers-s expression was detected using a monoclonal antibody directed against the v5 tag (invitrogen) or a polyclonal antibody directed against the s2 subunit of the mers-s protein (sino biological). for detection of mers-s protein in infected cells, caco-2 and vero b4 cells were infected with mers-cov (human betacoronavirus 2c emc/2012) at a multiplicity of infection (moi) of 0.01 and 5, respectively. at 24 hours after infection, the cells were harvested and treated with nupage lds sample buffer (invitrogen), boiled for 20 minutes at 95°c, and analyzed by western blot as described above. as a loading control, the membranes were incubated with anti-β-actin antibody (sigma). to assess the role of proprotein convertase activity in mers-s processing in transfected 293t cells, the cells were transfected with plasmids encoding mers-s, ebov-gp, and lasv-gpc or with empty plasmid. the medium was changed 6 hours after transfection, and pci (merck) was added to the fresh medium at the indicated concentrations. medium was replaced again 32 hours after transfection, and inhibitor was replenished. cells were lysed 48 hours after transfection, and cleavage of viral glycoproteins was detected by western blot, using a monoclonal antibody recognizing the v5 tag. as a loading control, an anti-β-actin antibody was used. to assess whether proprotein convertase activity is required for mers-s-driven cell-cell fusion, a previously described cellcell fusion assay was used [24] , which is based on mixing effectors cells expressing the trans-activator vp16 with target cells expressing luciferase under the control of a vp16-responsive promoter. specifically, 293t target cells that expressed mers-s or no glycoprotein and effector cells that were transfected to express dpp4 and/or tmprss2 were incubated with 1 µm pci. one day after transfection, the cells were mixed to allow cell-cell fusion, and medium was supplemented with pci at 1 µm final concentration. cell-cell fusion was quantified by determination of luciferase activity in cell lysates at 48 hours after cocultivation, using a commercially available kit (pjk). for analysis of the importance of proprotein convertase activity for mers-s-driven virus-cell fusion, a previously established pseudotyping strategy was used [14] : pnl4-3-luc-r − e − [26] vectors bearing mers-s, lasv-gpc, ebov-gp, or vsv-g or no glycoprotein were generated in the presence or absence of 1 µm pci. as target cells, 293t cells transfected with dpp4 encoding plasmid or empty plasmid, were seeded into 96-well plates and pretreated with 0.5 µm pci for 60 minutes at 37°c. the cells were then incubated with pseudotypes for 8 hours, followed by replacement of infection medium with culture medium containing inhibitor. transduction efficiency was measured after 72 hours by determining luciferase activities in cell lysates. to determine the role of proprotein convertase activity in infection with authentic mers-cov, caco-2 cells seeded in 24-well plates were incubated with dimethyl sulfoxide or rising concentrations of pci for 1 hour at 37°c and were then inoculated with mers-cov (moi 0.01 and 0.001 in quadruplicates) in the presence of inhibitor. after incubation for 30 minutes at 4°c, the cells were washed and again incubated in culture medium with the inhibitor. at 24 hours after infection, the cells were washed and harvested, and the pellet was lysed with ripa lysis buffer, supplemented with 4xnupage (invitrogen) and boiled for 20 minutes at 95°c. s protein expression in lysates was detected by western blot, using a polyclonal antibody directed against the s2 subunit of mers-s (sino biological). in parallel, for quantification of viral rna, 50 µl of the cell supernatant was dissolved in rav1 buffer (macherey-nagel) for rna extraction, followed by quantitative reverse-transcription pcr analysis, using the upe assay as previously described [29] . quantification of infectious particles was done by plaque assay, using vero b4 cells as published elsewhere [6] . briefly, 10-fold dilutions of supernatants were tested in duplicates, using cell monolayers of vero b4 cells. after 1 hour of virus adsorption, cells were washed and overlayed with a 1.2% avicel resin. after 3 days, the plates were fixed with 7% paraformaldehyde and stained with crystal violet solution. to investigate the influence of pci on the formation of mers-covinduced cytopathogenic effects, vero b4 cells were infected at an moi of 0.1 and fixed with 7% paraformaldehyde 42 hours after infection. mers-cov antigen detection was performed by incubation with a serum specimen from a patient with mers as described elsewhere [30] . bound antibodies were detected with a cyanine 2-labeled goat-anti human immunoglobulin g secondary antibody (dianova). nuclei were stained by mounting the slides with dapi containing prolong gold antifade mounting medium (life technologies). to investigate mers-s cleavage in virus producing cells, we determined whether the s protein is cleaved in transfected and infected cells. western blot analysis of s protein transfected 293t cells and mers-cov-infected vero b4 cells with an antibody specific to the s2 subunit of mers-s revealed 2 prominent s protein bands with molecular weights of 170 kda and 90 kda (figure 1 ), in keeping with our previous results [14] . the 170k-da band corresponds to uncleaved mers-s, while the presence of the 90-kda band indicates efficient processing of mers-s into an n-terminal s1 subunit (not detected) and a c-terminal s2 subunit (figure 1 ). several rxxr motifs located at the border of the s1 and s2 subunits are required for processing of mers-s inspection of the sequences located at the border of the s1 and s2 subunits of mers-s revealed the presence of 4 rxxr sequences (figure 2a) , which might represent recognition sites for proprotein convertases [22] . to determine the importance of these motifs for mers-s cleavage, we changed the arginine residues in 626-rxxr-629, 691-rxxr-694, 748-rxxr-751, and 884-rxxr-887 to alanine residues, creating the potential cleavage site mutants pcm1, pcm2, pcm3, and pcm4 (figure 2a ). all s protein mutants were expressed with the same efficiency as wild-type mers-s in transfected cells ( figure 2b ). the only exception was pcm2, for which consistently no expression was detected (not shown). importantly, mutation of potential cleavage sites 3 and 4 alone impacted s protein processing: the presence of the 90-kda band was reduced in cells expressing pcm3, and a band with a molecular weight slightly 293t cells were transfected with a plasmid encoding the mers-s protein or with empty plasmid (pcdna). vero b4 cells were either infected with mers-cov at a multiplicity of infection of 5 or mock infected. subsequently, the cells were lysed and analyzed by western blot, using a polyclonal antibody directed against the s2 subunit of mers-s. a β-actin antibody served as a loading control. similar results were obtained in 2 separate experiments. higher than 90 kda was observed upon expression of pcm4 ( figure 2b ). processing of pcm1 was comparable to that seen for mers-s wild type. however, combined mutation of potential cleavage sites 1 and 3 (pcm1 + pcm3) abrogated s protein processing ( figure 2b ), suggesting that also potential cleavage site 1 contributes to s protein cleavage in this experimental setting. similar effects were seen when potential cleavage sites 3 and 4 were simultaneously altered (pcm3 + pcm4) or when all 3 potential cleavage sites were mutated in parallel figure 2 . rxxr motifs located at the border between s1 and s2 are required for efficient processing of the middle east respiratory syndrome coronavirus spike protein (mers-s). a, the domain organization of the mers-s protein is schematically depicted. the mers-s sequence at the border between the s1 and s2 subunits is shown. rxxr motifs, which constitute potential cleavage sites, are highlighted, and the predicted start of the s2 subunit is underlined. the mutations introduced into the potential cleavage sites in mers-s are shown. b, 293t cells were transfected with expression plasmids coding for mers-s wild type and the indicated mers-s mutants equipped with a c-terminal v5 tag. transfection of empty plasmid (pcdna) served as negative control. expression of s proteins in cell lysates was determined by western blot, using a v5 tag-specific monoclonal antibody. expression of β-actin in cell lysates was assessed as a loading control. the results shown are representative for at least 3 independent experiments. abbreviations: ct, cytoplasmic tail; pcm, potential cleavage site mutant; rbd, receptor binding domain; sp, signal peptide; tm, transmembrane domain. (pcm1 + pcm3 + pcm4). collectively, these results indicate that mers-s cleavage is reduced or altered upon mutation of potential cleavage sites 1, 3, and 4. we next sought to identify the proteases responsible for mers-s cleavage. for this, we investigated whether s protein cleavage can be blocked by a cell-permeable tripeptide derivative containing an arg-x-arg motive, which is known to inhibit several proprotein convertases, including furin [31] . treatment with this pci reduced the production of the 90-kda band in cells transfected to express mers-s ( figure 3) . moreover, the presence of pci diminished processing of the zaire ebolavirus glycoprotein (ebov-gp) into gp1 and gp2 (figure 3) , which depends on the activity of the proprotein convertase furin [32] . in contrast, processing of the glycoprotein of lassa virus (lasv-gpc), which is mediated by the proprotein convertase ski-1/s1p [33] , was not suppressed (figure 3 ). this finding is in keeping with the known differences in substrate specificity (and thus inhibitor sensitivity) of ski-i/sip, compared with proprotein convertases with specificity for basic amino acids, like furin [34] . collectively, these results show that the activity of proprotein convertases is essential for mers-s processing in transfected cells. we next assessed whether proprotein convertase activity is required for s protein-driven cell-cell fusion. for this, we used a previously described assay [24] , which is based on directed expression of s protein and receptor/protease in effector and target 293t cells, respectively. expression of mers-s in effector cells allowed inefficient fusion with control-transfected 293t target cells, which are known to express low amounts of endogenous dpp4 [35] , and the efficiency of cell-cell fusion was markedly increased when target cells were transfected with dpp4 and/or tmprss2 encoding plasmid ( figure 4a ). however, the continuous presence of pci in target and effector cell cultures, before, during, and after mixing had no appreciable effect on fusion efficiency ( figure 4a ), suggesting that proprotein convertase activity is dispensable for mers-s-driven cell-cell fusion. to determine whether proprotein convertase activity is required for mers-s-dependent virus-cell fusion, we used a previously reported vector system [14] . notably, the addition of pci to cells producing mers-s-harboring lentiviral particles reduced transduction of 293t target cells by roughly 100-fold ( figure 4b ). in contrast, infectivity of particles bearing lasv-gpc or ebov-gp was not affected (figure 4b ), in keeping with the findings that processing of lasv-gpc is not inhibited by pci (figure 3 ) and that processing of ebov-gp by proprotein convertases is dispensable for gp-driven virus-cell fusion [36, 37] . of note, no inhibitory effect was detected when 293t cells transfected to express dpp4 were chosen as targets (figure 4b) , indicating that s protein processing in virus-producing cells might be dispensable for infectivity when target cells express robust amounts of dpp4. in sum, these results suggest that proprotein convertase activity is largely dispensable for mers-s-driven cell-cell and virus-cell fusion, at least when target cells produce high levels of dpp4. the lack of a prominent inhibitory effect of pci on mers-s-driven cell-cell and virus-cell fusion might be due to high levels of directed mers-s and dpp4 expression in these experimental systems. therefore, we assessed whether pci inhibits mers-cov spread in target cells expressing endogenous dpp4. pci treatment of caco-2 and vero b4 cells infected with mers-cov did not inhibit total mers-s expression but reduced s protein cleavage efficiently and in a concentration-dependent manner ( figure 5a and data not shown), confirming that mers-s is a substrate of proprotein convertases in infected cells. however, pci treatment did not reduce mers-cov propagation, as determined by the amount of viral rna ( figure 5b and data not shown) or infectious units ( figure 5b and data not shown) present in culture supernatants. similarly, treatment of mers-cov-infected cultures had little if any effect on the formation of cytopathic effects, as demonstrated by comparable destruction of the cell monolayer ( figure 5c ). thus, processing of mers-s by proprotein convertases is dispensable for mers-cov infectivity in cell cultures. the processing of the glycoproteins of human immunodeficiency virus (hiv) [19] and highly pathogenic avian influenza viruses [20] by proprotein convertases is essential for viral infectivity. as a consequence, antiviral strategies aiming at the inhibition of these enzymes are being developed [31] . our results add mers-s to the list of proprotein convertase substrates and show that several rxxr motifs within mers-s are required for efficient s protein processing. however, activity of proprotein convertases was dispensable for infectivity of mers-cov, indicating that, in the context of mers-cov infection, proprotein convertases do not constitute targets for antiviral intervention. the observations that tmprss2 activates mers-cov [14, 16, 17] and other coronaviruses [38] [39] [40] and that knock out of tmprss2 has no phenotype in the absence of infection [41] defined tmprss2 as a target for therapeutic intervention. however, tmprss2 might not be the only cellular protease that constitutes a potential therapeutic target in the context of coronavirus infection. thus, cleavage of mers-s during biogenesis in infected cells might be required for subsequent cleavage and activation of the s protein by tmprss2 during viral uptake into target cells. such a scenario would not be unprecedented, given that entry mediated by the glycoproteins of certain bunyaviruses depends on glycoprotein processing during synthesis in the constitutive secretory pathway and, as suggested by a recent report, during viral uptake into target cells [42] . alternatively, it is conceivable that processing of mers-s in infected cells is dispensable for virus-cell but required for cell-cell fusion, which results in formation of syncytia, a feature of mers-cov pathogenesis [5] . we found that mers-s is efficiently although not completely cleaved in s protein-transfected 293t cells and mers-cov-infected vero b4 and caco-2 cells, and inhibition experiments showed that proprotein convertase activity is essential for mers-s cleavage. the finding that mers-s is efficiently cleaved in transfected or mers-cov-infected cells is in keeping with several studies examining mers-s expression for other purposes [16, 43, 44] . one report showed that mers-s is not cleaved in transfected cells [17] ; however, a mers-s variant with a truncated cytoplasmic tail was examined, and it is conceivable that this deletion interfered with s protein exposure to or recognition by proprotein convertases. alternatively, the cells used for s protein expression might have produced only low amounts of proprotein convertases. robust s protein processing by proprotein convertases has also been reported for mouse hepatitis virus strain a59 [45, 46] and ibv [18, 47] , while cleavage of sars-s by these proteases is inefficient [48] [49] [50] . thus, mers-s belongs to the subgroup of coronavirus s proteins that are substrates of proprotein convertases, and mers-s processing in different cellular systems, including the colon-derived cell line caco-2, is robust. most proprotein convertases cleave the following motif: (r/k)-2nx-r↓, with n standing for 0-3 amino acids [34] . the finding that rxxr motifs located at the predicted border between s1 and s2 subunit are important for mers-s processing by proprotein convertases might therefore not be unexpected. nevertheless, it is noteworthy that at least 2 rxxr motifs had to be mutated in parallel to markedly reduce s protein processing. this suggests that the processing enzyme(s) can recognize >1 cleavage site. indeed, the s2 fragment produced upon cleavage of pcm4 showed a slightly increased molecular weight relative to the fragment generated from mers-s wild type, which would be in keeping with use of an upstream cleavage site, most likely potential cleavage site 3. alternatively, the integrity of the rxxr motifs located at the border of the s1 and s2 subunit might be required to present a single cleavage site in a protease sensitive configuration. interference with ebolavirus glycoprotein processing by proprotein convertases is compatible with robust viral spread in vitro and in vivo [36, 37] . in contrast, proprotein convertase activity is required for full activity of certain mhv and ibv s proteins in cell-cell and virus-cell fusion reactions [18, [45] [46] [47] . moreover, processing of the hiv envelope protein by proprotein convertases is essential for viral infectivity, and it has been suggested that processing of sars-s, although being inefficient, is still required for full viral spread and for the establishment of cytopathic effects in infected cultures [48] . the present study provides evidence that efficient blockade of mers-s processing by pci has no appreciable effects on s protein activity in cell-cell fusion assay and does not modulate mers-cov spread in susceptible cells. in contrast, inhibition of mers-s processing markedly reduced mers-s-driven fusion of virions with 293 t cells expressing low amounts of endogenous dpp4. it is therefore conceivable that processing of mers-s by proprotein convertases is required for optimal spread of mers-cov in target cells expressing low levels of dpp4. such a scenario would be in keeping with the observation that inefficient sars-s-driven cell-cell fusion due to limited receptor expression can be rescued by directed expression of s protein-activating proteases and vice versa [50] . in sum, our results identify mers-s as a substrate of proprotein convertases but indicate that the activity of these enzymes is dispensable for mers-cov spread in target cell lines 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vaccinia virus ankara efficiently induces virus-neutralizing antibodies cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion coronavirus ibv: partial amino terminal sequencing of spike polypeptide s2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m41 implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry different host cell proteases activate the sars-coronavirus spike-protein for cell-cell and virus-cell fusion acknowledgments. we thank stephan kallies for excellent technical assistance.financial support. this work was supported by fp7-emperie (contract 223498 to c. d. potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-312552-udky2ko7 authors: fouque, florence; gross, karin; leung, zee; boutsika, konstantina title: introduction to a landscape analysis of multisectoral approaches for prevention and control of infectious and vector-borne diseases date: 2020-10-29 journal: j infect dis doi: 10.1093/infdis/jiaa489 sha: doc_id: 312552 cord_uid: udky2ko7 the swiss development cooperation, canada’s international development research centre, the swiss tropical public health institute, and the unicef/united nations development programme (undp)/world bank/world health organization (who) special programme for research and training in tropical diseases (tdr) collaborated on a project to review, understand and promote the use of multisectoral approaches (msas) in the prevention and control of vector-borne diseases (vbds). the objectives of the project were to support a landscape analysis of how msas have been used in the prevention and control of vbds; to develop a theoretical framework for guiding the implementation of interventions; and to test the recommendations in real-life conditions. to realize these objectives, the project supported several activities, including commissioning a series of scientific reviews on msas in 5 thematic areas, sharing the key findings of these reviews in workshops and events, and developing a guidance framework for the implementation of msas. these activities have produced the theoretical framework that will be tested in real-life conditions through the support of case studies. the collaboration on implementing multisectoral activities against vbds will continue among tdr, the swiss tropical public health institute, and new partners such as the who water sanitation and hygiene group, undp, and un-habitat, in order to face the challenges identified and propose solutions tailored to specific contexts. the prevention and control of vbds require strong and adapted msas with the full participation of all relevant sectors. the burden of infectious diseases has drastically decreased in the past 50 years, but it still represents the major cause of premature death in the world and is expected to remain so until 2030, with 41 million deaths annually [1] . vector-borne diseases (vbds), including malaria and emerging arboviral diseases, account for about one-quarter of all infectious diseases [2] , and the significant progress against malaria are halting since a few years. further, the rapid expansion of other diseases. including those caused by arboviruses such as dengue, chikungunya, yellow fever, and zika, is shown by exponential increases in numbers of cases and fatalities. the emergence, transmission, and distribution of vbds are linked to a wide range of intertwined and partially overlapping factors that belong to multiple sectors-from the biological elements of the system, such as pathogen and vector characteristics, to social and global elements, such as poverty, human behavior, and climate change. it has become evident that the prevention and control of these diseases must be driven by more than a single approach, because transmission patterns are driven by vector-host-pathogen relationships in which natural conditions, human societies and vector parameters are dynamically interacting and changing. in this context, a multisectoral approach (msa) is required to effectively address these complex and dynamic transmission patterns. a first framework for msa against malaria was developed by the roll back malaria (rbm partnership) in collaboration with united nations development program (undp) through the multisectoral action framework for malaria (mafm), published in 2013 and revised in 2019 [3] . the mafm calls for action at several levels and in multiple sectors, globally and across international and intranational boundaries, and by different organizations. it emphasizes complementarity, effectiveness, and sustainability, and it involves new interventions as well as putting new life into those that already exist, coordinating and managing these in new and innovative ways. with the purpose to expand the mafm to other vbds, the swiss development cooperation (sdc) and the swiss tropical public health institute (swiss tph) developed a concept note on which the current project was based. the sdc, the international development research centre (idrc) from canada, the swiss tph, and the unicef/united nations development programme (undp)/world bank/world health organization (who) special programme for research and training in tropical diseases (tdr) agreed on a collaborative activity, started in late 2016, to better understand the landscape, the building blocks, and the processes of an msa for the prevention and control of vbds and to implement selected case studies to test these approaches. the objectives of this project were (1) to synthesize global knowledge on current multisectoral activities that are deployed in different regions against infectious diseases, (2) to draw theoretical processes and general recommendations from experiences, and (3) to test the recommendations into real-life conditions through case studies. the project was then structured to include different steps ( figure 1 ). the first step was the support of commissioned reviews on specific related topics; the second was the organization of joint meetings, workshops, and events to discuss and put together all relevant sectors; and the third was the development of a guidance framework for implementing of msas. these first 3 steps have been fully completed in the past 3 years and were used to draw the final step of the project. this last step is currently ongoing and will follow the implementation of case studies on msas against different diseases in low-and middle-incomes countries to test the theoretical framework. a call was launched in january 2017 to support 6 commissioned reviews on specific topics related to msas for the prevention and control of vbds. the overall objective of the call was to support a landscape analysis on examples of msas to prevent and control vbd transmission and to identify knowledge gaps. the commissioned reviews were mandated to investigate current knowledge and experiences on topics related to different sectors. the publications on some of the main findings from the reviews are included in the current supplement to the journal of infectious diseases, and the abstracts from the final technical reports are provided below. review 1: impact of the industrial sector on vbds, with the example of gold mining activities disrupting malaria ecosystems in africa and the americas. the objective of this review was to retrieve and analyze available data and information on the impact of industrial activities on vbds transmission, with a special focus on mining activities that can greatly disturb existing malaria ecosystems in africa, asia and latin america. the information presented in this review has already been made available to improve policies, practices, and research priorities going forward [4] . some key recommendations from the findings include establishing a national interministerial task force for vector control, promoting the building of suitable and location-specific multisectoral partnerships, mandating the use of health impact assessments and/or expanding the environmental impact assessments to health requirements, reaching the informal sector through local health posts and roaming healthcare workers, conducting risk mapping to determine transmission risks, ensuring community engagement and social outreach when implementing vector control programs, making health messaging and education a component of disease control efforts, and monitoring and evaluating vbd programs. including within the ecobiosocial context. weighted and prioritized integrated strategies for the prevention and control of vbds within the context of ecobiosocial approaches provided evidence for progressive implementation of more comprehensive ways to control vbds. high-level commitment of multiple ministries is central to the intersectoral interactions required to plan, fund, and implement prioritized activities outlined in the response. sustained political engagement will be required to maintain momentum for systems reforms required for adjustment to an integrated approach. prospective primary field studies are needed to generate evidence addressing the impact of integrated strategies to prevent and control vbds within the context of ecobiosocial approaches and the multisectoral participation. in addition, the usefulness of msa was found to go beyond current public health vbd threats to emerging and reemerging ones, especially viral diseases. population displacement and other forced movement patterns after natural disasters or armed conflicts or due to socioeconomic reasons were found to contribute significantly to the global emergence of aedes-borne viral disease epidemics. dengue and chikungunya epidemiology are critically affected by situations of displacement and forced movement patterns, within and across borders. in this respect, waves of human movements have been a major driver for the changing epidemiology and outbreaks of the disease on local, regional, and global scales. both emerging dengue autochthonous transmission and outbreaks in countries known to be nonendemic and cocirculation and hyperendemicity with multiple dengue virus serotypes have led to the emergence of severe disease forms, such as dengue hemorrhagic fever and dengue shock syndrome. the same was also found with atypical and severe forms of chikungunya when emerging in outbreaks due to human mobility and affecting nonimmune populations in new territories. the review was focusing on the vulnerable groups, such as the mobile and migrant population (mmp) in myanmar, which are now the focal malaria transmission groups, impeding malaria elimination in the country. to control malaria transmission and achieve subsequent malaria elimination, one of the interventions focused on increasing use of personal protective measures, such as insecticide-treated bed nets (itns) for the mmps in myanmar artemisinin resistance containment zones. the objectives of this study were to (1) identify which stakeholders were involved in intersectoral approaches to support the intervention of increasing access to and use of itns targeted at the mmps in these zones, (2) characterize the itn interventions targeted to these special groups of the population, and (3) analyze how the intersectoral collaboration was deployed and how this approach was supported in the target population. the findings show that interventions to distribute itns for the prevention of malaria were supported by multiple stakeholders (local, national, nongovernmental organizations, and others); however, it was not described how the intersectoral collaboration was working. nevertheless, the net ownership and rates of use at the end of the project did not met the who targets for myanmar. further data were missing to specifically assess the role of the different stakeholders involved in the interventions. this review clearly demonstrates some gaps in looking at multisectoral collaboration, as well as the absence of specific indicators to show how msa was working and whether the failure was due to faulty implementation of the approach or to other factors. although who is recommending intersectoral collaboration as one of the key elements of integrated vector management and assumed this would make an important contribution to vbds control and elimination, there is limited evidence comparing the effect and contribution of intersectoral approaches with those of the health sector only. for that purpose, a systematic review from more than 40 years of scientific literature was undertaken to develop an evidence-based framework of intersectoral collaboration and assess its effectiveness in sustaining the prevention and control of vbds. among the 50 articles included in the analysis, 19 were categorized as of moderately strong quality. all articles compared preintervention and postintervention outcomes against disease or vector variables (with the intervention being intersectoral collaboration). three articles included outcome variables on intersectoral collaboration and participation indicators. however, analysis by different sectors or different activities was retrieved. only 1 article compared cost data for communityintersectoral intervention for indoors residual spraying (irs) and traditional "vertical" irs. six factors extracted from 47 studies were identified as influencing the effectiveness of intersectoral collaboration. the main ones were using approach factor (in 37 studies out of 47), resources (in 34 studies out of 47), relationships (in 33 studies out of 47), and management (in 29 studies out of of 47). the recent global strategy of vbd control and prevention encourages intersectoral collaboration as an approach to achieving cost-effective and efficient results from an intervention. the review showed that although intersectoral collaboration has played an important role in achieving reduction of vbds or vector densities, very few studies have measured how much intersectoral collaboration contributed to this impact, and the relevant indicators are missing [5] . review 6: scoping review of intersectoral collaborations to prevent and control vbds. this review synthesizes evidence for models of intersectoral collaborations for the prevention and control of vbds. half of the articles were about malaria control in the african region. the other half of the publications retrieved were on the prevention and control of dengue, with interventions based in asia and latin america. among the many gaps or challenges that impeded successful implementation and lowered the chances of project sustainability, most notably, the disconnect between different stakeholder responsibilities was often encountered and exemplified. the observation of a lack of communication between multilateral organizations and local governments, for whatever reason, is of great concern with a top-down approach that makes stakeholder relationships more susceptible to disconnection from field reality. the ownership of the programs by the community was an issue, as multiple cases cited the lack of understanding, interest, and initiative as a reason for discontinuity. this observation emphasizes the needs of msas, including local and community sectors. the lack of research capacity, including baseline data, skilled and knowledgeable staff, and models for data analysis that could be contextualized to local needs, was evident for both malaria and dengue control programs. the overall results show the need for a comprehensive framework for an effective and sustainable msa to prevent and control vbds. because both intersectoral collaboration and vbds are broad topics that hinge on social and economic development, the issues of financing, investment in human resource development, and supply of materials should be addressed through the collaboration. the delphi-validated statements summarizing recommendations for msas are globally applicable, but they need to be contextualized to a national and even municipal level. the workshop was attended by about 40 participants and had a specific session with who member states. some activities were recognized by the participants as priority activities to be undertaken once the commissioned reviews have produced their results, through final technical reports and publications. these activities included specific work to support the msa for prevention and control of vbds and recommendations at different levels, including global, cross-border, national, and local. the recommendations from the workshop included the establishment of a guiding and advocacy framework as well as support for case studies to collect evidence on msa potential and functioning, including collection and sharing of data, analysis, cost estimates, overall health impacts, and risks for potential outbreaks. the recommendations for international agencies and national/local levels included a requirement for assessment of vector control needs; coordination of health impact assessment; and operationalization of the msa at the country level, creating a coordinated system that is ready to respond to different vbd situations, including when displaced people are affected or spread vbds. the aims of the session were an introduction to the rationale of msas for vbds; the presentation of key results from the commissioned reviews by the speakers; a discussion on successes, challenges, gaps, needs, and opportunities; and illustration of the potential beneficial impact of follow-up activities. the session began with a general introduction on the burden of vbds, the available evidence that msas are needed to prevent and control vbds needs msas, and the need to know where we are in conceptualizing and implementing these approaches. rashad abdul-ghani (phd) presented the review on chikungunya virus as a globally (re)emerging and rapidly expanding epidemic threat driven by human mobility patterns, showing that the expansion of this virus is strongly linked to human movement, which in turn is related to social, economic, political (multisectoral) factors. the warning that what is happening with chikungunya virus may also happen with any other virus, maybe more virulent, truly anticipated the current coronavirus disease 2019 pandemic. alfonso rodriguez morales (phd) presented the integrated strategies for the prevention and control of vbds within the context of ecobiosocial approaches, showing how to move from a single-oriented control of vbds (vector control for example) to a multisectoral one. the single-oriented control take into account only one sector and one approach such health sector and vector control, opposite to a multisectoral one taking into consideration more than one sector such health and water and more than a single approach such as vector control and water management. the added value of integrating traditional vector control activities (health system) with newer technologies (community involvement, social approaches) was discussed, along with how these activities and approaches can be synergized and the associated challenges. also discussed were recommendations on the way forward and potential opportunities for multisectoral research and action to help prevent and control vbds. jana fitria kartika sari presented intersectoral collaboration for the prevention and control of vbds, supporting the implementation of a global strategy, with some examples of success when working outside the health sector (eg, including education, agriculture, etc) and the remaining knowledge gaps. the final discussion introduced the global vector control response, which advocates for msas among the 4 pillars of the response [5] . diseases, " new orleans, louisiana, 31 october 2018. the annual meetings of the american society of tropical medicine & hygiene attract thousands of participants, so this was an ideal forum to expose this work to a wider audience before the planned peer-reviewed publication. the poster summarized the progress and suggested the expected outcomes and the next steps. this document [6] was produced following one of the main recommendations from the reviews and exchanges with the stakeholders. the document starts with 2 introductory chapters on vbd basics and msas. the determinants of vbds are described and grouped into the following categories: pathogen and vector related, environmental and agroecological, economic and social, and health system related. together, these determinants go beyond the ministries of health and the health sector, concerning many other sectors and stakeholders. existing prevention and control methods were laid out with challenges highlighted, among which was the weakness of the health sector alone. there are opportunities for better coordinated actions, such as potential synergy with the global momentum of the sustainable development goals, multiple entry points for interventions across sectors and diseases, and empowerment through more research. the historical background of msa was traced to reveal this inevitable growing consensus. examples of existing msa case studies were extracted from the commissioned reviews and briefly discussed. chapters 3 and 4 present the conceptual framework and its components. the conceptual framework is named bet-for base, energy, and technical elements-and includes 7 components: pillars, dimensions, levels, resources, sectors, domains and enablers. these components envelope the ingredients to include in a customized and tailored msa. chapter 5 outlines the coordination pathway from step 1 to step 6. roles of nongovernment sectors and bodies are discussed with a focus on nongovernmental and international organizations, private sectors, and communities. because financing and legislation are an indispensable part of the entire coordination pathway, funding mechanisms and the types of norms and policies needed during a multisectoral collaboration are included in this chapter. the guidance also emphasizes integration and synergy with the existing institutional structure for msa within the country as well as with global multinational and multisectoral efforts, such as those under the health and environment linkages initiative, sustainable development goal-related programs, and the one health initiative. chapter 6 covers sectoral guidance. a sectoral pathway is intended to assist government ministries to plan and initiate their work according to an msa, from defining the vision to sectoral monitoring and evaluation, including important steps such as aligning msa activities with the sector's existing activities. a nonexhaustive list of key sectors is included, along with health: environment, water and sanitation, agriculture and aquaculture, energy, housing, education and research, finance, and legislature. the concluding chapter of the guidance document highlights the need for a system to monitor and evaluate these approaches and the interventions. the activities developed around the analysis of msas for prevention and control of vbds have resulted in new evidences retrieved from the analyses of the available publications and findings on this topic. several thousands of articles were retrieved, screened, selected, and analyzed through the work done by the 6 commissioned reviews. the findings were published and some of them are included in this special issue. the main results were discussed over several events and exchanges with stakeholders from different sectors and levels (from global to local). among the main challenges identified to building up an effective and efficient msa, the lack of pathway and framework was considered critical, but the development of such a theoretical framework was feasible and was then achieved. this guidance document has now been published and needs to be tested and improved in real-life conditions. although the guidance is primarily directed to decision makers, with specific relevance for governmental sectors, the framework can be tailored to suit the needs of subnational and decentralized stakeholders. the purpose of this framework is not only to support supraministerial leaders and health sector but also to enhance the capacity of decision makers in other sectors to achieve in a collective effort efficient prevention and control of vbds. because the guidance document aims to delineate "how to," apart from describing the essential components, recommendations are providedfor instance, on how to mobilize political will, how to enhance commitment and coordination among sectors, and how to strengthen community engagement. these elements will be studied in the following step of the project within specific cases of multisectoral collaboration to control specific diseases, such as malaria and arboviral diseases. for that purpose, research proposals will be supported through a partnership with one of the most relevant sectors linked to health and vbds, that is the water, sanitation, and hygiene (wash) sector. a proposal was developed in partnership with the who wash group to reduce wash-related disease of poverty with a primary focus on vbds, through the following activities: (1) refining and promoting research for impact on multisectoral action for health, (2) increasing the impact of joint convening of wash and health sectors, and (3) supporting the strengthening of health systems to better address infectious diseases of poverty in general and vbds in particular. the project will include 2 work packages: work package 1 on strengthening the prevention and control of diseases of poverty through multisectoral collaboration, based on the latest research findings and who wash norms, and work package 2 on strengthening health systems to better address infectious diseases of poverty through improved wash in healthcare facilities and enhanced capacity to manage wash services and engage in good hygiene practices. the partnership around the multisectoral activities will continue between tdr, the swiss tph, and new partners, such as undp and un habitat for building stronger recommendations and better addressing the numerous challenges identified. from past experiences and evidences, it has become clear that the prevention and control of vbds cannot be achieved and sustained through single-sector intervention(s) and without the full commitment of not only the responsible entities but also the communities involved. what msas are targeting is exactly this full participation of all relevant sectors according to their own incentives and needs. the international bank for reconstruction and development/ the world bank world health organization. global vector control response 2017-2030. world health organization roll back malaria partnership to end malaria and united nations development programme. multisectoral action framework multisectoral approach to the prevention and control of vector-borne diseases: a conceptual framework the impact of industrial activities on vector-borne disease transmission intersectoral collaboration for the prevention and control of vector borne diseases to support the implementation of a global strategy: a systematic review key: cord-327673-3uem0e22 authors: qin, gang; mao, huawei; zheng, jian; sia, sin fun; liu, yinping; chan, ping-lung; lam, kwok-tai; peiris, j. s. malik; lau, yu-lung; tu, wenwei title: phosphoantigen-expanded human γδ t cells display potent cytotoxicity against monocyte-derived macrophages infected with human and avian influenza viruses date: 2009-09-01 journal: j infect dis doi: 10.1086/605413 sha: doc_id: 327673 cord_uid: 3uem0e22 backgroundinfluenza virus is a cause of substantial annual morbidity and mortality worldwide. the potential emergence of a new pandemic strain (eg, avian influenza virus) is a major concern. currently available vaccines and anti-influenza drugs have limited effectiveness for influenza virus infections, especially for new pandemic strains. therefore, there is an acute need to develop alternative strategies for influenza therapy. γδ t cells have potent antiviral activities against different viruses, but no data are available concerning their antiviral activity against influenza viruses methodsin this study, we used virus-infected primary human monocyte-derived macrophages (mdms) to examine the antiviral activity of phosphoantigen isopentenyl pyrophosphate (ipp)–expanded human vγ9vδ2 t cells against influenza viruses resultsvγ9vδ2 t cells were selectively activated and expanded by ipp from peripheral blood mononuclear cells. ipp-expanded vγ9vδ2 t cells efficiently killed mdms infected with human (h1n1) or avian (h9n2 or h5n1) influenza virus and significantly inhibited viral replication. the cytotoxicity of vγ9vδ2 t cells against influenza virus–infected mdms was dependent on nkg2d activation and was mediated by fas–fas ligand and perforin–granzyme b pathways conclusionour findings suggest a potentially novel therapeutic approach to seasonal, zoonotic avian, and pandemic influenza—the use of phosphoantigens to activate γδ t cells against influenza virus infections proportion (2%-10%) of t lymphocytes in the blood and peripheral organs of most adult animals and humans [6] [7] [8] . in humans, vg9vd2 t cells make up the majority of peripheral blood and lymphoid organ gd t cells [9] . human gd t cells share characteristics of t cells, natural killer (nk) cells, and antigen-presenting cells [6] [7] [8] . therefore, gd t cells are thought to represent one of the first lines of the host immune defense. the antiviral activities of gd t cells have been demonstrated in different models [10] [11] [12] . in the mouse model, gd t cells were shown to contribute to recovery from influenza pneumonia [13, 14] , but no data are available on the contribution of gd t cells at early stages of influenza virus infections. activated mouse gd t cells showed profound cytotoxicity against hemagglutinin (h1 or h3) expressing target cells in a non-major histocompatibility complex-restricted manner [15] . however, it is still unknown whether human gd t cells have antiviral activities against human or avian flua viruses or what their underlying mechanisms are. vg9vd2 t cells are specifically activated in an hla-unrestricted manner by small nonpeptidic phosphoantigens, which are metabolites of isoprenoid biosynthesis pathways in all organisms [16] . the most potent phosphoantigen is hydroxydimethylallyl-pyrophosphate, produced through a nonmevalonate pathway in microorganisms such as mycobacteria. natural isopentenyl pyrophosphate (ipp), an intermediate produced through the mevalonate pathway that also leads to cholesterol synthesis in mammalian cells, was found to selectively activate and expand human vg9vd2 t cells in vitro or in vivo [17, 18] . vg9vd2 t cells expanded by synthetic phosphoantigens were demonstrated to have antiviral potential against other viruses [19, 20] , but no data are available concerning their antiviral activities against human and avian flua viruses. one characteristic of the host immune response to flua virus infection is the influx of both macrophages and t lymphocytes into the lungs [21] . we recently demonstrated that the macrophage is one of the major target cells for avian h5n1 virus in human lungs apart from alveolar epithelial cells [22] . moreover, h1n1 and h5n1 viruses have been shown to replicate efficiently in both human primary lung epithelial cell cultures and macrophages in vitro [23] . using the model of flua virusinfected monocyte-derived macrophages (mdms), we have found that macrophages infected with human and avian flua viruses express differential proinflammatory cytokines and chemokines [23, 24] and exhibit differential apoptosis-inducing capability in t cells through the tumor necrosis factor-related apoptosis-inducing ligand (trail) pathway [25] . in the present study, we used the similar virus-infected mdms model to examine the cytotoxicity of phosphoantigenexpanded vg9vd2 t cells against flua viruses. specifically, we demonstrated for the first time that ipp-expanded vg9vd2 t cells could efficiently kill mdms infected with human (h1n1) and avian (h9n2 and h5n1) flua viruses and inhibit virus replication. the cytotoxicity of ipp-expanded vg9vd2 t cells was dependent on nkg2d activation and mediated by perforin-granzyme b and fas-fas ligand (fasl) pathways. our findings suggest that phosphoantigen could be used to activate vg9vd2 t cells against flua infections, in particular for avian flua virus infections. peripheral blood mononuclear cells (pbmcs) were isolated from buffycoat preparations of blood from healthy donors from the hong kong red cross by ficoll-paque (pharmacia) gradient centrifugation, as described elsewhere [26] . the research protocol was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster. pbmcs were cultured in roswell park memorial institute (rpmi) 1640 medium supplemented with 10% fetal bovine serum. ipp (sigma) was added on days 0 and 3, to a final concentration of 6 mg/ml. recombinant human interleukin 2 (il-2) (invitrogen) was added every 3 days beginning on day 3, to a final concentration of 500 iu/ml. after being cultured for 14 days, the cells were purified by negative selection with a tcrg/d + t cell isolation kit (miltenyi biotec), in accordance with the manufacturer's instructions. culture of mdms. human mdms were generated from mononuclear cells, as described elsewhere [25] . briefly, adherent monocytes were seeded in 96-well flat-bottomed plates at cells/well or in 24-well plates at cells/well. they 5 5 1 ϫ 10 5 ϫ 10 were then re-fed by rpmi 1640 supplemented with 5% autologous serum and allowed to differentiate to macrophages for 14 days. the purity of monocytes, as determined by flow cytometry with anti-cd14 monoclonal antibody, was consistently 190%. virus preparation, titration, and infection. human flua h1n1 (a/hong kong/54/98) and avian flua h9n2 (a/quail/ hong kong/g1/97) and h5n1 (a/hong kong/483/97) viruses were used. all the viruses were cultured in madin-darby canine kidney cells (american type culture collection), as described elsewhere [25] . the virus titer was determined by daily observation of cytopathic effect, and the median tissue culture infective dose was calculated according to the reed-muench formula. differentiated mdms on day 14 were infected by flua viruses at a multiplicity of infection (moi) of 2. after 1 h of viral adsorption, the cells were washed with phosphate-buffered saline to remove unadsorbed virus. cytotoxicity assay. vg9vd2 t cells (effector) were cocultured with h1n1-, h9n2-, or h5n1-infected mdms (target) at different effector-to-target (e:t) ratios for 4-6 h. afterward, nonadherent cells were harvested directly. adherent cells were detached with 0.25% trypsin-ethylenediaminetetraacetic acid. all of the adherent and nonadherent cells were then stained with anti-cd3 to identify vg9vd2 t cells and ethidium homodimer 2 (ethd-2) to identify dead cells [27] . the cytotoxicity of vg9vd2 t cells against virus-infected mdms was assessed by flow cytometry as the percentage of ethd-2 + cells in the cd3 ϫ population. to evaluate cell-cell contact requirement for vg9vd2 t cell cytotoxicity, a transwell system was used (24 wells; pore size, 0.4 mm; millipore). mdms (target) in the bottom well were infected with h1n1 or h9n2 virus at an moi of 2. vg9vd2 t cells (effector) were added directly into the bottom wells or into transwell inserts at an e:t ratio of 10:1. in some inserts, the same amount of virus-infected mdms was added to activate vg9vd2 t cells. after 6 h, the mdms in the bottom wells were harvested and analyzed for cell death, as described above. blocking assay. vg9vd2 t cells (effector) were cocultured with h1n1 virus-infected or h9n2 virus-infected mdms (target) at an e:t ratio of 10:1 for 6 h. the neutralization antibodies anti-nkg2d (10 mg/ml; 1d11, bd biosciences), anti-fasl (10 mg/ml; 100419, r&d systems), anti-trail (10 mg/ ml; rik-2, r&d systems) and their respective isotype controls were added in the coculture for blocking nkg2d-, fasl-, and trail-mediated pathways, respectively [28] . for blocking perforin and granzyme b, the perforin inhibitor concanamycin a (cma) (1 mg/ml; sigma) and the granzyme b inactivator bcl-2 (1 mg/ml; r&d systems) were used, as described elsewhere [29] . cytotoxicity was analyzed by flow cytometry, as described below, and calculated as the percentage of inhibition relative to that in controls. flow cytometry. cells were stained for surface markers with the following antibodies: anti-cd3 (hit3a), anti-t cell receptor (tcr)-g9 (b3), anti-tcr-d2 (b6), anti-nkg2d (1d11), anti-trail (rik-2), anti-cd107a (h4a3) (bd biosciences), anti-tcr-gd (5a6.e9), anti-cd14 (tuk4), and anti-fasl (alf-2.1) (invitrogen). for intracellular staining, cells were fixed, permeabilized, and then stained with anti-perforin (pfp, dg9) and anti-granzyme b (grb, gb11) antibodies (bd biosciences) or their respective isotype controls. all samples were acquired using a bd facsaria cell sorter (bd biosciences) and were analyzed by means of flowjo software (version 8.8.3; tree star). ) were infected 5 1 ϫ 10 with h1n1, h9n2, or h5n1 virus at an moi of 2. one hour later, unadsorbed virus was washed away carefully and the mdms were cultured alone or with vg9vd2 t cells for 6 1 ϫ 10 48 h. the cells and supernatant were then harvested, and total rna was extracted by means of trizol ls reagent (invitrogen), in accordance with the manufacturer's instructions. complementary dna was synthesized with oligo(dt) [12] [13] [14] [15] [16] [17] [18] primer and superscript ii reverse transcriptase (invitrogen). viral matrix gene copies were quantified on the basis of a sybr green fluorescence signal after polymerase chain reaction (forward primer, 5 -cttctaaccgaggtcgaaacg-3 ; reverse primer, 5 -ggcattttggacaaagcgtcta-3 ) by means of the abi prism 7700 sequence detection system (applied biosystems). results were expressed as the number of target gene copies per mdms. 5 1 ϫ 10 statistical analysis. data were expressed as means ‫ע‬ standard errors of the mean. statistical significance was determined by the student t test or nonparametric tests using graphpad prism software (version 5). differences were considered significant at . p ! .05 consistent with previous findings [18, 19] , we found that ipp and il-2 could selectively expand vg9vd2 t cells. freshly iso to determine whether there was a decreased viral load in virus-infected mdms after coculture with vg9vd2 t cells, h1n1-, h9n2-, or h5n1-infected mdms (target) were cultured alone or with ipp-expanded vg9vd2 t cells (effector) at an e:t ratio of 10:1. after 2 days of coculture, h1n1, h9n2, and h5n1 flua virus matrix gene copies from whole virusinfected mdms and culture supernatants were significantly re vg9vd2 t cells is dependent on cell-cell contact, we used a transwell culture system. as shown in figure 4a and 4b, vg9vd2 t cells lost their cytotoxicity during h1n1 or h9n2 virus infections when vg9vd2 t cells were physically separated from virus-infected mdms. however, when virus-infected mdms were put with vg9vd2 t cells, cytotoxicity toward targets in the bottom wells was also observed, although their cytolytic activities were much lower than those in the direct cell-cell contact coculture. these data suggest that the cytotoxicity of vg9vd2 t cells is dependent on cell-cell contact and requires activation of virus-infected mdms; diffusible soluble factors, such as granules released from activated vg9vd2 t cells, may also be involved. we then sought to confirm granule release during the killing of flua virus-infected mdms by vg9vd2 t cells. the expressions of cd107a (lysosome-associated membrane protein 1), a marker associated with the degranulation of nk cells and cytotoxic t lymphocytes (ctls) [30] , were significantly upregulated in vg9vd2 t cells after coculture with h1n1-and h9n2-infected mdms for 4 h, compared with mock-infected mdms ( figure 4c ). these results suggest that virus-infected mdms trigger more intensive granule release from vg9vd2 t cells. because ipp-expanded vg9vd2 t cells also expressed high or medium levels of nkg2d, fas, and trail, we further determined whether nkg2d, fas-fasl, and trail pathways were involved in their cytotoxicity. using neutralizing antibodies for nkg2d and fasl, we found that blockades of nkg2d and fasl significantly inhibited the cytolytic activities of vg9vd2 t cells against h1n1-infected mdms (64.08% ‫ע‬ inhibition by nkg2d blocking; by fasl 9.15% 41.34% ‫ע‬ 9.51% blocking). in contrast, there was no significant change in their cytolytic activity against h1n1-infected mdms after treatment with trail-blocking antibody (figure 5a). similar results were also found during the killing of h9n2-infected mdms by vg9vd2 t cells ( figure 5b ). these results demonstrate that both the nkg2d and the fas-fasl pathway are also involved in the killing of mdms infected with human and avian flua viruses by vg9vd2 t cells. to further confirm the involvement of cytolytic granule release in the killing of virus-infected mdms by vg9vd2 t cells, the perforin-specific inhibitor cma and granzyme b inactivator bcl-2 were used. as shown in figure 5a , the cytolytic activities of vg9vd2 t cells against h1n1-infected mdms were sigfigure 5 . dependency on nkg2d activation and mediation by fas-fas ligand (fasl) and perforin-granzyme b pathways for vg9vd2 t cell cytotoxicity. vg9vd2 t cells (effector) were cocultured with h1n1 virus-infected (a) or h9n2 virus-infected (b) human monocyte-derived macrophages (mdms) (target) at an effector-to-target ratio of 10:1 for 6 h. the perforin inhibitor concanamycin a (cma), the granzyme b inactivator bcl-2, anti-nkg2d (ankg2d), anti-tumor necrosis factor-related apoptosis-inducing ligand (atrail), and anti-fasl (afasl) blocking antibodies or their respective isotype controls were used as described in methods. cytotoxicity was analyzed by flow cytometry and calculated as the percentage of inhibition relative to that of controls. data are means from 4 experiments; error bars represent standard errors of the mean. * for the comparison with isotype p ! .05 control. gigg, goat immunoglobulin g; migg1, mouse immunoglobulin g1; migg2b, mouse immunoglobulin g2b. nificantly abrogated after cma treatment (57.25% ‫ע‬ 9.77% inhibition) or bcl-2 treatment ( inhibition). 28.42% ‫ע‬ 4.41% similar results were also observed during the killing of h9n2infected mdms by vg9vd2 t cells (figure 5b). these results indicate that the perforin-granzyme b pathway is involved in the cytotoxicity of vg9vd2 t cells against mdms infected with human and avian flua viruses. gd t cells have been reported to play an important role in the defense against pathogens and tumors, and they have broad antiviral activities against different viruses [10, 12, 31, 32] . in human in vitro systems, gd t cells have been shown to have potent cytolytic activities against virus-infected cells, suppressing the replication of human immunodeficiency virus, hepatitis b virus, herpes simplex virus, vaccinia virus, human cytomegalovirus, and severe acute respiratory syndrome coronavirus [20, [33] [34] [35] . in the present study, we focused on the cytotoxic properties of gd t cells in influenza virus infection, because cell-mediated cytotoxicity is the major mechanism to eliminate virus-infected cells and thus to eliminate potential sources of new virus. in particular, as a component of innate immunity, gd t cells may act as early responders in viral control and clearance, compared with specific ctl activity. we showed that ipp-expanded vg9vd2 t cells could kill ∼50% of human h1n1 virus-infected mdms after 6 h of coculture. most importantly, similar potent cytotoxic activities of these cells against mdms infected with the newly emerged avian flua h5n1 virus and its precursor h9n2 virus were also found after 4-6 h of coculture. furthermore, we found that ipp-expanded vg9vd2 t cells significantly inhibited h1n1, h9n2, and h5n1 viral replication by eliminating virus-infected mdms. to the best of our knowledge, ours is the first study to demonstrate that ippexpanded gd t cells have rapid and potent antiviral activity against both human and avian flua viruses. consistent with findings of other in vitro studies [17, 18] , we found that the phosphoantigen ipp could selectively activate and expand human vg9vd2 t cells from pbmcs in the presence of il-2. during 2 weeks of stimulation by ipp and il-2, vg9vd2 t cells were expanded by ∼36-fold. in fact, phosphoantigens have demonstrated the potential to facilitate largescale in vitro expansion of functional gd t cells for use in adoptive immunotherapy for tumors [36] and infectious diseases in different models [19, 20] . although the lack of murine counterparts of vg9vd2 t cells has dramatically hampered efforts to understand the in vivo roles of phosphoantigens in vg9vd2 t cells, the selective activation and expansion of these cells by phosphoantigens were also confirmed in some in vivo models, such as cynomolgus monkeys and severe combined immunodeficient mice engrafted with human peripheral blood leukocytes (human pbl-scid mice) [17, 37] . therefore, phosphoantigens such as ipp could be an alternative for treating human and avian flua infections via targeting vg9vd2 t cells. indeed, synthetic phosphoantigens named therapeutic amino-bisphosphonates (pamidronate and zoledronate) have been commonly used clinically to treat osteoporosis through vg9vd2 t cell-mediated lysis of osteoclasts [38, 39] . one concern regarding gd t cell-based immunotherapy as induced by phosphoantigens is whether these cells can traffic to infected sites, such as the lungs, during the flua virus infection. because gd t cells express chemokine (c-c motif) receptor 5 and are capable of diapedesis [40] , these cells should be able to migrate to inflammatory sites. a more recent study in a macaque model of mycobacterium tuberculosis infection demonstrated that phosphoantigen-specific gd t cells could accumulate at all inflammatory sites in lymphoid and nonlymphoid tissues (including the lungs) [41] , suggesting that the strategy of targeting gd t cells by phosphoantigens may be feasible for the treatment of human and avian flua virus infections. use of nonhuman primate models or human pbl-scid mice to further evaluate gd t cell-based immunotherapy for influenza virus infections in vivo may accelerate its future clinical application. although nkg2d was originally described as an activating receptor for nk cells, it has also been recognized as a potent costimulatory receptor of the cytotoxic functions of human vg9vd2 t cells [42, 43] . it has recently been demonstrated that nkg2d can directly activate vg9vd2 t cells and trigger their release of cytolytic granules through recognition of the nkg2d ligand [44] . in humans, it has been identified that stress-inducible major histocompatibility complex class i-related proteins a and b and members of the ul16-binding protein family (ulbp1-4 and raet1g) are the ligands for nkg2d [45] . in different tumor models, nkg2d-mediated cytotoxicity was reported to be involved in the lysis of tumor cells by gd t cells [18, 46] . in the present study, we showed that most ipp-expanded vg9vd2 t cells expressed nkg2d and that the cytotoxicity of vg9vd2 t cells against h1n1-and h9n2-infected mdms was significantly blocked by nkg2d neutralizing antibody, suggesting that the killing of influenza virus-infected cells by vg9vd2 t cells requires nkg2d activation and recognition. that only influenza virus-infected mdms expressed up-regulated major histocompatibility complex class i-related protein b, an nkg2d ligand [47] , could also explain why ippexpanded vg9vd2 t cells killed only h1n1-, h9n2-, and h5n1-infected mdms but not mock-infected mdms in the present study. the fas-fasl-mediated pathway was also shown to be involved in the killing of listeria monocytogenes-infected macrophages by murine gd t cells in vivo [48] . we found that ippexpanded vg9vd2 t cells expressed high levels of fas and that blockade of the fas-fasl pathway significantly inhibited the cytotoxicity of vg9vd2 t cells against h1n1-and h9n2-infected mdms, indicating that the fas-fasl-mediated pathway is also involved in the killing of influenza virus-infected cells by vg9vd2 t cells. we recently demonstrated that trail was significantly upregulated by both h9n2 and h5n1 avian flua viruses in human mdms, compared with h1n1, and that avian flua virus-infected mdms could induce t cell apoptosis through the trail pathway [25] . the present study, however, did not show involvement of the trail pathway during the killing of h1n1or h9n2-infected mdms by vg9vd2 t cells; blockade of the trail pathway did not reduce the cytotoxicity of vg9vd2 t cells against either h1n1-or h9n2-infected mdms. using the transwell system, we demonstrated that the killing of flua virus-infected mdms by vg9vd2 t cells was dependent on cell-cell contact and required the activation of virus-infected cells. in addition, cd107a expression was increased only in vg9vd2 t cells cocultured with flua virus-infected mdms and not in those cocultured with mock-infected mdms, supporting the hypothesis that granule release from vg9vd2 t cells requires virus-infected cell activation. using the perforin inhibitor cma and the granzyme b inactivator bcl-2, we confirmed that perforin and granzyme b facilitated the cytolytic responses of vg9vd2 t cells to flua virus-infected mdms, which is consistent with the findings of other studies in tumor and other virus-infected cells [18, 20, [33] [34] [35] . therefore, human vg9vd2 t cells closely resemble nk and cd8 + t cells in their cytotoxic function, using a predominantly granule-exocytosis mechanism to kill virus-infected cells. in summary, we have demonstrated for the first time that vg9vd2 t cells can recognize and efficiently kill mdms infected with human and avian flua viruses and thus contribute to virus clearance. the cytotoxicity of vg9vd2 t cells against flua virus-infected mdms is dependent on nkg2d activation and is mediated by the fas-fasl and perforin-granzyme b pathways. our study suggests a novel approach of using phosphoantigens to activate vg9vd2 t cells against flua virus infections-newly emerged avian flua virus infections in particular. h5n1 influenza: a protean pandemic threat the next influenza pandemic: lessons from hong kong avian flu to human influenza prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) gammadelta t cells link innate and adaptive immune responses professional antigen-presentation function by human gammadelta t cells the function of gammadelta t cells in innate immunity adaptive immune response of vgamma2vdelta2+ t cells during mycobacterial infections gammadelta t cells: functional plasticity and heterogeneity antiviral reactivities of gammadelta t cells vgamma9vdelta2 t cell-mediated non-cytolytic antiviral mechanisms and their potential for cell-based therapy tcrgammadelta cells and viruses activation of cytokine genes in t cells during primary and secondary murine influenza pneumonia heterosubtypic immunity to influenza a virus in mice lacking 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anti-tumoral responses of polyclonal phosphoantigen-reactive vgamma9vdelta2 t lymphocytes recognition and destruction of virus-infected cells by human gammadelta ctl shared reactivity of vd2 neg gd t cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells characterization of tumor reactivity of human vgamma9vdelta2 gammadelta t cells in vitro and in scid mice in vivo in vivo immunomanipulation of vgamma9vdelta2 t cells with a synthetic phosphoantigen in a preclinical nonhuman primate model vgamma2vdelta2 t-cell receptor-mediated recognition of aminobisphosphonates gamma/delta t-cell stimulation by pamidronate patterns of chemokine receptor expression on peripheral blood gammadelta t lymphocytes: strong expression of ccr5 is a selective feature of vdelta2/vgamma9 gammadelta t cells immune distribution and localization of phosphoantigen-specific vgamma2vdelta2 t cells in lymphoid and nonlymphoid tissues in mycobacterium tuberculosis infection activation of nk cells and t cells by nkg2d, a receptor for stress-inducible mica mica engagement by human vgamma2vdelta2 t cells enhances their antigen-dependent effector function activation of vgamma9vdelta2 t cells by nkg2d nkg2d ligands: key targets of the immune response lysis of a broad range of epithelial tumour cells by human gammadelta t cells: involvement of nkg2d ligands and t-cell receptor-versus nkg2d-dependent recognition cytokine and contact-dependent activation of natural killer cells by influenza a or sendai virus-infected macrophages fas-fas ligand interactions are essential for the binding to and killing of activated macrophages by gammadelta t cells key: cord-306983-6w2fvtfy authors: wang, siye; le, trong quang; kurihara, naoki; chida, junji; cisse, youssouf; yano, mihiro; kido, hiroshi title: influenza virus—cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza date: 2010-10-01 journal: j infect dis doi: 10.1086/656044 sha: doc_id: 306983 cord_uid: 6w2fvtfy background. severe influenza is characterized by cytokine storm and multiorgan failure with edema. the aim of this study was to define the impact of the cytokine storm on the pathogenesis of vascular hyperpermeability in severe influenza. methods. weanling mice were infected with influenza a wsn/33(h1n1) virus. the levels of proinflammatory cytokines, tumor necrosis factor (tnf) α, interleukin (il) 6, il-1β, and trypsin were analyzed in the lung, brain, heart, and cultured human umbilical vein endothelial cells. the effects of transcriptional inhibitors on cytokine and trypsin expressions and viral replication were determined. results. influenza a virus infection resulted in significant increases in tnf-α, il-6, il-1β, viral hemagglutininprocessing protease trypsin levels, and viral replication with vascular hyperpermeability in lung and brain in the first 6 days of infection. trypsin upregulation was suppressed by transcriptional inhibition of cytokines in vivo and by anti-cytokine antibodies in endothelial cells. calcium mobilization and loss of tight junction constituent, zonula occludens-1, associated with cytokineand trypsin-induced endothelial hyperpermeability were inhibited by a protease-activated receptor-2 antagonist and a trypsin inhibitor. conclusions. the influenza virus-cytokine-protease cycle is one of the key mechanisms of vascular hyperpermeability in severe influenza. cular hyperpermeability and multiorgan failure in severe influenza remains unclear. significant increases in levels of proinflammatory cytokines such as tumor necrosis factor (tnf) a, interleukin (il) 6, and il-1b (ie, cytokine storm) affect host survival both positively and negatively [5] [6] [7] . the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and multiorgan failure [8] . in addition, influenza a virus infection upregulates several cellular proteases, including ectopic trypsin [9] and matrix metalloprotease (mmp) 9 [10] . ectopic trypsin, like tryptase clara [11] , mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin [12] , which is crucial for viral entry and replication [13] [14] [15] [16] [17] and subsequent tissue damage in various organs [9, 16, 17] . influenza a virus infection significantly upregulates trypsin in endothelial cells and in hippocampal neurons [9] . because trypsin efficiently converts pro-mmp-9 to active mmp-9 [18] , induction of both proteases synergistically degrades basement membrane proteins, potentially destroying tight junctions and the blood-brain barrier, followed by multiorgan failure [19, 20] . the aim of the present study was to define the pathogenic impact of cytokine storm in influenza a virus infection and the molecular mechanisms by which proinflammatory cytokines cause vascular dysfunction in animal models and in human vein endothelial cells. the results pointed to the role of the influenza virus-cytokine-protease cycle as one of the main mechanisms of vascular dysfunction in severe influenza. specified pathogen-free 3-week-old weanling c57bl/6crslc female mice were obtained from japan slc. under ketamine anesthesia, 250 or 500 plaque-forming units (pfu) of influenza a/wsn/33(h1n1) [21, 22] in 15 ml of saline or saline alone as the vehicle was instilled intranasally in mice. mice ( ) also received inhibitors against nuclear n p 10 factor-kappa b (nf-kb), such as pyrrolidine dithiocarbamate (10 mg/kg) and n-acetyl-l-cysteine (10 mg/kg) [23, 24] , and inhibitor against activator protein 1, nordihydroguaiaretic acid (2.5 mg/kg) [25] , intraperitoneally. these inhibitors were administrated once daily for 4 days immediately after viral infection (day 0). virus titers were determined in madin-darby canine kidney cells [11] . all animals were treated in accordance with the guidelines of the animal care committee of the university of tokushima. cell culture. human umbilical vein endothelial cells (lonza) were grown using the protocol supplied by the manufacturer. the cells were infected by influenza a virus wsn at a multiplicity of infection of 0.5 or treated with recombinant human il-6, tnf-a, and il-1b (10 ng/ml of each) (peprotec) in the presence or absence of antibodies against these cytokines (abcam). evaluation of vascular permeability. vascular permeability was analyzed by the evan's blue extravasation method [26] . one hour after intraperitoneal injection of 400 ml of 2% (w/ v) evan's blue dye in saline, the whole body was perfused with saline through the cardiac ventricle. the leakage of dye was detected macroscopically and by fluorescence microscope. enzyme-linked immunosorbent assay (elisa). the levels of il-6, tnf-a, and il-1b in tissue homogenates and plasma were measured using cytokine elisa kits (bd biosciences). western blotting and gelatin zymography. tissues were homogenized with 3 volumes of tris-hcl, ph 6.8, containing 2% sodium dodecyl sulphate and 0.5 m nacl, and centrifuged at 12,000 g for 30 min. human endothelial cells were lysed in radioimmune precipitation buffer (nacalai tesque) at 4њc. these extracts (30 mg protein) were electrophoresed and transferred to polyvinylidene difluoride membranes. rabbit antizonula occludens-1, anti-occludin antibodies (zymed), and anti-actin antibody (chemicon) were used. immunoreactive bands were detected using chemiluminescence (amersham bio-sciences). for gelatin zymography, the extracts (50 mg protein) were subjected to electrophoresis on 10% gelatin zymogram gels (invitrogen) as reported previously [9] . immunohistochemical staining. immunohistochemical staining was conducted as described elsewhere [9] . lung and brain sections were reacted overnight with polyclonal antibodies against human influenza a, b virus (takara) at 4њc, washed, and then reacted for 1 h at room temperature with a biotinylated second antibody. the sections were counterstained with mayer's hematoxylin. permeability assay. human endothelial cells grown to confluence on 12-well tissue culture plates with falcon cell culture inserts (1.0 mm), were exposed to the cytokines for 12 h in the presence or absence of 50 mm of aprotinin (nacalai tesque). changes in the monolayer permeability were analyzed and quantified as clearance of fluorescein isothiocyanate-dextran from the upper chamber to lower chamber as reported previously [27] . total rna was isolated from human endothelial cells using an rneasy mini kit (qiagen) and reverse transcribed using oligo primers and superscript iii rt (gibco brl) for complementary dna synthesis. the following primer pairs were used to amplify human trypsin (hprss): hprss (forward primer [f], 5'-atccaggtgagactgggagagc-aca-3', nucleotide (nt) 222-246, and reverse primer [r], 5'-gtagaccttggtgtagactccaggc-3', nt 692-716) and those of viral ns1 as reported elsewhere [28] . rt-pcr and quantification of gene expression by real time-pcr were performed using fast start sybr green master (roche diagnostics) on an abi prism 7300 system [28] . human endothelial cells were cultured on glass chamber slides until confluence. after washing twice with calcium-and magnesiumfree phosphate-buffered saline (pbsϫ), the cells were incubated with cytokines (10 ng/ml for each cytokine) for 10 h with or without pretreatment for 30 min with 20 mm protease-activated receptor (par) 2 antagonist peptide, fsy-nh 2 [29] , or 50 mm aprotinin at 37њc. the cells were also activated with 1 mg/ml trypsin or 10 mm par-2 agonist peptide [30] for 30 min. the cells were also treated for 5 h with 10 nm calcium ionophore a23187 (calbiochem) or 2 mm cacl 2 . for imaging, 10 mm fluo-3/am (invitrogen) was introduced into the cells by incubation for 30 min. the cells were then washed twice with pbsϫ and incubated with 5 mm glucose in pbsϫ at 37њc. intracellular calcium ([ca 2+ ] i ) levels were analyzed using a confocal laser scanning microscope (model cm1900; leica). statistical analysis. results are presented as mean value ‫ע‬ standard error of the mean (from 3-5 independent experiments). differences between groups were examined for statistical significance by the paired t test or 1-way analysis of variance. the wilcoxon test for comparisons of kaplan-meier survival curves was used. a p value !.05 was considered to be statistically significant. a virus wsn to study the pathogenic effects of cytokine storm on vascular dysfunction. the levels of tnf-a and il-6 in the lungs, the site of initial virus infection, were increased persistently for 6 days, and levels of il-1b peaked at days 4-6 after infection ( figure 1a ). because these cytokine responses are associated with activation of the transcription factors nf-kb and activator protein 1 [7, [31] [32] [33] , we treated mice once daily for 4 days with anti-oxidant inhibitors: pyrrolidine dithiocarbamate and n-acetyl-l-cysteine against nf-kb activation, and nordihydroguaiaretic acid against activator protein 1 activation. pyrrolidine dithiocarbamate and nordihydroguaiaretic acid significantly suppressed the upregulation of tnf-a and il-1b ( ), and n-acetyl-l-cysteine suppressed tnf-a ( p ! .001 p ! ) and il-6 ( ) at day 4 after infection ( figure 1a ). .001 p ! .01 gelatin zymography showed upregulation of ectopic trypsin in mice lung, brain, and heart during infection for 6 days ( figure 1b ). trypsin induction was inhibited by treatment with pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, and nordihydroguaiaretic acid, probably via blockade of nf-kb and activator protein 1 binding in the promoter region of the gene (s. r. talukder, unpublished data). viral rna replication in various organs at day 4 after infection was suppressed by 11 order of magnitude by pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, and nordihydroguaiaretic acid ( figure 1c ). suppression of viral multiplication and induction of cytokines and trypsin by treatment with pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, and nordihydroguaiaretic acid significantly improved the survival of mice at day 14 after infection (ie, the late stage of infection) ( figure 1d ). the kinetics of viral replication monitored by viral ns1 gene showed that the level of viral rna was the highest at day 4 after infection and decreased at day 6 in these organs (figure 2a ). to determine the pathogenesis of tissue injury, the viral protein accumulation in the lung and brain at day 4 after infection was analyzed by immunohistochemical staining ( figure 2b ). viral protein was detected in alveoli and terminal bronchioles in the lung and was also detected in the brain, particularly in the hippocampus, neocortex, brainstem, and brain capillaries. vascular hyperpermeability is one of the main complications of organ injury in severe influenza. vascular permeability was analyzed by infiltration of evans blue dye in the lung and brain after infection ( figure 2c and 2d) . in contrast to no dye infiltration in uninfected animals, infected mice showed a progressive increase in vascular permeability in the lung and brain at day 4 after infection. fluorescence microscopy showed leakage of dye from the blood vessels in these organs ( figure 2d ). to elucidate the mechanisms underlying vascular dysfunction in the brain, changes in the levels of tight-junction proteins, intracellular zonula occludens-1 and transmembrane occludin, and the matrix protein laminin were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day 4 after infection, which were partly rescued by pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, or nordihydroguaiaretic acid ( figure 2e ). no other tight-junction protein, claudin-5, or matrix fibronectin and type iv collagen was affected (data not shown). to clarify the linkage between upregulated cytokines and trypsin and vascular hyperpermeability after viral infection, the relationships among these findings were examined in human endothelial cells. viral infection significantly increased tnf-a and il-6 levels (2.7fold and 7.1-fold, respectively) but not il-1b levels in the culture media in a time-dependent manner over a 24-h period ( table 1) . influenza a virus infection upregulated human trypsin/ hprss gene by approximately 2-fold in the cells after infection for 6-12 h ( figure 3a ). to analyze the linkage between cytokines and trypsin in the cells, changes in the expression of hprss gene were analyzed after exposure to 10 ng/ml tnfa, il-6, and il-1b instead of viral infection for 6 h ( figure 3b ). all tested cytokines tended to upregulate hprss expression levels, especially tnf-a ( ) and il-1b ( ), although p ! .01 p ! .05 less effectively than did viral infection, and the upregulation was inhibited by simultaneous treatment of the respective neutralizing antibodies (100 ng/ml) with these cytokines (p ! for tnf-a; for il-1b). .05 p ! .01 treatment of the cells for 12 h with tnf-a, il-6, and il-1b markedly suppressed tight-junction protein zonula occludens-1 levels and occluding levels slightly, and loss of these proteins were abrogated by simultaneous treatment of the cells with 50 mm of the nonpermeable trypsin inhibitor aprotinin ( figure 4a ). furthermore, cytokine treatment disrupted the continuous and linear arrangement of zonula occludens-1 among the cells, and aprotinin inhibited the disruption ( figure 4b ). accordingly, treatment with cytokines, especially il-1b and tnf-a, tended to increase endothelial cell monolayer permeability and this effect was blocked by 50 mm of aprotinin ( ) ( figure p ! .05 4c) . these findings suggest that cytokines upregulate trypsin in vascular endothelial cells and that secreted trypsin plays an (original magnification, ϫ200) . b, immunoreactive deposits in the lung (original magnification, ϫ200). c, viral antigen (arrowheads) in epithelial cells of respiratory bronchioles and infiltrated leukocytes in alveoli (original magnification, ϫ400). d, no immunoreactive deposits in the brain before infection (original magnification, ϫ200). e, virus antigen in the cornu ammonis (ca) 1 and ca-2 and in the stratum granulosum of the dentate gyrus (dg) of the hippocampus (original magnification, ϫ200). f, virus antigen (arrowheads) in the enlarged image of ca-1 (original magnification, ϫ400). scale bars are 100 mm.c, vascular permeability in the lung and brain analyzed by evan's blue dye extravasation before (wsn-d0) and after infection at day 4 (wsn-d4). d, fluorescent micrographs of evan's blue leakage from capillaries in the brain and lung before and after infection at day 4. e, loss of tight-junction proteins, zonula occludens (zo) 1 and occludin, and laminin in the brain analyzed by western immunoblotting at day 4 after infection and its restoration by pyrrolidine dithiocarbamate (pdtc), n-acetyl-l-cysteine (nac), and nordihydroguaiaretic acid (ndga) treatments. the levels before infection are shown as control (ctr). important mechanistic role in the loss of zonula occludens-1 and increased permeability. 2+ ] i through g protein-coupled receptors, leading to cytoskeletal reorganization in the microvascular endothelium and consequent increase in permeability and tissue edema [34] . trypsin receptor par-2 is a g protein-coupled receptor activated by trypsin and tryptase and plays an important role in increasing [ca 2+ ] i [35] . to investigate the mechanisms underlying vascular hyperpermeability in severe influenza, we treated human endothelial cells with cytokines, trypsin, and par-2 agonist peptide in pbsϫ and then measured [ca 2+ ] i ( figure 5 ). marked [ca 2+ ] i mobilization was found after treatment of the cells with trypsin, par-2 agonist peptide, and cacl 2 for 10 h, whereas calcium ionophore a23187 decreased [ca 2+ ] i . treatment with tnf-a, il-1b, and il-6 also increased [ca 2+ ] i , and the mobilization was suppressed by pretreatment of the cells with par-2 antagonist, fsy-nh 2 , or aprotinin for 30 min. these results suggest that [ca 2+ ] i mobilization by proinflammatory cytokines through activation of trypsin and its receptor par-2 is one of the main mechanisms underlying increased endothelial cell permeability. the present study reports several new observations: (1) proinflammatory cytokines, tnf-a, il-1b, and il-6, when upregulated by influenza a virus infection, induce trypsin expression in various organs and human endothelial cells; (2) the upregulated trypsin induces [ca 2+ ] i mobilization via activation of the par-2, followed by loss of zonula occludens-1 and vascular hyperpermeability; (3) inhibitors of nf-kb and activator protein 1 effectively suppress the upregulation of proinflammatory cytokines and trypsin and improve the survival rates of infected mice. based on these results, we propose the influenza viruscytokine-protease cycle hypothesis as one of the mechanisms of vascular dysfunction in multiorgan failure with cytokine storm in severe influenza and influenza-associated encephalopathy ( figure 6 ). the significance of proinflammatory hypercytokinemia, or cytokine storm, in the pathogenesis of influenza a virus infigure 6 . the hypothesis of influenza virus-cytokine-protease cycle, which may affect the pathogenesis of vascular hyperpermeability and tissue destruction in severe influenza. ap-1, activator protein 1; bbb, blood-brain barrier; par-2, protease-activated receptor 2; zo-1, zonula occludens-1. fection remains unclear. the positive effects include that cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, the negative effects of excess cytokines include the fact that the hyperinflammatory process evoked by viral infection [8, 36] may become harmful through intracellular activation of nf-kb, activator protein 1, and the janus kinase-signal transducers and activators of transcription signaling pathways [31-33, 37, 38] . the in vivo experiments presented here showed that nf-kb and activator protein 1 inhibitors markedly suppress the expression of cytokines and trypsin, viral replication, and endothelial dysfunction and result in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production and activation of vasodilatory mediators, such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs [8] (figure 6 ). the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyperpermeability fol-lowing the cytokine storm remain unclear. tnf-a upregulation alters the cellular redox state, reduces the expression of 4 complex i subunits by increasing mitochondrial o 2 ϫ production and depleting adenosine triphosphate (atp) synthesis, decreases oxygen consumption (resulting in mitochondrial damage) [8, 39] , and increases [ca 2+ ] i [40] . atp depletion dissociates zonula occludens-1 from the actin cytoskeleton and thereby increases junctional permeability [41] . the present results allow us to propose a new mechanism of junctional permeability regulation: upregulated trypsin by influenza a virus and/or proinflammatory cytokines induces increase in [ca 2+ ] i and loss of zonula occludens-1 in endothelial cells via par-2 signaling. in contrast to the marked upregulation of cytokines in the lungs ( figure 1a) , upregulation of cytokines in the brain was mild (data not shown), which suggests that the bloodbrain barrier destruction is the result of systemic effects of cytokines produced in the lung in severe influenza. anti-cytokine antibodies and trypsin inhibitors may effectively suppress junctional permeability. endothelial dysfunction induced by the influenza virus-cy-tokine-protease cycle in the early stage of severe influenza may also affect various circulating factors, coagulation factors, and complement systems, as well as vascular interacting cells, such as neutrophils, macrophages, and lymphocytes. multiorgan failure is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h5n1 and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and the formation of oxidized phospholipids, which induce lung injury via toll-like receptor 4 signaling pathway [42] . in addition to these data, upregulated trypsin and proinflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of influenza a virus infection. further studies are required on the role of the influenza virus-cytokine-protease cycle in the pathogenesis of multiorgan failure, 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weseslindtner, lukas title: performance of severe acute respiratory syndrome coronavirus 2 antibody assays in different stages of infection: comparison of commercial enzyme-linked immunosorbent assays and rapid tests date: 2020-05-30 journal: j infect dis doi: 10.1093/infdis/jiaa305 sha: doc_id: 334988 cord_uid: brumg6jh we comparatively assessed sensitivities and specificities of 4 commercial enzyme-linked immunosorbent assays (elisas) and 2 rapid tests in 77 patients with polymerase chain reaction–confirmed severe acute respiratory syndrome coronavirus 2 infection, grouped by interval since symptom onset. although test sensitivities were low (<40%) within the first 5 days after disease onset, immunoglobulin (ig) m, iga, and total antibody elisas increased in sensitivity to >80% between days 6 and 10 after symptom onset. the evaluated tests (including igg and rapid tests) provided positive results in all patients at or after the 11th day after onset of disease. the specificities of the elisas were 83% (iga), 98% (igg), and 97% (igm and total antibody). we comparatively assessed sensitivities and specificities of 4 commercial enzyme-linked immunosorbent assays (elisas) and 2 rapid tests in 77 patients with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection, grouped by interval since symptom onset. although test sensitivities were low (<40%) within the first 5 days after disease onset, immunoglobulin (ig) m, iga, and total antibody elisas increased in sensitivity to >80% between days 6 and 10 after symptom onset. the evaluated tests (including igg and rapid tests) provided positive results in all patients at or after the 11th day after onset of disease. the specificities of the elisas were 83% (iga), 98% (igg), and 97% (igm and total antibody). keywords. sars; coronavirus; antibodies; immunoassay. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a new betacoronavirus, is currently causing a massive pandemic with severe consequences for the health-care systems worldwide [1, 2] . although polymerase chain reaction (pcr)based tests quickly became the cornerstone of sars-cov-2 diagnosis, the potential of antibody tests has not been comprehensively evaluated. depending on respective infection stages, antibody assays could nonetheless significantly complement pcr-based testing [3, 4] . multiple commercial enzyme-linked immunoassays and rapid tests (lateral flow immunoassays) have recently become available, but their diagnostic ability has to be thoroughly evaluated and compared before they can be widely used in the clinical setting [5, 6] . in the current study, we compared the diagnostic ability of 4 enzyme-linked immunosorbent assays (elisas), which assess sars-cov-2-specific antibodies of different immunoglobulin (ig) classes (euroimmun sars-cov-2 iga and igg and wantai sars-cov-2 igm and total antibody), and 2 rapid tests (wantai sars-cov-2 ab rapid test and hangzhou alltest biotech 2019-ncov igg/igm rapid test) in 77 patients with symptomatic sars-cov-2 infection. the study included serum/plasma samples from 77 symptomatic patients with acute sars-cov-2 infection (29 female, 48 male, median age, 63 years; age range, 15-92 years; 1 sample per patient) diagnosed by means of positive pcr from nasopharyngeal swab/respiratory secretion samples. in addition to swab/ respiratory secretion samples, these serum/plasma samples were sent to the center for virology of the medical university of vienna between 27 february and 30 march 2020 for diagnostic testing and were subgrouped for this study by the interval between blood sample collection and initial onset of symptoms, as reported by the patients. occurrence of symptoms was evaluated using a study protocol based on the world health organization guidelines for diagnosing coronavirus disease 2019. the majority of patients reported fever, cough, headache, and general weakness. notably, many patients did not have the subjective feeling of dyspnea, although hypoxemia was diagnosed. written consent was obtained from the patients, and the study was approved by the ethics committee of the medical university of vienna (approval nos. ek 2156/2019 and ek 2283/2019). serum samples from 100 individuals without sars-cov-2 infection (60 female, 40 male; median age, 49 years; range, 2-93 years) served as controls. they comprised (1) symptomatic individuals whose samples were obtained during the same observational period as samples from infected patients, but in whom absence of sars-cov-2 was confirmed by pcrnegative swab samples (n = 30); (2) healthy volunteers with consecutive pcr-negative swab samples (n = 30); (3) stored serum samples from individuals with previous pcr-confirmed coronavirus oc43 infections (n = 10; median interval between infection and sampling, 306 days; range, 4-1452 days) and (4) serum samples from patients with pneumonia collected before december 2019 (n = 30). sars-cov-2 nucleic acid was extracted using the nuclisens easymag extractor, according to the manufacturer's instructions (biomerieux). sars-cov-2 real time taqman pcr was performed with world health organization-recommended primers and probe located in the e-gene, as described elsewhere [1] . sensitive detection was confirmed using a proficiency panel from instand. anti-sars-cov-2 antibodies were assessed using (1) euroimmun sars-cov-2 iga and igg elisas (euroimmun), (2) wantai sars-cov-2 igm and total antibody elisas (beijing wantai biological pharmacy), (3) the wantai sars-cov-2 ab rapid test (also from beijing wantai), and (4) the 2019-ncov igg/igm rapid test (hangzhou alltest biotech). all tests were performed as recommended by the manufacturers [3, 4] . the euroimmun sars-cov-2 iga and igg elisas use the recombinant structural protein (s1 domain) of the spike protein as antigen. the wantai sars-cov-2 total antibody elisa is based on a double-antigen sandwich principle that detects total antibodies against the sars-cov-2 spike protein receptor binding domain. the recombinant protein is used as the immobilized and the horseradish peroxidase-conjugated antigen. the igm elisa uses the same horseradish peroxidase-conjugated receptor binding domain antigen as the total antibody elisa. results by euroimmun elisa (iga and igg) were classified as negative when the antibody ratio was <0.8, as borderline with a ratio from 0.8 to 1.1, and as positive with a ratio >1.1. wantai elisa results (igm, total antibody) were interpreted as negative with a ratio <0.9, borderline with a ratio from 0.9 to 1.1, and positive with a ratio >1.1. rapid tests were filled with 10 μl of serum/plasma using a pipette and interpreted by the same laboratory-experienced person after incubation for 10 minutes (2019-ncov igg/igm rapid test) or 15 minutes (wantai rapid test). all performed rapid tests provided positive control bands and were considered valid. in some patients, test bands were weak (with weaker band intensities than clearly positive tests with strong bands) or very weak (bands even weaker, but still recognizable with the naked eye). all tests with (still) visible bands were considered positive. of the 77 patients with pcr-confirmed sars-cov-2 infection, 30 individuals (12 female, 18 male; median age, 58 years; age range, 15-83 years) provided serum/plasma samples that were obtained at symptom onset or 1-5 days after the onset of disease (group 1). fifteen of these patients (50%) were hospitalized owing to moderate or severe illness severity, and 15 (50%) were dismissed to home care. from 25 patients (9 female, 16 male; median age, 68 years; range, 22-92 years), serum/plasma samples were obtained between 6 and 10 days after onset of disease (group 2). twenty-three of these patients (92%) were hospitalized, and 2 (8%) were dismissed to home care after blood sample collection. finally, 22 patients (4 female, 18 male, median age, 64 years, range, 26-79 years) provided a serum/ plasma sample at or after day 11 after the onset of symptoms (group 3). the median interval between onset of symptoms and sample acquisition in these patients was 15 days (range, 11-29 days). except for 1 individual (a healthcare worker identified by a screening test), blood samples were obtained from all patients during hospitalization (95.4%). virus concentration in nasopharyngeal swab/respiratory secretion samples differed significantly among these groups ( as shown in figure 1a , the euroimmun iga and igg elisas tested positive in 9 (30%) and 1 (3.3%) of the 30 individuals from group 1, in 21 (84%) and 10 (40%) of the 25 from group 2, and in all 22 patients (100%) from group 3. the wantai igm and total antibody elisas tested positive in 8 (26.7%) and 11 (36.7%) of the 30 individuals from group 1. both tests provided positive results in 23 of 25 patients (92%) from group 2 and in all 22 (100%) from group 3 ( figure 1a ). individual antibody concentrations among the different groups are also shown in figure 1a . the wantai rapid test tested positive in 6 of 30 individuals (20%) from group 1, in 20 of 25 (80%) from group 2, and in all 22 (100%) from group 3 ( figure 1b figure 2 , test specificities were determined in 100 non-sars-cov-2-infected controls. specificities were 83% and 98% for the euroimmun iga and igg and 97% for the wantai igm and the total antibody elisas, respectively (figure 2a) . the wantai rapid test displayed a specificity of 98%, and the 2019-ncov igg/igm rapid test a specificity of 99% for igm and 100% for igg, respectively ( figure 2b ). the current study provides the first comparative data on the sensitivity and specificity of 4 commercially available elisas and 2 rapid tests in 77 patients with sars-cov-2 infection. we demonstrate that the sensitivities of the evaluated anti-sars-cov-2 igm and iga elisas were low within 5 days after disease onset but subsequently increased to 84% for the euroimmun iga and 92% for the wantai igm elisa between 6 and 10 days after onset of symptoms [3, 7, 8] . we furthermore observed very high sensitivities for all tests (including igg and total antibody elisas) in the later phase of infection (beyond the 11th day after onset of symptoms, with a median interval of 15 days between onset of symptoms and sample acquisition). of note, all samples from the later phase of the infection displayed significant igm, iga, and igg titers (exceeding the respective cutoffs), and the majority (86%) of these samples were obtained within 21 days after the onset of disease (maximum, 29 days). however, although test specificities for igm, igg and total antibodies were ≥97%, specificity for the iga assay was only 83%. although data provided by the study indicate that the evaluated igm or iga assays should not substitute for pcr-based diagnosis early after onset of symptoms, the high sensitivities we demonstrate for all evaluated tests beyond the 11th day after symptom onset highlights the possibility that these assays might significantly aid the diagnosis in later stages of infection-for example, in patients with pneumonia who have lower virus concentrations in the upper respiratory tract (possibly causing false-negative pcr results from pharyngeal swab samples) [9] . although we obtained comparable results for the rapid tests we evaluated, it should be considered that these tests were performed under optimal laboratory conditions (with pipetting of exact serum volumes and interpretation by an experienced laboratory technician under the same conditions), which might not necessarily reflect their ability in the point-of-care setting. our observation, however, also indicates that rapid tests from different manufacturers may differ significantly in their diagnostic performance, especially in early stages of infection, and should therefore be particularly evaluated [1, 5] . importantly, the majority of patients in our cohort who provided serum/plasma samples during the later phase of infection (at or beyond the 11th day after onset of disease) were hospitalized at this time point. because antibody titers have been shown to be correlated with disease severity, the sensitivity of the evaluated tests could thus differ significantly in individuals with mild or asymptomatic courses of infection, calling for further studies with asymptomatic individuals and patients with mild disease [3, 7] . high antibody levels, which we observed in this cohort of mainly hospitalized patients during the late phase of the infection, might also have affected the good test performance of the rapid tests we evaluated. in summary, although our study has the limitation of a relatively small sample size, it nonetheless provides comparative data on the early available commercial elisas, indicating a high potential of the evaluated tests for sars-cov-2 diagnosis, especially in symptomatic patients and progressed stages of infection. financial support. this work was supported by the medical scientific fund of the mayor of the city of vienna. potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr note from the editors: world health organization declares novel coronavirus (2019-ncov) sixth public health emergency of international concern antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients performance of vivadiag covid-19 igm/igg rapid test is inadequate for diagnosis of covid-19 in acute patients referring to emergency room department development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis virological assessment of hospitalized patients with covid-2019 profiling early humoral response to diagnose novel coronavirus disease (covid-19) temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study key: cord-334242-m5dr19v4 authors: teran, luis m.; seminario, maria cristina; shute, janis k.; papi, alberto; compton, steven j.; low, j. lorraine; gleich, gerald j.; johnston, sebastian l. title: rantes, macrophage-inhibitory protein 1α, and the eosinophil product major basic protein are released into upper respiratory secretions during virus-induced asthma exacerbations in children date: 1999-03-01 journal: j infect dis doi: 10.1086/314618 sha: doc_id: 334242 cord_uid: m5dr19v4 the presence of cytokines and the toxic eosinophil granule product major basic protein (mbp) was investigated in nasal aspirates from children with naturally occurring virus-induced asthma exacerbations and compared with levels in nasal aspirates taken from the same children when asymptomatic. increased levels of mbp accompanied by increased levels of the chemokines rantes and macrophage-inhibitory protein 1α were observed in nasal aspirates from children during the virus-induced exacerbations. granulocyte-macrophage colony-stimulating factor was mostly undetectable in samples obtained during both symptomatic and asymptomatic periods. interleukin-5 levels were low, but tended to increase in samples from symptomatic children. these data confirm that the eosinophil product mbp and the eosinophil chemoattractant chemokines rantes and macrophage-inhibitory protein 1α are increased in upper respiratory viral infections associated with asthma exacerbations and suggest an important role for these chemokines in regulating eosinophil influx and activation. these chemokines may represent targets for therapeutic intervention in virus-induced asthma exacerbations. granules such as major basic protein (mbp) and eosinophil cationic protein (ecp) . we recently showed that naturally occurring upper respiratory viral infections in asthmatic children induce the release of interleukin (il)-8 into nasal secretions and that il-8 is associated with the presence of neutrophil-derived proteins, suggesting that il-8 may be important in neutrophil recruitment and activation [5] . however, no studies have investigated the possible role of eosinophils in the pathogenesis of naturally occurring asthma exacerbations associated with respiratory viral infections. because molecules promoting eosinophil recruitment and activation are likely targets for the development of new therapies for virus-induced asthma, we investigated whether eosinophil chemoattractants, such as granulocyte-macrophage colony-stimulating factor (gm-csf) and il-5, and the chemokines rantes and macrophage-inhibitory protein 1a (mip-1a) [6] [7] [8] are implicated in eosinophil recruitment and activation in naturally occurring upper respiratory viral infections in children with acute asthma exacerbations. clinical samples. diluted nasal aspirate samples were taken from children in the southampton area during acute naturally occurring colds associated with exacerbations of asthma as part of another study of 108 children aged 9-11 years [1] . for the present study, we obtained 35 nasal aspirate samples during periods of acute asthma exacerbation from 26 children (16 boys, 10 girls; 14 atopic, 12 nonatopic). we obtained another 26 samples when the children were asymptomatic. the "acute exacerbation samples" were selected if the child had a proven viral infection and sufficient nasal aspirate remained in both acute and asymptomatic samples after completion of the previous study for the planned assays. methods of sample collection have been described [1] . virus detection methods. rhinoviruses were detected by polymerase chain reaction (pcr) as previously reported [1] . we detected other respiratory viruses by cell culture on five cell lines, immunofluorescence microscopy, and serology. differentiation of rhinoviruses from enteroviruses was done by acid lability testing [9] . full details of the methods have been published [1] . cytokine and mbp measurements. we measured rantes, mip-1a, and il-5 in nasal aspirates by elisa according to the manufacturer's protocol (r&d systems, abingdon, uk). the lower limit of detection of rantes, mip-1a, and il-5 in nasal aspirates was 10, 15, and 1 pg/ml, respectively. in the gm-csf elisa, we used paired antibodies specific for gm-csf from pharmingen (cambridge, uk). the elisa was performed ac-cording to the manufacturer's protocol. the lower limit of detection was 8 pg/ml. eosinophil mbp in nasal aspirates was measured in duplicate using a two-site ria as previously described [10] . the lower limit of detection for mbp was 8.8 ng/ml. statistical analysis. we used the mann-whitney u test to examine differences in cytokine levels in nasal aspirate samples obtained when symptomatic and asymptomatic, and to examine rantes and mip-1a levels between atopic and nonatopic children in symptomatic samples. spearman rank correlations were used to examine the relationships between mbp, rantes, and mip-1a levels and respiratory symptom scores during an acute asthma exacerbation. cytokine and mbp levels were reported and analyzed as measured in the diluted nasal aspirate samples. the 26 children had 35 acute asthma exacerbations. rhinoviruses were detected in 25 (71%), influenza virus type a in 5 (14%), parainfluenza virus type 2 in 3 (9%), adenovirus in 1 (3%), and coronavirus 229e in 1 (3%). rhinovirus was also detected in 2 asymptomatic samples, although only by very weak pcr signals. significant episodes of upper respiratory symptoms were reported in 33 (94.2%) of the 35 exacerbations. lower respiratory episodes characterized by cough, wheeze, or shortness of breath of at least moderate severity were recorded in 25 (71%), and 20 (57%) had a significant drop in peak flow (pef). all episodes were defined as previously reported [1] . the median maximal percentage fall in pef from baseline during these episodes was 33% (equivalent to a fall 1102 l/min [1] ). figure 1 shows symptoms, pef, and viruses identified in 2 study children to illustrate the nature of the clinical episodes analyzed in this study. rantes and mip-1a levels in nasal aspirate samples. levels of rantes and mip-1a in samples obtained during periods of acute exacerbation were significantly higher (median, 43 pg/ml [range, !10-1185] and 123 pg/ml [range, !15-2500], respectively) than samples obtained when the children were asymptomatic (median !10 pg/ml [range, !10-55] and !15 pg/ ml [range, 29.2-280.0], respectively; ; figure 2). when p ! .001 samples from atopic and nonatopic children were compared, concentrations of rantes and mip-1a were 3-fold higher in nasal aspirates from atopic children than from nonatopic children during acute exacerbations of asthma (median rantes levels: 101.3 vs. 33 pg/ml, respectively; median mip-1a: 123 vs. 39 pg/ml, respectively); however, this difference did not reach statistical significance. gm-csf, il-5, and mbp levels in nasal aspirates. concentrations of gm-csf ( ), mbp ( ), and il-5 n ϭ 23 n ϭ 13 ( ) were measured in nasal aspirates if sufficient material n ϭ 11 remained. gm-csf concentrations were mostly undetectable in samples from both symptomatic and asymptomatic children, and median values were below the limit of detection (8 pg/ml) of the gm-csf elisa. il-5 levels tended to increase in the nasal aspirates of symptomatic when compared with asymptomatic children (median, 1.5 pg/ml [range, !1.0-31.4] vs. !1.0 pg/ml [range, !1.0-12.1]); however, this difference failed to reach statistical difference ( ). p ϭ .1 mbp levels were significantly higher in samples from acute exacerbation episodes (median, 60 ng/ml; range, !8.8-335) than from asymptomatic periods (median, !8.8 ng/ml; range, !8.8-212; ; figure 2 ). there were no significant corre-p ! .001 lations between mbp and rantes levels ( , , r ϭ .11 p ϭ .7 ) or between mbp and mip-1a levels ( , , n ϭ 13 r ϭ .43 p ϭ .14 ) in nasal samples from periods of acute exacerbation. n ϭ 13 correlation of mbp levels with respiratory symptoms. to investigate the possible relationship between eosinophil influx and activation and local respiratory symptoms, we sought correlations between the levels of mbp and severity (as previously defined [1] ) of upper respiratory tract symptoms recorded during the episode. there was no significant correlation between the severity of upper respiratory symptoms and mbp levels ( , , ) . similarly, no significant correlation r ϭ ϫ.2 p ϭ .4 n ϭ 13 was found between the severity (also previously defined [1] ) of lower respiratory symptoms and mbp concentrations (r ϭ , , ). .24 p ϭ .5 n ϭ 9 in this study, we observed increased levels of the eosinophil chemoattractants rantes and mip-1a and of the eosinophil product mbp in nasal aspirates from children during periods of proven virus-induced asthma exacerbations. these data implicate these chemokines in eosinophil recruitment and acti-vation in virus-induced asthma and suggest that they or their receptors may represent promising targets for the development of therapies for virus-induced asthma. respiratory syncytial virus induces selective production of the rantes by upper airway epithelial cells in vitro [11] . in the present study, we showed that viral infection of the upper respiratory tract induces release of the chemokines rantes and mip-1a and release of the eosinophil product mbp in vivo. this is of interest, as both of these chemokines are eosinophil chemoattractants. while mip-1a is less potent than rantes as an eosinophil chemoattractant, it is an important mediator in the inflammatory response to viral infection [12] . furthermore, in a mouse model, absence of ccr1, which binds mip-1a most strongly, resulted in reduced host defense to infection and a tilt in favor of a th2-type response to infection [13] . given the role of th2 cells in asthma, these properties suggest that both mip-1a and rantes may be important mediators of virus-induced asthma exacerbations. this observation is supported by the finding that levels of these cytokines in atopic children were 3-fold greater than in nonatopic children. we have observed increases in chemokine and mediator levels in the upper airway in children with simultaneous virus-related colds and asthma exacerbations. the known properties of these chemokines strongly suggest that they are likely to be important in eosinophil recruitment and activation in the upper airway, and therefore by implication, are likely to be important in the eosinophil recruitment and activation known to be important in the pathogenesis of virus-induced asthma exacerbation. these data suggest that studies investigating the importance of these chemokines in the lower airway should be done to test this hypothesis. the lack of correlation between chemokine levels and either eosinophil mediator levels or respiratory symptoms raises some doubts about the relevance of these chemokines to eosinophil recruitment and activation in the airway. however, such correlations are difficult to find in clinical studies where many variables may affect the strength of a relationship between two biologic measurements. for example, other chemokines such as eotaxin or il-8 may also be involved, perhaps in concert with rantes and mip-1a. furthermore, the finding of a correlation is still not proof of a causal relationship between two variables. in our view, the known biologic functions of these chemokines and the presence of increased eosinophil mediators is sufficient to support our hypothesis that these chemokines are important in eosinophil recruitment and activation in vivo. further studies with chemokine antagonists will be required to test the strength of this relationship. the cytokines and the eosinophil product mbp detected in the nasal aspirates could be derived from local production or from serum via vascular leakage. we believe that the high levels detected (especially considering that mucus was diluted 20-40 times before storage and analysis) suggest that local production predominates. gm-csf and il-5 are two other cytokines that play an important role in leukocyte trafficking. gm-csf is chemotactic for both neutrophils and eosinophils, while il-5 is important in eosinophil hematopoiesis, priming, and chemoattraction [6, 7] . concentrations of gm-csf in nasal secretions were mostly undetectable, suggesting that this cytokine plays a minor role in the pathogenesis of naturally occurring virus-induced asthma exacerbations. this finding is consistent with previous reports showing that gm-csf plays a minor role in upper respiratory viral infections [14, 15] . in a small group of children from whom enough clinical material remained, we also investigated the presence of il-5. although levels of il-5 tended to be increased in the nasal aspirates from symptomatic children, there were no significant differences between symptomatic and asymptomatic children. the role of il-5 in viral exacerbations of asthma must be further studied in greater numbers of children. in summary, we found increased levels of the chemokines rantes and mip-1a in nasal secretions during exacerbations of asthma associated with upper respiratory viral infection in children compared with asymptomatic samples from the same children. gm-csf was not detectable in most nasal aspirates, but il-5 concentrations tended to increase in samples from symptomatic children. this study also found increased levels of the eosinophil granule protein mbp during virus-induced exacerbations of asthma. these findings suggest that the development of new drugs to neutralize the effects of rantes and mip-1a on eosinophils may affect virus-induced asthma exacerbations. community study of role of virus infections in exacerbations of asthma in 9-11 year old children respiratory viruses and exacerbations of asthma in adults lower airways inflammation during rhinovirus colds in normal and asthmatic subjects a common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects role of nasal interleukin-8 in neutrophil recruitment and activation in children with virus-induced asthma neutrophil and eosinophil chemotaxins in asthma recombinant human interleukin-5 is a selective eosinophil chemoattractant the chemokines: their potential role in allergic inflammation diagnostic procedures for viral, rickettsial and chlamydial infections analysis of pregnancy-associated major basic protein levels throughout gestation respiratory syncytial virus induces selective production of the chemokine rantes by upper airway epithelial cells requirement of mip-1a for an inflammatory response to viral infection impaired host defense, hematopoiesis, granulomatous inflammation and type 1-type 2 cytokine balance in mice lacking cc chemokine receptor 1 nasal cytokines in common cold and allergic rhinitis nasal cytokine production in viral acute upper respiratory infection of children we thank philip pattemore, gwen sanderson, and sandy smith for help with sample collection and virus detection. key: cord-339271-t7cxqkp1 authors: pan, yanfeng; yu, xue; du, xinwei; li, qingqing; li, xianyang; qin, tao; wang, miaomiao; jiang, minlin; li, jie; li, weiguo; zhang, qian; xu, zhiwei; zhang, lu title: epidemiological and clinical characteristics of 26 asymptomatic sars-cov-2 carriers date: 2020-04-22 journal: j infect dis doi: 10.1093/infdis/jiaa205 sha: doc_id: 339271 cord_uid: t7cxqkp1 background: we retrospectively analysed 26 persistently asymptomatic severe acute respiratory syndrome coronavirus 2 (sars-cov-2) carriers. methods: epidemiological and clinical characteristics from the 26 asymptomatic patients with positive results for sars-cov-2 rna testing were obtained. results: twenty-two patients (84.6%) correlated with clustering occurrence. the median period from contact to diagnosis and the last positive nucleic acid test was 19 (8–24 days) and 21.5 days (10–36 days), respectively. the median period from diagnosis to negative nucleic acid test was significantly different between patients with normal or atypical chest computed tomography (ct) findings (n=16, 61.5%; 7.5 days [2–20 days]) and patients with typical ground-glass or patchy opacities on ct(n=10, 38.5%; 12.5 days[8–22 days]; p<0.01). seven patients (70.0%) with initial positive nucleic acid test results had a negative result simultaneously with improved ct findings. obvious improvement in ct findings was observed in three patients (30.0%) despite positive nucleic acid test results. conclusion: in asymptomatic patients, changes in biochemical and inflammatory variables are small and changes on chest ct can occur. it is worth noting the long existence of sars-cov-2 in some asymptomatic patients and false-negative results need to be considered in sars-cov-2nucleic acid test. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the causation agent of a novel respiratory infectious coronavirus disease (termed covid-2019), which has disseminated quickly among people. since its initial outbreak in wuhan, china was reported in december 2019, there have been 1279722 cases worldwide up to april 7, 2020 [1, 2] . covid-19 is highly contagious with rapid transmission, and people are general susceptible to this new coronavirus. patients with sars-cov-2 infection may show covid-19-related symptoms. asymptomatic carriers have also been found [3] . for instance, there are reports documenting a substantial fraction of truly asymptomatic individuals with covid-19 from the diamond princess cruise ship (approximately 18%) and japanese evacuees out of wuhan [4, 5] . there are a few other case reports in various journals in table 1 [3, [6] [7] [8] [9] [10] . these reports mainly focus on the estimated asymptomatic proportion, mode of transmission, some radiological findings, and solutions to identify and isolate asymptomatic patients. emerging evidence suggests that patients with clinical manifestations are contagious, with analysis of their clinical characteristics [11, 12] . however, there are few studies reported the epidemiological and clinical characteristics of asymptomatic patients. a few studies have described the clinical characteristics of asymptomatic patients, but some of the asymptomatic patients in these studies were asymptomatic at the time of diagnosis but later developed symptoms, so they cannot truly represent the clinical characteristics of asymptomatic patients [13, 14] . here, we identified a total of 26 persistently asymptomatic patients with positive test results for sars-cov-2 nucleic acid to determine the clinical characteristics and asymptomatic carrier transmission of covid-19 infection. a total of 26 hospitalized patients with a sars-cov-2 epidemiological history and positive sars-cov-2 nucleic acid test results were identified to analyze the epidemiological and clinical characteristics of covid-19infected asymptomatic carriers. these patients' sputum and throat swab were sampled for sars-cov-2 confirmation and tested using reverse transcription polymerase chain reaction (rt-pcr). an epidemiological history was defined as a close contact with a diagnosed or possible case of covid-19 or had a history of residence in or travel to wuhan. informed consent was obtained from each registered patient and this study was reviewed and approved by the medical ethics committee of zhengzhou university. a c c e p t e d m a n u s c r i p t 5 detailed information regarding the epidemiological history and information of other family members of each patient was obtained. we also collected information regarding patient age, sex, previous medical history, clinical manifestations, laboratory tests, chest computed tomography (ct) scans, treatment measures, and prognosis. normally distributed continuous variables are presented as mean ± standard deviation (sd) and were compared using the t-test. non-normally distributed data are described as the median and were analyzed using the rank sum test of two groups. categorical variables are show as percentages. all statistical analysis was performed using the spss software version 22.0. a total of 26 patients with asymptomatic sars-cov-2 infection were collected in this study, of whom 16 (61.5%) were men and 10 (38.5%) were women. the median age was 29.5 years (2 to 80 years) and the age of 18 patients (69.2%) ranged from 20 to 60 years. two patients had a history of smoking (both for over 20 years), with an average of 20 cigarettes per day. two patients had a history of hypertension and one had coronary heart disease. regarding the source of the infection, six patients (23.0%) has a history of travel to wuhan, with no clear contact with a source of infection. exact contacts with confirmed or possible patients were found in eighteen cases (69.2%), and the contact history was unknown in two patients (7.8%). among patients with a clear source of infection, 10 cases (55.6%) had previously talked to or had a meal with a confirmed covid-19 patient, and eight cases (44.4%) lived with a confirmed patient. the sources of infection in these cases included confirmed patients with clinical symptoms (n=16, 88.8%), a possible covid-19 patient with clinical symptoms (n=1, 5.6%), and a confirmed patient without clinical symptoms (n=1, 5.6%). in addition, 22 cases (84.6%) were a c c e p t e d m a n u s c r i p t 6 found to be correlated with a local cluster. the close contacts of these 26 enrolled patients were traced, and only two sars-cov-2-infected patients were identified. in the six patients with a recent history of travel to wuhan, the median period from leaving wuhan to a covid-19 diagnosis was 15 days (14-19 days). in 18 patients with a clear source of infection, the median period from contact with a confirmed or possible case to the confirmed to diagnosis was 19 days (8-24 days). this period was longer than 14 days in 14 cases (77.8%) and over 20 days in four cases (22.2%) ( figure 1 ). the initial leukocyte counts were slightly lower than the normal value in two patients, slightly higher in one patient, and normal in the other patients. the lymphocyte count was normal in 26 patients. regarding coagulation function, there was an elevated platelet count in four patients, higher prothrombin time in one patient, and increased d-dimer in four patients. procalcitonin was slightly elevated in three cases, all of which were less than 1 ng/ml, and c-reactive protein was normal in 26 patients. additionally, investigations revealed three patients with reduced albumin, four patients with mildly abnormal transaminase, and two patients with slightly elevated creatinine levels. five patients had slightly lower serum sodium concentration and one patient had higher serum potassium concentration. lactate dehydrogenase and creatine kinase levels were slightly elevated in three patients and one patient, respectively. there was no statistical significance between the initial and following results for all the above indicators (p>0.05). being sars-cov-2-free was defined as two consecutive negative nucleic acid results with an interval of at least 24 hours. the period from diagnosis to being confirmed sars-cov-2-free was 10 days in 15 patients, 20 days in 23 patients, and more than 20 days in three patients (20 days, 20 days, and 22 days, respectively). the exact time of contact with a confirmed or possible covid-19 case was clear in 18 patients. the median period from contact to the last positive covid-19 nucleic acid test was 21.5 days (10 to 36 days) and in four patients this was over 30 days. in a case originating from wuhan, the period from leaving wuhan to the last positive covid-19 nucleic acid test was 35 days (figure 1 ). there were positive test results for sars-cov-2 following a negative result in two patients. in particular, in one patient, the sars-cov-2 nucleic acid tests repeatedly had positive and negative results (figure 1 ). a c c e p t e d m a n u s c r i p t 7 chest ct scans were performed an average of 3-4 days after the initial ct examination and each patient had at least two ct scans. the results of the first ct scan of the 26 patients were as follows: nine patients (34.6%) with normal ct scans, 10 patients (38.5%) with typical manifestations (patch-like, ground-glass opacities distributed in the extrapulmonary zone), seven patients with changes in a unilateral lung, and three patients with changes in bilateral lungs. in seven patients (26.9%), ct scans showed atypical ct manifestations (chronic inflammation and small cord shadows), including a small strip shadows in three patients (11.5%), small nodular shadows in one patient, thickened bronchial vascular bundles in two patients, and a small manifestation of chronic inflammation in one patient. in patients with normal ct scans on admission, the chest ct manifestations did not significantly change during hospitalization and seven patients with atypical ct manifestations also showed no significant changes on chest ct scans. for patients with typical chest ct manifestations, improved chest cts were observed in seven patients and three patients showed improvement after an initial progression of lesions ( figure 2 ). patients with normal or atypical chest ct findings had a relatively short period from diagnosis to continuous negative covid-19 nucleic acid results, with a median period of 7.5 days (2-20 days). in patients with typical patchy or ground-glass opacities, the median period was 12.5 days (8-22 days). this difference was statistically significant (p <0.01). of the 10 patients with typical chest ct manifestations, seven patients showed improved chest ct results that synchronized with the negative sars-cov-2 nucleic acid test result, while three patients had a marked improvement in ct results without a negative sars-cov-2 nucleic acid test result. all patients were isolated and treated with one or more antiviral drugs. recombinant human alpha-1b interferon nebulized inhalation (5 million u bid for adults and 3 million u bid for children) was administered in 25 cases. twenty-two patients received oral lopinavir/ritonavir (400 mg/ 100 mg bid for adults, 200 mg/ 50 mg qd for children). five adult patients were treated with oral arbidol (200 mg tid) and one patient developed purulent tonsillitis during hospitalization and was treated with antibiotics. one patient developed nausea during hospitalization, which may have been caused by lopinavir/ritonavir. another case developed a transient fever, which was related to suppurative tonsillitis. this patient felt better after antibiotic treatment. neither of the two symptoms was associated with sars-cov-2 infection. the remaining cases were asymptomatic during hospitalization. discharge criteria for covid-19 were as follows: 1) normal body temperature for more than 3 days; 2) significantly improved respiratory symptoms; 3) significantly improved chest radiography; and 4) two consecutive negative sars-cov-2 nucleic acid test results (sampling interval at least 1 day). all 26 patients were discharged from the hospital. the period from admission to discharge was ranged from 10 to 24 days, with a median of 13 days. a c c e p t e d m a n u s c r i p t 9 sars-cov-2, a new coronavirus that recently appeared, has rapidly spread across many countries and regions around the world. this new coronavirus is reported to be more contagious than atypical pneumonia (sars) and middle eastern respiratory syndrome coronavirus (mers) [15] [16] [17] . covid-19 is mainly seen a mild to moderate conditions, with a mortality rate of 3.2% [11] . isolating sources of infection is an important measure to control this current epidemic [2, 18] . infected individuals with clinical symptoms can be tracked according to their clinical symptoms, while those who are asymptomatic, especially those without an epidemiological history, are difficult to track. these asymptomatic cases may also capable of transmitting sars-cov-2, which could cause difficulties in covid-19 prevention and control. at present, there are few studies of asymptomatic disease, most of which focus on the proportion of asymptomatic patients and the evidence of asymptomatic transmission. the description of clinical features of asymptomatic patients is technically a description of asymptomatic disease at the time of diagnosis, with some patients going on to develop symptoms. there is also little research on asymptomatic epidemiology. the infectiousness and effect of the asymptomatic epidemiological characteristics and disease clinical characteristics of dynamic change are not clear. therefore, this study focused on persistently asymptomatic cases of covid-19. by tracing and isolating the contacts of people who were diagnosed or possible covid-19 patients, this study identified a total of 26 asymptomatic patients. asymptomatic carriers were found to be of any age, mainly concentrated in the 20-60 years age range and are characterized by local clusters. disease transmission from two asymptomatic patients was included in this study. infected patients may have clinical symptoms or may be asymptomatic. the study found that asymptomatic cases were mostly infected by symptomatic patients and a few were infected by asymptomatic patients. only two asymptomatic of the 26 enrolled asymptomatic patients infected others. therefore, we speculate that asymptomatic carriers may be less infectious than symptomatic patients. in asymptomatic carriers, there was a long period between contact with the infection source to diagnosis, with a median of 19 days. these asymptomatic carriers were identified only when they contacted diagnosed or possible covid-19 patients, suggesting the difficulty in detecting these asymptomatic carriers. the period from diagnosis to negative nucleic acid test tests was within 20 days for 23 (88.5%) patients ( figure 1 ). the duration of sars-cov-2 infection in asymptomatic patients is unclear. the median period from contact with diagnosed or possible cases to the last positive nucleic acid test was 21.5 days (10-36 days). most patients will miss the a c c e p t e d m a n u s c r i p t real last positive assays since nucleic acid tests for sars-cov-2 were not performed every day, so this time might be shorter than the actual time. the period was longer than 30 days in four patients, of whom one patient talked with a covid-19 patient for 1 hour, without contact with any other confirmed or possible patients contacted, 35 days previously (figure 1 ). in addition, this period of 35 days was for a patient with a wuhanrelated history (figure 1) . therefore, asymptomatic patients may carry sars-cov-2 for more than one month, suggesting that this virus is carried in asymptomatic patients for a long period. in two patients, there was a positive nucleic acid test after an initial negative result and one patient had recurrent alternation of positive and negative results. this phenomenon may be caused by false-negative results of assays and detection can be affected by factors including the specimens, reagents, and operations. thus, positive nucleic acid tests cannot be used as the only criterion for covid-19 diagnosis. standardized operation and repeated verifications are required to improve the positive rate. patients with normal chest cts had a shorter period from diagnosis to being sars-cov-2-negative than those with typical ct changes. in some patients, conversion of nucleic acid test results from positive to negative was synchronized with an improvement in chest ct findings, but this change may also occur later than the improvement on ct. therefore, ct improvement cannot be used to indicate that a patient is not infectious. indices of blood cell counts, liver and renal function, and inflammation in asymptomatic patients did not change significantly during the disease. reduced lymphocyte and leukocyte counts are characteristics of clinically symptomatic covid-19, but these changes were not common in the asymptomatic patients in this study. chest ct in asymptomatic patients can be normal or present with typical ground-glass opacities, usually limited to a unilateral lung but sometimes occurring in bilateral lungs. three patients with asymptomatic infections developed lung infections during treatment and then improved. although these asymptomatic patients appear normal, some relatively light damage may have already been produced. this study also has some limitation. for example, only 26 patients were included in this study. in addition, this retrospective analysis did not include daily nucleic acid tests for sars-cov-2. large-scale prospective studies are needed to validate our findings. in summary, asymptomatic infections can occur at any age and appear to be correlated with local clusters of diseases. asymptomatic transmission may be less contagious but should still be recognized as an infection source for covid-19 transmission. there were no significant changes in the biochemical and inflammatory variables in these patients. however, changes on chest ct may occur in these patients. it is necessary to note a c c e p t e d m a n u s c r i p t multiple patchy ground glass opacities can be seen in the right lung on february 6. the infection progressed more than before on february 11. on february 16, the signs of viral pneumonia in the right lung improved slightly, and on february 26, the chest ct improved. a c c e p t e d m a n u s c r i p t the wuhan sars-cov-2 -what's next for china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia transmission of 2019-ncov infection from an asymptomatic contact in germany estimation of the asymptomatic ratio of novel coronavirus infections (covid-19) estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship covid-19: identifying and isolating asymptomatic people helped eliminate virus in italian village radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study incidental findings suggestive of covid-19 in asymptomatic patients undergoing nuclear medicine procedures in a high prevalence region asymptomatic cases in a family cluster with sars-cov-2 infection delivery of infection from asymptomatic carriers of covid-19 in a familial cluster clinical characteristics of coronavirus disease 2019 in china clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province clinical outcome of 55 asymptomatic cases at the time of hospital admission infected with sars-coronavirus-2 in shenzhen clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china a novel coronavirus emerging in china -key questions for impact assessment world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) a c c e p t e d m a n u s c r i p t transmission of 2019-ncov infection from an asymptomatic contact germany camilla rothe, et.al [3] bmj identifying and isolating asymptomatic people italian village michael day [6] lancet infect dis radiological findings in both symptomatic and asymptomatic patientswuhan, china heshui shi, et.al [7] j nucl med nuclear medicine services in asymptomatic patients brescia, italy domenico albano, et.al [8] lancet infect dis guangdong, china pan x,et al [9] int j infect dis delivery of infection from asymptomatic carriers of covid-19 in a familial cluster chengdu, china ye f,et al [10] a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-350749-ihkxouz8 authors: panda, aditya k; tripathy, rina; das, bidyut k title: plasmodium falciparum infection may protect a population from severe acute respiratory syndrome coronavirus 2 infection date: 2020-07-29 journal: j infect dis doi: 10.1093/infdis/jiaa455 sha: doc_id: 350749 cord_uid: ihkxouz8 nan to the editor-we read with great interest the article published by nickbakhsh et al [1] describing epidemiological evidence of interaction between seasonal coronaviruses and other co-circulating viruses in a united kingdom population. the authors have suggested that prior exposure of children to coronavirus oc43 offers protection against severe covid-19 phenotype by possible crossimmunity. these observations encouraged us to investigate the possible role of plasmodium infection on coronavirus disease 2019 (covid-19) infection or severity. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection and mortality rates are variable in different countries. several factors may help account for the variability in the virus infection or mortality rate, such as age, sex, comorbidity, or genetic makeup. recent studies indicated that protozoan infections may offer some protection against various positive-strand rna viruses. prior exposure to plasmodia significantly suppressed chikungunya virus-associated pathogenesis, characterized by reduced viral load and improved joint inflammation [2] . furthermore, coinfections of a rodent plasmodium strain and lactate dehydrogenase-elevating virus offered protection against experimental cerebral malaria and experimental autoimmune encephalomyelitis [3] . based on these observations on plasmodium infection and positive-strand rna viruses, we hypothesized that there could be a possible association between malaria and sars-cov-2 infection. to validate our observation, we investigated the prevalence of covid-19 in the plasmodium falciparum-endemic area of odisha, india, odisha is highly endemic for p. falciparum infection. we obtained the annual parasite index (api) of p. falciparum for the last 10 years (2010-2019) from the national vector borne disease control program and covid-19 infection status in odisha from the government of odisha website (see https://health. odisha.gov.in/covid19-dashboard. html). api and covid-19 data from 30 districts were analyzed and shown in figure 1 . a significant negative correlation (spearman r = -0.37; p = .04; n = 30) was observed between 10-year average api scores and the number of covid-19 cases detected. malaria is known to stimulate b cells resulting in hyper-gammaglobulinemia [4] and multiple cross-reactive antibodies are often produced, which could be protective. a recent study further highlighted the production of immunoglobulin g (igg) and immunoglobulin m (igm) against p. falciparum merozoite, which persists for an extended period and protects the host from clinical malaria [5] . it has also been well established that infection by some enveloped viruses triggers naturally occurring antibodies to activate the complement system, leading to lysis of the virus [6] . antibodies against disaccharide galactose α-(1,3)-galactose (α-gal) are most prevalent and constitute about 2% of total igg and igm. previously, we have demonstrated high levels of antibodies to α-gal (igg and igm) in healthy subjects in malaria-endemic areas (igg: 144.62 ± 47.23; igm: 19.93 ± 15.08 enzyme-linked immunosorbent assay [elisa] units) compared to residents of nonendemic regions (igg: 30.15 ± 9.3; igm: 8.5 ± 6.1 elisa units) [7] . these are polyspecific antibodies capable of interacting with multiple antigens. although the presence of α-gal on the surface of sars-cov-2 has not been reported, the anti-gal antibodies, being polyspecific, can cross-react with multiple epitopic determinants [8] . furthermore, in our earlier study (unpublished), we observed high levels of specific antimalarial antibodies (pfp0, pfemp, resa, msp-1, and msp-3) in residents of malaria-endemic areas. the cross-reactivity of these antibodies, along with anti-gal antibodies and covid-19, needs to be investigated for possibly demonstrating a cause-effect relationship. further validation of this hypothesis might be established from other p. falciparum-endemic areas of the world. epidemiology of seasonal coronaviruses: establishing the context for the emergence of coronavirus disease 2019 plasmodium co-infection protects against chikungunya virus-induced pathologies a virus hosted in malaria-infected blood protects against t cell-mediated inflammatory diseases by impairing dc function in a type i ifn-dependent manner to b or not to b: understanding b cell responses in the development of malaria infection igm in human immunity to plasmodium falciparum malaria natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity immunological correlates in plasmodium falciparum infection with special reference to cerebral malaria naturally-occurring anti-alphagalactosyl antibodies in human plasmodium falciparum infections-a possible role for autoantibodies in malaria covid-19 coronavirus pandemic department of health and family welfare, government of india key: cord-007026-ejv0gidp authors: coleman, kristen k; wong, chui ching; jayakumar, jayanthi; nguyen, tham t; wong, abigail w l; yadana, su; thoon, koh c; chan, kwai peng; low, jenny g; kalimuddin, shirin; dehghan, shoaleh; kang, june; shamsaddini, amirhossein; seto, donald; su, yvonne c f; gray, gregory c title: adenoviral infections in singapore: should new antiviral therapies and vaccines be adopted? date: 2020-02-15 journal: j infect dis doi: 10.1093/infdis/jiz489 sha: doc_id: 7026 cord_uid: ejv0gidp background: a number of serious human adenovirus (hadv) outbreaks have been recently reported: hadv-b7 (israel, singapore, and usa), hadv-b7d (usa and china), hadv-d8, -d54, and -c2 (japan), hadv-b14p1 (usa, europe, and china), and hadv-b55 (china, singapore, and france). methods: to understand the epidemiology of hadv infections in singapore, we studied 533 hadv-positive clinical samples collected from 396 pediatric and 137 adult patients in singapore from 2012 to 2018. genome sequencing and phylogenetic analyses were performed to identify hadv genotypes, clonal clusters, and recombinant or novel hadvs. results: the most prevalent genotypes identified were hadv-b3 (35.6%), hadv-b7 (15.4%), and hadv-e4 (15.2%). we detected 4 new hadv-c strains and detected incursions with hadv-b7 (odds ratio [or], 14.6; 95% confidence interval [ci], 4.1–52.0) and hadv-e4 (or, 13.6; 95% ci, 3.9–46.7) among pediatric patients over time. in addition, immunocompromised patients (adjusted or [aor], 11.4; 95% ci, 3.8–34.8) and patients infected with hadv-c2 (aor, 8.5; 95% ci, 1.5–48.0), hadv-b7 (aor, 3.7; 95% ci, 1.2–10.9), or hadv-e4 (aor, 3.2; 95% ci, 1.1–8.9) were at increased risk for severe disease. conclusions: singapore would benefit from more frequent studies of clinical hadv genotypes to identify patients at risk for severe disease and help guide the use of new antiviral therapies, such as brincidofovir, and potential administration of hadv 4 and 7 vaccine. human adenoviruses (hadvs) cause diverse illnesses that often vary by hadv species (a-g) and type (>90 genotypes have been described) [1] . human adenovirus types 3, 4, 7, 21, 14, and 55 have been associated with severe acute respiratory disease [2] [3] [4] [5] . although a number of hadv vaccine types have been or are under current study, live enteric-coated vaccines against hadv-b7 and hadv-e4 have been used with remarkable effectiveness and safety among us military trainees [6] . clinical epidemiological data regarding hadv infections are relatively sparse for southeast asia (sea). however, existing data suggest that hadv-b3 is ubiquitous and more often associated with milder illness when compared with recent hadv-b7 infections in singapore, malaysia, taiwan, and china [7] [8] [9] [10] [11] [12] [13] [14] . unlike hadv-b3 and hadv-b7, outbreaks of hadv-e4 have not been as commonly reported globally until recently. upon its discovery in the 1950s [15] , hadv-e4 was largely considered restricted to and controlled by vaccine in the us military population, with rare detections among civilians, such as school children in the netherlands in 1958 [16] , users of a private swimming pool in the usa in 1977 [17] , and conjunctivitis patients in japan in 1979 [18] . more recently, surveillance for hadv has improved, and evidence of hadv-e4 infections among civilian populations in the usa, italy, west africa, china, hong kong, taiwan, india, malaysia, and singapore have been increasingly reported [8, [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . due to the historical lack of hadv genotyping surveillance, it is unknown whether hadv-e4 is a new or re-emerging pathogen in sea. in an effort to epidemiologically describe clinical hadv genotypes in singapore, we used a hexon and fiber genotyping algorithm [29] to retrospectively and prospectively study hadv infections among patients from 2 large public hospitals in singapore. institutional review board (cirb reference 2015/2773) to study archived and prospectively collected hadv-positive specimens. clinical samples previously collected from hadv-positive patients admitted to singapore general hospital (sgh) and kk women's and children's hospital (kkh) between 2012 and 2015 were preserved at −80°c and transferred to duke-nus laboratory of one health research for study. clinical records were accessed only to determine clinical sample type. from 2015 to 2018, informed consent was sought from all patients with laboratory-confirmed adenovirus infection at sgh and kkh. a patient with laboratory-confirmed adenovirus infection was defined as a person of any age seeking healthcare and who had evidence of adenovirus infection by polymerase chain reaction (pcr), real-time pcr, commercial direct fluorescent antibody (dfa), culture, specific monoclonal antibody assay, or other clinically approved adenovirus tests. the clinical samples (nasal, pharyngeal, nasopharyngeal, throat, bronchoalveolar, blood, serum, conjunctival, or urine) were first clinically screened with commercial dfa or seeplex rv15 ace pcr targeting influenza a and b virus, parainfluenza 1-4 virus, respiratory syncytial virus, metapneumovirus, rhinovirus, enterovirus, human coronavirus oc43 and 229e/nl63, adenovirus, and bocavirus. residual samples were then preserved at −80°c. clinical records were reviewed and specimen information forms were completed. human adenovirus-positive samples were transported on dry ice to the sgh department of microbiology where they were divided into two 0.5-ml aliquots (1 kept at sgh and 1 transferred to duke-nus's laboratory of one health research). human adenovirus-positive patients with an influenza virus codetection were excluded from our study because these samples were required for study at singapore's national public health laboratory. human adenovirus culture was attempted at duke-nus. a 500-μl aliquot of each sample was inoculated into a549 cells with dulbecco's modified eagle's medium 2% (v/v) fetal bovine serum and incubated at 37°c while controlling for bacterial contamination. inoculated shell vials were observed for cytopathic effect 72 hours after inoculation and daily afterward for 10 days. observation of cytopathic effect was used to score hadv-positive culture. at duke-nus, deoxyribonucleic acid (dna) was extracted from the culture supernatants of hadv-positive samples. for culture-negative hadv samples, dna was extracted directly from the original clinical sample. total dna was extracted using the qiaamp dna blood mini kit (qiagen, inc., valentica, ca) and typed using conventional pcr [29, 30] [29] was independently performed, and in-house sequencing was performed using the bigdye terminator v3.1 cycle sequencing kit (applied biosystems) on the abi prism 3130 genetic analyzer (applied biosystems). results from the typing efforts at each institution were then compared and the hadv genotype was confirmed. deoxyribonucleic acid extracted from culture supernatants from 10 culture-positive hadv samples were sent for wholegenome sequencing at the duke-nus genome biology facility. a qubit high sensitivity assay was used to measure dna concentrations (ng/μl) in each sample before library preparation using the nextera xt dna library prep kit. samples were then studied using the miseq 250 × 2 bp (500 cycle) nano kit v2. the quality of short ngs reads were initially checked by fastqc and followed by the removal of the illumina adaptors using trimmomatic [31] . the trimmed reads were then mapped against the reference genomes (hadv-c or hadv-e) using geneinous r9.0.3 (biomatters ltd., auckland, new zealand). a total of 10 new adenovirus genomes were generated from this study and compared with all available hadv-c and hadv-e sequences of fiber, hexon, and penton base and full genomes from genbank. for each dataset, multiple sequence alignment was initially performed using mafft [32] followed by manual alignment. gene and full genome phylogenies were initially reconstructed using fasttree [33] , and the outliers were removed for subsequent analysis. for hadv-c viruses, 4 final datasets were further subsampled to 84 fiber sequences (1759 bp in length), 72 hexon sequences (2928 bp), 66 penton base sequences (1725 bp), and 46 full genomes (36 445 bp). for hadv-e viruses, another 4 final datasets were also subsampled to 46 fiber sequences (1278 bp in length), 39 hexon sequences (2811 bp), 39 penton base sequences (1608 bp) and 37 full genomes (36 397 bp). for each dataset, maximum likelihood (ml) phylogenies were reconstructed using raxml v8.0 [34] , and branch support was assessed using 1000 bootstrap (bs) replicates. to detect potential recombinant events, full genome datasets were analyzed separately for hadv-c and hadv-e, because these were the species identified from the subset of isolates (n = 10) that were fully sequenced. human adenovirus-c datasets consisted of 4 novel genomes from singapore and 22 representative virus isolates from different genotypes (hadv-c1, hadv-c2, hadv-c5, and hadv-c6). human adenovirus-e datasets consisted of 6 novel genomes and 40 representative virus isolates of hadv-e4. the datasets were analyzed using different methods as implemented in recombination detection program (rdp4) [35] . these methods included rdp, geneconv, bootscan, maxchi, chimaera, siscan, and 3seq and their p values estimated. the breakpoints positions and their major and minor parents were identified for putative recombination strains. for each novel genome, a subsampled dataset was subsequently analyzed in rdp to determine their recombination events. to further confirm detected recombination events, independent phylogenetic analyses were performed using ml method as described above. epidemiological data were imported and categorized for statistical analyses using stata, version 15.0 (statacorp, college station, tx). logistic regression was performed to examine hadv genotype frequencies by time period. pearson's χ 2 and fisher's exact tests were used to examine potential risk factors (gender, age group, immunosuppression, and hadv genotype) for associations with severe disease (prolonged hospitalization, prolonged fever, or intensive care unit admission). stepwise, unconditional logistic regression using a saturated model and backward elimination (exclusion p > .05) was used to further examine these risk factors. clinical samples were studied from 533 hadv-positive patients admitted at either of the 2 hospitals from 2012 to 2018. partial genome (hexon and fiber) sequencing was attempted on all 533 samples, 458 (86%) of which were successfully genotyped. the most prevalent genotypes identified were hadv-b3 (35.6%), hadv-b7 (15.4%), and hadv-e4 (15.2%) followed by hadv-c2 (11.8%) and hadv-c1 (4.9%). see figure 1 for hadv genotype frequencies by year. among the 300 retrospective cases (245 pediatric and 55 adult), hadv-b7 was most prevalent from 2012 to 2013, which was consistent with previous literature for singapore [36] . a total of 233 (151 pediatric and 82 adult) prospective hadvpositive patients provided informed consent, and their samples (89% respiratory) and clinical data were included in the study (table 1) . twenty-two immunocompromised patients were included in the study and described separately ( table 2) . human adenovirus-b3 (26.2%) was most prevalent among prospective cases, followed by hadv-e4 (25.8%) and hadv-b7 (18.5%) ( table 1) . lastly, immunocompromised patients (adjusted odds ratio [aor], 11.4; 95% confidence interval [ci], 3.8-34.8) and patients infected with hadv-c2 (aor, 8.5; 95% ci, 1.5-48.0), hadv-b7 (aor, 3.7; 95% ci, 1.2-10.9), hadv-e4 (aor, 3.2; 95% ci, 1.1-8.9), or an unknown hadv genotype (aor, 5.7; 95% ci, 2.0-16.2) were found to have an increased risk for severe disease (table 3) . (table 4) . overall, 137 (25.7%) of the clinical samples were collected from adults, of which genotypes hadv-b7 (28.5%), hadv-e4 (21.2%), and hadv-c2 (3.6%) were most prevalent. among the adult samples, 55 (40.1%) were retrospectively collected from 2012 to 2013, whereas the remaining 82 (59.9%) were prospective samples from 2015 to 2018. no significant differences in hadv genotype frequencies were found between retrospective and prospective adult patients. in 2016, we noticed an increase in hadv-e4 infections. to learn whether the observed increase might reflect a novel hadv-e4 strain, 6 hadv-e4 sample genomes were sequenced (3 from severe and 3 from mildly ill patients). another 4 hadv species c samples were chosen for sequencing based on their discrepant hexon and fiber genotyping results between laboratories. based on the full genomes of hadv-c (figure 2 ), 3 isolates-sg05, sg08, and sg09-belonged to c1 and were strongly monophyletic, with 100% bs support. these new isolates were also sister to a group of hadv strains from the united states. contrarily, isolate sg06 was nested within c2 lineage (ml bs = 100%) and closely related with a beijing hadv-c2 strain (human/chn/bj04/2012) collected from an infant during a hadv-c epidemic in beijing in 2012-2013 [37] as well as earlier c2 strains from the united states isolated from 1992 to immunocompromised within 6 months before clinical sample collection. c hospitalized for more than 7 consecutive days. 2004. it is notable that sg06 was isolated from a 2-year-old patient requiring oxygen therapy. hexon, fiber, and penton base topologies (supplemental figures 1 and 2) indicated that isolates sg05 and sg09 were most closely related, with strong bs support (87%, 88%, and 99%, respectively). however, penton base phylogeny (supplemental figure 2 ) demonstrated that isolate sg08 was related to c2 lineage, although this lacks statistical support, as opposed to c1 based on full genomes. more notably, the penton base phylogeny demonstrated that isolates sg05 and sg09 were closely related to c2 and c6. clinical descriptions of the singapore hadv-c isolates and their genbank accession numbers can be found in supplemental table 1 and figure 2 . in contrast to hadv-c, the full genomes of hadv-e ( figure 3 ) clearly indicated 6 new isolates (sg01, sg03, sg04, sg07, sg10, and sg11) from singapore grouped within e4, with strong bs support (ml bs = 100%). these results were concordant with fiber and penton base phylogenies (supplemental figures 3 and 4) , indicating that these e4 isolates were genetically identical (100% in both genes) and appeared to be clonal. however, the hadv-e hexon phylogeny (supplemental figure 3) indicated that the 6 e4 isolates were relatively segregated, despite being largely monophyletic. these 6 isolates also exhibited 99.8%-100% nucleotide similarities, suggesting that some genetic variations existed among them. clinical descriptions of the hadv-e isolates and their genbank accession numbers can be found in supplemental table 1 and figure 3 . global mutational analysis using genome mutation mapper ([gmm]; unpublished beta software) was then used to investigate nucleotide-level variations (single-nucleotide polymorphisms and insertions/deletions) between the 6 hadv-e4 isolates (sg01, sg03, sg04, sg07, sg10, and sg11) and a 2002 hadv-e4 reference field strain [38] . based on the genotyping failed. f gmm results, sg01 and sg10 are clones, "lineal" to sg04 and sg07, which are clonal. clones sg01 and sg10 are also lineal to sg03, which is lineal to sg11. it is notable that sg11 has both an insertion and a deletion near the 30.7-kda gene. in addition, to provide nexus with the earlier reports of hadv-e4 in the literature and to compare genome types [5, 38] , in silico restriction enzyme fragment analyses were performed using snapgene (version 4.3), revealing that sg03 and sg11 are novel separate genome type variants, with differing drai and bamhi patterns and given designations "4a1 drai/ bamhi v1" and "4a1 drai/bamhi v2". the others are genome typed as hadv-e4a1. these patterns support the lineages described by the gmm results. for reference, the 2002 hadv-e4 field strain is genome type hadv-e4a1 [5] , and the patterns for the prototype strains differ from the hadv-e4a strains across all restriction enzymes applied, with one exception, a match of hadv-e4p mil and the hadv-e4a strains using ecori. our rdp analyses detected recombination events in 4 new hadv-c isolates from singapore. recombination analysis of sg05 isolate clearly identified 2 parental strains (bj04 and c1), and the estimated breakpoint was located at the position 17874 (supplemental figure 5a , supplemental table 2 ). to confirm this result, phylogenetic analysis was independently performed for the gene regions before and after the breakpoint. the ml topologies (supplemental figure 5b) indicate that sg05 recombinant was descended from c1, but it also comprised genetic components from the bj04 recombinant strain. the bj04 strain was crucially important [37] because it has been shown to bear gene sequences from 3 different genotypes: hadv-c1 at (position 1-18076, comprising e1a, e1b, penton base), hadv-c2 (18077-33397, comprising hexon and fiber), and hadv-c6 (33398-end, comprising the e4 gene region). the e4 region of sg05 recombinant also contained c6 because this region was recombined with the bj04 strain. another recombinant, sg09, was found to be almost identical (99.9% nucleotide identity) to sg05, therefore they both displayed the same breakpoint location (supplemental table 2 ) and rdp graph (data not shown) as well as the phylogenetic position as expected (supplemental figure 1 ). these findings indicate that sg05 and sg09 isolates were recombinants of c1 and bj04 parental strains. recombination analysis of isolate sg06 (supplemental figure 6a ) suggested that this strain was recombinant of c2 and bj04 parental strains, although the sg06 backbone was largely derived from c2. the ml phylogenies (supplemental figure 6b) confirmed, with strong bs support (ml bs = 100%), that sequence position 12502-35973 of sg06 isolate was nested within c2, whereas sequence position 7536-12501 of sg06 isolate was closely related with the bj04 and bj09 strains. in contrast, the after stepwise logistic regression using saturated model and backward elimination of covariates with p > .05. c immunocompromised within 6 months before clinical sample collection. figure 7a ) displayed the parental c1 subtype as the main backbone, although recombination with c6 was found towards the end of the full genome. the rdp results were also concordant with the 2 ml phylogenies that are incongruent with each other (supplemental figure 7b ). all recombination results were statistically significant (p < .05) (supplemental table 2 ). in marked contrast, no recombination events were detected in the 6 hadv-e isolates. this is the first large-scale study to systematically describe clinical hadv genotypes in singapore. our results highlight the prevalence of hadv-b3 and an increase in morbidity due to hadv-e4 and hadv-b7 pediatric infections in singapore. more important, patients with hadv-c2, hadv-e4, and hadv-b7 were more likely to have severe disease. hence, it seems prudent for public health officials and clinicians to consider using antiviral therapies and hadv vaccines in singapore. for instance, brincidofovir, an experimental antiviral therapy, is being used in the usa against severe hadv infections with good outcomes [39] [40] [41] . in addition, a very effective "adenovirus type 4 and type 7 vaccine, live, oral" has been used among us military personnel since 1971. the new version of this vaccine, used since 2011, has resulted in a dramatic decline of respiratory infections among us military recruits [42] . although this nonattenuated vaccine is not indicated to prevent infections for other populations (concerns for safety among the immunocompromised), other vaccines [6] are being developed that might prove useful in singapore. given the lack of hadv surveillance in sea, it is unknown whether hadv-e4 is a new or re-emerging pathogen in the region. the high prevalence of hadv-e4 infections identified in our study is intriguing. the hadv-e4 genes and genome have been shown to be almost identical to several chimpanzee adenoviruses, including the sadv-e25 genome at 91% and the sadv-e26 at 92%, with the 1952 prototype as reference [38, 43] . these values are consistent with hadv-e4 being the only hadv amongst several chimpanzee adenoviruses phylogenetically grouped within hadv species e, supporting a zoonotic origin, consistent with current hypotheses that cross-species infections may lead to the emergence of novel human pathogens [44] . furthermore, sequence analysis of 2 recent circulating hadv-e4 strains revealed a recombination event that provides a conserved dna replication motif (nf-i), likely from a respiratory pathogen of hadv species b, that is present in almost all hadvs but absent in sadvs as well as the hadv-e4 prototype and contemporaneously circulating isolates, including the "vaccine" strain [38, 45] . this hadv-like motif, as well as the conserved core origin and nf-iii motif, were present in all 6 of the fully sequenced singapore hadv-e4 genomes. the nf-i human transcription factor and its binding have been extensively characterized molecularly and biochemically as required for optimal hadv dna replication [46, 47] . this putative genome-based host adaptation may be the "tipping point" that has allowed hadv-e4 entry into a global general population, which is presumably immunologically naive to its epsilon (hexon) antigen, because hadv-e4 had been known to circulate sporadically [20, [48] [49] [50] . the recombination event is likely a recent one, with the earliest appearance in 1978 (kx384956 v0014/france/1978) as noted in a resurvey [23] of 2 sets of recently released genomes that include archived and currently circulating military and civilian hadv-e4 isolates [20, 49] , and it may be important as an example of a molecular evolution pathway by which zoonosis may be a source of emergent human pathogens [44] . our rdp analyses detected separate recombination events in each of our 4 hadv-c isolates, which suggests the frequent exchange of genetic components among different genotypes. sg05, sg09, and sg06 were shown to be recombinants of a clinically relevant parental strain, bj04, a hadv-c2 strain isolated from an infant during a 2012-2013 epidemic in beijing [37] . it is notable that sg06 was the only hadv-c2 strain fully sequenced in our study and was isolated in 2016 from a severely ill 2-year-old singapore patient requiring oxygen therapy. given the observed increased risk for severe disease among the 6 prospective pediatric patients infected with hadv-c2, 4 infections of which occurred within a 1-month period, we speculate that these patients might represent a cluster. although our study revealed important findings regarding the epidemiology and clinical severity of hadvs in singapore, we were limited in our approach because we did not rule out all possible coinfections (other than influenza virus). it is also possible that some of the hadv infections were not associated with the acute clinical presentations. our study could have been strengthened by testing blood (or another sterile compartment such as csf) from all study patients or by testing matched patient controls to demonstrate a stronger probability of disease association. however, the prevalent genotypes identified (ie, hadv-b3, hadv-b7, and hadv-e4) were genotypes that are commonly known to cause acute respiratory infections. finally, mixed hadv infections could not be ruled out as the cause of the untypable patient samples (14%). this theory is supported by the observed increased risk for severe disease among patients infected with an "unknown" hadv genotype, as well as the high prevalence (40.9%) of unknown hadv genotype infections among immunocompromised patients in our study. another reason for an untypable sample is inadequate hadv dna for amplification, resulting in an unknown hadv genotype classification. the majority of these untypable samples were adult samples, which is consistent with the theory that infected adults shed less virus when compared with children. our study demonstrates how hadv genotyping can be performed in a clinical setting and can be useful in detecting novel hadv incursions. for singapore and other countries considering new hadv treatment and control measures, we strongly recommend periodic but routine hadv genotype surveillance with a goal of collecting the hadv prevalence data necessary to make informed decisions. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole 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louis a; wahl, victoria; hevey, michael; dabisch, paul title: airborne sars-cov-2 is rapidly inactivated by simulated sunlight date: 2020-06-11 journal: j infect dis doi: 10.1093/infdis/jiaa334 sha: doc_id: 332303 cord_uid: 0bbw64p5 aerosols represent a potential route of transmission of covid-19. this study examined the effect of simulated sunlight, relative humidity, and suspension matrix on the stability of sars-cov-2 in aerosols. both simulated sunlight and matrix significantly affected the decay rate of the virus. relative humidity alone did not affect the decay rate; however, minor interactions between relative humidity and the other factors were observed. decay rates in simulated saliva, under simulated sunlight levels representative of late winter/early fall and summer were 0.121±0.017 min(-1) (90% loss: 19 minutes) and 0.379±0.072 min(-1) (90% loss: 6 minutes), respectively. the mean decay rate without simulated sunlight across all relative humidity levels was 0.008±0.011 min(-1) (90% loss: 125 minutes). these results suggest that the potential for aerosol transmission of sars-cov-2 may be dependent on environmental conditions, particularly sunlight. these data may be useful to inform mitigation strategies to minimize the potential for aerosol transmission. a c c e p t e d m a n u s c r i p t for aerosol transmission to occur, viruses within aerosol particles must remain infectious between generation and inhalation by a susceptible host. loss of infectivity during this period will decrease the likelihood of aerosol transmission. van doremalen et al. [15] have reported that sars-cov-2 is detectable in aerosols for several hours in darkness at room temperature. similar results have been reported previously for other coronaviruses under similar conditions [16, 17] . environmental conditions, including relative humidity and sunlight, have been shown to influence the decay rate of infectious viruses in aerosols [16, [18] [19] [20] [21] [22] . however, no such data on the influence of these factors on the aerosol persistence of sars-cov-2 exist. therefore, the present study examined the influence of both simulated sunlight and relative humidity on the stability of sars-cov-2 in aerosols generated from virus suspended in different liquid matrices. the data generated will further our understanding of factors which have the potential to influence aerosol transmission of sars-cov-2 and could be utilized to inform mitigation strategies for aerosol transmission of virus during the current pandemic. vero cells (atcc ccl-81) were grown at 37 °c and 5% co 2 in culture medium, consisting of minimum essential medium (mem, gibco) supplemented with 10% heat-inactivated fetal bovine serum (fbs, hyclone or atlanta biologicals), 2mm glutamax (gibco), 0.1 mm nonessential amino acids solution (gibco), 1mm sodium pyruvate (gibco), and 1% antibiotic-antimycotic solution (gibco). a passage four isolate of sars-cov-2 (betacov/usa/wa1/2020) was obtained from bei resources and passaged twice in vero cells to produce a stock of virus that was concentrated by tangential flow filtration and frozen at -80°c until use. for aerosol tests, aliquots of the concentrated virus were thawed and diluted 1:10 in either fresh culture medium or simulated saliva, formulated as described in the astm standard for measuring virus decontamination efficacy [23] , but prepared with kh 2 po 4 a c c e p t e d m a n u s c r i p t and k 2 hpo 4 at final concentrations of 15.4 mm and 24.6 mm, respectively. diluted virus aliquots were prepared daily from frozen stocks and kept on ice between tests. titers of infectious virus in aerosol samples were determined by microtitration assay on confluent monolayers of vero cells in 96-well plates. plates were incubated at 37°c and 5% co 2 , with cytopathic effect read four days postinfection and viral titers calculated according to the method of kärber and spearman [24, 25] . the ph and solids content of viral suspensions diluted in each matrix was measured in triplicate using a sevenexcellence ph meter (mettler-toledo) and ma35 moisture analyzer (sartorius ag), respectively. protein content was quantified using a pierce bca protein assay kit (23225, thermo fisher scientific) with an albumin standard curve. the assay was read on a spectramax m5 plate reader (molecular devices). two different environmentally controlled rotating drum aerosol chambers, with volumes of 16-l and 208-l, were used in the present study to expose aerosols containing sars-cov-2 to controlled levels of temperature, relative humidity, and simulated sunlight. the environmental control systems were similar for both drums, and have been described previously for one of these drums [19] . briefly, the temperature of the air inside the drum was regulated by a temperature-controlled glycol solution circulated through channels in the walls of the drums. relative humidity was controlled by adjusting the balance of dry and humid air entering the drum prior to tests, during filling, and as makeup air when aerosol samples were collected from the drums. temperature and relative humidity probes in the interior of each drum were used to record the values of these parameters in ten-second intervals over the course of each test. for each test, the mean and standard deviation were determined for these parameters using data from the beginning of the first aerosol sample to the end of the final sample. for a subset of tests, sars-cov-2 aerosols were exposed to simulated sunlight generated by a solar simulator (newport oriel) equipped with a 320-nm highpass filter (wg320 filter pn sl07614, solar a c c e p t e d m a n u s c r i p t light co.) through a fused-silica window on one face of the chambers. tests were conducted at one of two intensity levels, with spectra designed to represent the ultraviolet (uv) range (280-400 nm) of natural sunlight. the two spectra used in the present study, referred to hereafter as high-intensity and mid-intensity, have similar uv irradiances to model spectra from the national center for a c c e p t e d m a n u s c r i p t virus-containing aerosol particles of respiratory origin have been found in a range of particle sizes from sub-micron to several microns in diameter [8, [26] [27] [28] . in the present study, the target mass median aerodynamic diameter (mmad) was 2 µm, an approximate mid-point of the range of relevant possible sizes. aerosols were generated into an external stainless steel plenum attached to each drum using an air assist nozzle (iaza5200415k; lee company). the nozzle was supplied with dry, compressed air at 45 psig and supplied with the viral suspension at 200 to 300 µl/min using a syringe pump. aerosol was drawn from the external plenum into the drums. the filling time differed for the two drums due to the difference in volume and was 30 seconds for the smaller drum and 60 seconds for the larger drum. following filling, aerosols were allowed to mix in the drum for thirty seconds prior to collection of the first sample. aerosols were then aged in the drums for up to 60 minutes. five samples of the aerosol present in a drum were collected over the course of each test. the test duration and sample intervals were determined based on the anticipated decay rate for a given set of environmental conditions. at each sampling time point, a ten second sample was collected using an aerodynamic particle sizer (aps; model 3321, tsi inc.) to measure the mass concentration and size distribution of the aerosol in the drum. immediately following the aps sample, a 20 to 60 second sample was collected onto a 25 mm gelatin filter (pn 225-9551; skc, inc.) in a delrin filter holder (pn 1109; pall corp.) operated at 5 l/min. the gelatin filter was immediately removed from the holder and dissolved in 10 ml of culture medium to re-suspend the collected virus. relative humidity-conditioned makeup air entered the drum during both aps and gelatin filter sampling to maintain the relative humidity and neutral pressure in the chamber. tests were conducted in both suspension matrices across a range of relative humidity levels (20, 45, and 70%) and simulated sunlight intensities (darkness, mid-intensity, and high-intensity). a 2x2 full factorial design with a center-point was utilized to examine the effect of each parameter, as well as a c c e p t e d m a n u s c r i p t interactions between parameters, on the decay rate of aerosolized sars-cov-2. tests were conducted at all combinations of the low and high levels of both factors, as well as at the mid-point levels of both factors. this experimental design is an efficient approach that allows examination of the impact of relative humidity and simulated sunlight, as well as potential interactions of these factors, while minimizing the total number of tests required. additional tests were conducted without simulated sunlight at target relative humidity values of 37 and 53% to examine the effect of relative humidity under temperature and light conditions relevant to indoor environments in greater detail. three to six replicate tests were performed for each combination of suspension matrix and environmental condition. all tests were conducted at a target temperature of 20°c. the aerosol concentration of infectious sars-cov-2 within the drum at each time point, in tcid 50 /lair, was calculated as the total amount of virus collected by the gelatin filter divided by the amount of air sampled. the aerosol mass concentration within the drum at each time point, in mg/m 3 , was calculated from the data collected by the aps. for each test, time-series log 10 transformed viral and mass aerosol concentration data were fit using linear regression in microsoft excel (v. 2016). the slopes of these regression lines represent the decay rates of infectious virus and total aerosol mass in the chamber, respectively. in the published literature, decay is often reported as the decay constant from a one-phase exponential fit [16, 19, [29] [30] [31] [32] . to allow a direct comparison to these values, the slope was converted from log base 10 to log base e, as this value is equivalent to the decay constant from a one-phase exponential decay fit of the data. the mean mmad and geometric standard deviation (gsd) at the first sample collected across all tests in simulated saliva were 1.96 ± 0.05 µm and 1.62 ± 0.04, respectively. for tests in culture medium, these values were 1.98 ± 0.08 and 1.60 ± 0.04, respectively. a small downward shift in the mmad occurred over the course of each test due to a more rapid physical loss of larger particles in the size distribution. as a result, the mean mmad of aerosols generated from simulated saliva and culture medium at the final sample were 1.78 ± 0.14 and 1.88 ± 0.13, respectively. decay data for sars-cov-2 in aerosols are shown in figure 2 , figure 3 , and table 1 . average decay constants for infectivity ranged from near zero for tests without simulated sunlight to 0.48 min -1 , or 38%/min, for tests with high-intensity simulated sunlight at 70% relative humidity. stepwise regression analysis demonstrated that k infectivity was dependent on the simulated sunlight intensity and the suspension matrix (p<0.0001 and p=.0004, respectively), but not relative humidity (p=0.0946). interactions between suspension matrix and simulated sunlight intensity (p<0.0001), suspension matrix and relative humidity (p=0.0017), and simulated sunlight intensity and relative a c c e p t e d m a n u s c r i p t humidity (p=0.0463), were also significant. while the effect of suspension matrix was statistically significant, the magnitude of the effect of simulated sunlight was much greater, as suggested by a greater standardized regression coefficient (-0.117 for simulated sunlight vs. 0.022 for matrix). the overall adjusted r 2 for the model was 0.88. the present study examined the influence of simulated sunlight and relative humidity on the stability of sars-cov-2 in aerosols generated from virus suspended in either simulated saliva or culture medium at 20°c. simulated sunlight rapidly inactivated the virus in aerosols in either suspension matrix , with half-lives of less than 6 minutes and 90% of the virus inactivated in less than 20 minutes for all simulated sunlight levels tested. there was a small but statistically significant reduction in decay rate under high-intensity sunlight when the virus was suspended in culture medium compared to simulated saliva, suggesting that the matrix in which the virus is suspended may also be an important factor to consider when examining the persistence of sars-cov-2 in an aerosol. while it has been reported previously that uvc can inactivate aerosolized coronaviruses [33] , the present study is the first to demonstrate that simulated sunlight, with uva and uvb levels similar to natural sunlight, is also able to inactivate airborne coronaviruses. it should be noted that many additional factors beyond the relative stability of the virus in an aerosol contribute to the potential for aerosol transmission of disease. these include the amount of virus present in an aerosol, the size and infectious dose of aerosol particles, the distance and airflow dynamics between infected and uninfected individuals, the presence of mitigation measures such as personal protective equipment. therefore, while the results of the present study provide novel data regarding the stability of sars-cov-2 aerosols in the environment, additional data are needed to provide a comprehensive assessment of the potential for aerosol transmission. a c c e p t e d m a n u s c r i p t relative humidity alone did not significantly affect decay of the virus, although there were interactions identified between relative humidity and the other factors. however, the magnitude of these interactions was minor compared to the magnitude of the effect of simulated sunlight. the half-lives estimated from the mean decay constants across all relative humidity levels without simulated sunlight present were 55 and 86 minutes for aerosols generated from virus suspended in culture medium and simulated saliva, respectively. the half-life from the present study for culture medium is similar to the value of 1.1 hours reported recently for sars-cov-2 in darkness and 65% relative humidity by van doremalen et al. [20] . the prolonged persistence of sars-cov-2 under conditions representative of indoor environments highlights the need for additional studies to better understand the potential sources of aerosols and viral load present in these settings. it has been previously reported that other coronaviruses were significantly less stable at higher relative humidities, with the half-life for human coronavirus 229e decreasing from 67.3 ± 8.2 hours to 3.3 ± 0.2 hours for relative humidity levels of 50 and 80%, respectively [16] . while a similar effect was not observed for sars-cov-2 in the present study, it is possible that the shorter test durations used in the present study precluded detection of this effect of relative humidity. it is possible that additional tests of longer duration without simulated sunlight would allow a better assessment of the effect of relative humidity on sars-cov-2 in aerosols, but the results of the present study suggest that any such effect would be relatively minor in comparison to the effect of sunlight. previous studies have also demonstrated that numerous other factors can influence the survival of microorganisms in aerosols. in particular, temperature has been shown previously to affect the survival of coronaviruses, including mers, in aerosols [16, 17] . furthermore, while the stability sars-cov-1 and sars-cov-2 in aerosols were shown to be similar under a single set of conditions [15] , other studies have demonstrated that the aerosol stability can vary between related viruses [34] [35] [36] [37] . therefore, additional testing incorporating a range of relevant temperatures and additional isolates a c c e p t e d m a n u s c r i p t of sars-cov-2 should be conducted to better estimate the range of potential decay rates associated with sars-cov-2. it was necessary to concentrate the viral stock used in the present study to ensure that quantifiable concentrations of virus were present in aerosols. however, the addition of the concentrated viral stock to the simulated saliva significantly altered the properties of the simulated saliva, specifically the fractional solids and protein content. thus, while a small difference in decay was observed between the simulated saliva and culture medium in the presence of simulated sunlight, it is possible that the viral suspension diluted in simulated saliva is not representative of the composition of expelled particles in infected individuals. previous studies have shown that particle composition can affect the decay rate of infectious viruses in aerosols [38] [39] [40] . therefore, additional studies world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units detection 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procedures for surfaces when challenged with droplets containing human pathogenic viruses beitrag zur kollektiven behandlung pharmakologischer reihenversuche. naunyn-schmiedebergs archiv für experimentelle pathologie und pharmakologie 1931 the method of 'right and wrong cases'('constant stimuli') without gauss's formulae influenza virus aerosols in human exhaled breath: particle size, culturability, and effect of surgical masks measurements of airborne influenza virus in aerosol particles from human coughs effect of relative humidity on the survival of airborne unicellular algae the use of a rotating drum for the. study of aerosols over extended periods of time factors affecting the viability of air-borne bacteria: i. bacteria aerosolized from distilled water resistance of aerosolized bacterial viruses to four germicidal products effect of ultraviolet germicidal irradiation on viral aerosols the influence of relative humidity on the aerosol stability of different strains of foot-and-mouth disease virus suspended in saliva influenza a of human, swine, equine and avian origin: comparison of survival in aerosol form the survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol decay of influenza a viruses of human and avian origin the effect of relative humidity on the survival of airborne semliki forest virus influenza virus infectivity is retained in aerosols and droplets independent of relative humidity survival of airborne ms2 bacteriophage generated from human saliva, artificial saliva, and cell culture medium a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t table 1 . summary of sars-cov-2 decay at 20°c in aerosols. decay constants (k infectivity ), decay rate, and half-life calculated from the mean k infectivity values are summarized as a function of matrix and simulated sunlight level. decay constants and rates are presented as the arithmetic mean ± standard deviation of each data set. results across different relative humidity levels were pooled since relative humidity was determined not to be a significant factor affecting decay. data from fifty-six tests are included; three tests were not included due to poor linear regression fits of the time-series viral aerosol concentration data. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-329061-1xut73dq authors: bhatt, pravin n.; percy, dean h.; jonas, albert m. title: characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group date: 1972-08-17 journal: j infect dis doi: 10.1093/infdis/126.2.123 sha: doc_id: 329061 cord_uid: 1xut73dq the virus that causes sialodacryoadenitis in rats has been isolated in mice and in primary cultures of rat-kidney cells and has been characterized as a heat-labile rna virus that is sensitive to lipid solvents and is relatively stable at ph 3.0. this virus is antigenically related to the virus of hepatitis in mice and to coronavirus of rats. the range of hosts of this agent appears to be narrow. on the basis of available biologic characteristics, it has been placed in the coronavirus group. cated that neither source had detectable serum antibodies to sda virus and that both were susceptible to infection with this agent. rats were inoculated intranasally with 0.1 ml of virus-infected salivary-gland suspension, observed daily for evidence of overt illness, and sacrificed at various intervals. all rats were maintained in rigid plastic isolators with high-efficiency air filters. tissue culture. three cell lines, baby-hamster kidney (bhk-21), vera, and hep-2, and primary monolayer cultures of rat embryo, rabbit kidney, rhesus-monkey kidney, guinea-pig-embryo skin, muscle, and kidney were used as previously described [l ] . monolayers obtained from explant cultures of submaxillary, parotid, harderian, and exorbital glands of germfree rats, monolayers of trypsin-dispersed brain cells of infant mice, and a line of polyoma-transformed mouse cells (py-al/n) [6j were also tested. at a later stage in the study, primary rat-kidney (prk) cultures prepared from kidneys of weanling charles river cd germfree or conventional inbred dark agouti fda) rats were used. inoculated tubes were kept in a roller drum at 37 c and observed for cytopathic effect (cpe) at intervals of two to four days for at least 21 days. the fluid medium was changed when necessary. in the absence of cpe, a blind passage was made between the eighth and 16th day after inoculation. cultures for passage were observed for one to two weeks for development of cpe, and, in the absence of cpe, vera, bhk-21, and pmk cultures were challenged on the 12th day after inoculation with chandipura [8j virus, an arbovirus of the vesicular stomatitis viral group, for determination of interference. in addition, the fluid from each of the second-passage cultures was inoculated ic into infant mice, and the mice were observed for 21 days. in some instances tissueculture fluid from inoculated tubes was passaged into infant mice without further passages in tissue culture. monolayer cultures of prk, infant-mouse brain, and py-al/n cells were also examined by indirect immunofluorescence for the presence of viral antigen [5] . characterization of the virus. for determination of the effect of 5-bromodeoxyuridine (5-budr), lipid solvent, low ph, and various temperatures, methods described by bhatt et al. [9j were used. the hemagglutination method will be bhatt, percy, and jonas described under results. staining with acridine orange was done according to the method of hsiung [10] . preparation of immune sera. hyperimmune sera were prepared in rats and mice by repeated inoculation of a suspension of salivary glands from infected rats and of brains from infected mice, respectively. complement-fixation test. complement-fixing antigen was prepared by sucrose-acetone extraction from infected brains of two-to four-day-old mice. polyvalent mouse-hepatitis cf antigen prepared in tissue culture was obtained from microbiological associates. the cf test was performed by the micromethod [l l ] using two units of complement and four to eight units of antigen. serum of mice immune to sda strain 681 was tested against 118 viral antigens by the cf test. neutralization test. the neutralization (n) test with sera immune to murine viruses was performed in infant mice, and these animals were observed for 14 days after inoculation. prk cultures were used for cross-n tests, using sda strain 681 and parker's rcv. sera were inactivated at 56 c for 30 min. cultures were examined on the third and fifth days after inoculation. the titers of antibody and virus were calculated by the method of reed and muench [12j. fluorescent-antibody method. pieces of mouse brain 2-4 mm thick were quick-frozen in a dry ice-alcohol bath and stored at -83 c. sections 6-8f!m thick were cut in a cryostat, two sections were mounted per slide, and then the slides were fixed in acetone at 25 c for 15-20 min and dried at 37 c for 15 min. sections were stained immediately or stored at -25 c for 1-30 days before use. tissue-culture cover slips were similarly prepared but at times were kept in chilled acetone at -25 c for 18 hr. the section and cover-slip preparations were reacted with sera immune to virus for 20 min at 25 c and then exposed to mouse or rat antiglobulin conjugate for 20 min. phosphate-buffered saline (pbs) was used for washing. preparations were examined with a carl zeiss microscope fitted with an hbo 200 w/4 supermercury lamp, a ug-5 exciter filter, and a 47/65 barrier filter. histopathology. histologic examination of tissue from inoculated rats included harderian, exorbital, parotid, and submaxillary glands. in suckling mice, coronal sections of brain and serial transverse sections of the thoracic and abdominal regions were examined. attempts to induce disease in weanling mice. sixty female mice, three-to four-weeks old, were given 2.5 mg of cortisone im twice a week beginning a week before inoculation and continuing until the end of the experiment. twenty mice each were inoculated ic and ip with 0.03 ml and 0.1 ml of viral suspension containing 2 x 1()3·9 and 6.3 x 10 3 . 9 infant mouse ld50 (imld50), respectively. twenty control mice were inoculated with diluent, 10 by the ic route and 10 by the ip route. another group of controls was neither inoculated nor given cortisone. six mice from each group were killed for histologic studies on the seventh day after inoculation and two were killed on the 14th day after inoculation. remaining mice were observed until the 21st day after inoculation, when the experiment was terminated. a complete necropsy was done on each mouse. induction of sialodacryoadenitis in susceptible rats by mouse-brain-adapted virus. eleven rats, weighing 250 g and from a colony known to be susceptible to sda virus, were inoculated by the intranasal route with fourth passage, infectedmouse-brain material. the inoculum contained approximately 6.6 x 10 3 . 8 imld 50 of virus. rats were sacrificed on the fifth, sixth, and eighth days after inoculation. harderian and submaxillary glands were processed for isolation of virus and histologic examination, whereas parotid gland was collected for histologic examination only. adaptation to mice and related observations. a 10% suspension of infected salivary glands was inoculated ic into one-day-old mice. one mouse was sick on the fifth day after inoculation, eight more were sick on the seventh day after inoculation, and six on the eighth day after inoculation. some of these animals were killed, and tissues were harvested for passages and histologic study, but mice that were sick but not killed died on the 10th day after inoculation. one mouse was unaffected and survived until the 21st day, when it was discarded. the disease was characterized by ataxia and uncoordination, followed by paresis, paralysis, and death. the same pattern of illness was observed on further passages. by the fifth mouse-brain passage, the incubation period was 125 shortened to two to three days. there was usually a random pattern of illness and death from two to eight days and occasionally up to 10 days after inoculation. the pattern has remained unchanged for 29 passages with this strain of virus. one other observation made during the first passage in mice and amply confirmed during subsequent work was emaciation of sick mice as compared to uninoculated control mice of the same age. these differences were more marked in mice that were two to four days old or older when inoculated. other significant observations can be summarized as follows: (l) the agent of sda does not cause detectable illness in weanling (three-to four-week-old) mice when inoculated ic or ip or in infant mice inoculated ip. (2) a comparative titration was done in mice two days old, 13 days old, and 22 days old that were inoculated ic with viral stock passaged 12 times in mouse brain. titers were 10 4 . 00, 10 4 . 25 , and <10 2 . 0 imld50/0.015 mi, respectively. (3) virus has undergone 29 serial ic passages in zero-to six-day-old mice, the cumulative dilution of which exceeds 10-100. (4) the titer of virus between the fifth and 29th passage in mouse brain has remained relatively stable at 10 3 . 5_105.0 imld50/0.015 ml (usually around 1q3·7 imld50/0.015 ml). (5) the original salivary-gland suspension was titrated in one-day-old mice and had a titer of 1()3·6 imld 50/o.015 ml. (6) when inoculated intranasally into susceptible rats, mouse-brain-adapted virus produced sialodacryoadenitis. (7) brains from two uninoculated mice (two days old) were harvested as controls; seven serial ic passages of this material were made at intervals of six to seven days in mice three to four days old. no agent pathogenic for mice was isolated from these control animals. histopathologic and immunofluorescent observations in inoculated mice. in general, histologic changes observed in the central nervous system of inoculated mice were characterized by diffuse and focal neuronal degeneration with minimal inflammatory cell response. regions of brain most frequently involved were the cortices of the occipital and parietal lobes. other foci of neuronal destruction were scattered elsewhere in the central nervous system; there was relatively little destruction 0 some important observations are summarized as follows. (l) cultures were most sensitive when used within a week after seeding; then sensitivity decreased. the cpe was delayed and less extensive in older cultures. (2) development of virus in prk cells was monitored by cpe, detection of viral antigen by indirect immunofluorescence, and quantitation of infectious virus in prk tubes. results are presented in table 1. significantly, detectable viral antigen developed by 12 hr and was followed by release of infectious virus into the medium. cpe was detected at 24 hr. beyond 24 hr, quantitation of viral antigen was difficult due to lysis of cell sheets, and after 36 hr, titer of infectious virus decreased. (3) the sensitivity of inoculation of mice ic with strain 681 virus was compared with that of inoculation of prk cultures. titers obtained with a mouse-brain-adapted virus were 2.5 x 10 4 imld50 in mice and lox 10 4 . 3 tcid50 in prk cultures. similar differences were also noted in other experiments. characterization of the agent. the effect of 5-budr on viral multiplication was determined by the method of bhatt et al. [9] . chandipura virus was used as rna control (p. n. bhatt, unpub-hour in the cerebellum. affected neurons were pyknotic and densely eosinophilic. in addition, there was a scattering of shrunken, densely staining astrocytes in these areas. occasionally there was hypertrophy and hyperplasia of capillary endothelial cells and minimal perivascular cuffing with mononuclear cells. sometimes a few polymorphonuclear leukocytes were scattered in areas of destruction. spinal cord, salivary glands, lung, heart, liver, kidney, spleen, and intestine were histologically normal. immunofluorescence procedures detected viral antigen in regions where frank cellular necrosis was seen by standard histologic techniques. in addition, intense staining was observed in neuronal cytoplasm of scattered cells that were intact and not associated with frank necrosis. serial coronal sections had immunofluorescence staining in dorsal cortical areas, the ventral portion of ammon's horn, the hypothalamus, and the brain stem, but fluorescence was rarely found in cerebellar folia and white matter. a ttempts to adapt the agent to monolayer cell cultures. the original salivary-gland suspension inoculated onto various monolayer cell cultures produced no detectable cpe up to 21 days after inoculation. when blind passages were made and cultures were challenged with chandipura virus [8] , interference was not observed. fluid from the second passage in tissue culture was inoculated ic into infant mice; the results were negative. attempts were made to propagate mouse-brainadapted virus to cell-culture systems, such as monolayers obtained from explant cultures of parotid, harderian, exorbital, and submaxillary glands of germfree rats and monolayers of trypsindispersed infant-mouse-brain cultures. there was no detectable cpe. similar results were obtained with the py-al/n cell line. infectious virus or viral antigen was not detected when tissue-culture fluids from infected-mouse-brain and py-al/n cultures were inoculated ic into infant mice or when monolayers were examined by indirect immunofluorescence. however, prk cultures showed cpe characterized by formation of multinucleated giant cells, which were seen as highly reflective masses. these cells fell off the glass wall a few hours later and were seen floating in medium. tissue-culture fluids of these cultures contained virus as detected in infant mice, and cultures were positive for viral antigen by indirect immunofluorescence. sensitivity of sda virus to a lipid solvent was also tested. the titer of virus was 10 3 . 8 and < 10 2 . 0 imld50/0.015 ml for controls and chloroformtreated samples, respectively. the test was repeated with similar results, and it was concluded that the agent is sensitive to lipid solvents. effect of low ph on infectivity. the test was performed as described by leibhaber [13] . tenfold serial dilutions of infected-mouse-brain suspension kept at different ph values were made in eagle's minimal essential medium in earle's base with 3% fetal bovine serum (fbs). the ph of each dilution was adjusted to approximately 7.0 by addition of tris, and this solution was inoculated into mice. end points of infectivity were calculated by the method of reed and muench [12] . the titers of infectious virus detected in pbs after incubation for 3.0 hr at 25 c was 103. 6 imld50/0.015 ml, whereas at ph 7.0 and ph 3.0 it was 10 3 . 9 imld50/0.015 m1 and lq2·8 1mld50/ 0.015 ml, respectively. thus infectious virus was relatively stable at low ph. effect of temperature on infectivity. the effect of a temperature of 37 c on infectious virus was determined as outlined by bhatt et al. [9] . to determine the effect of a temperature of 56 c, infectivity was determined at intervals of 0, 5, and 10 min. an aliquot of viral stock was kept at 4 c, and infectivity was determined on days 0, 7, and 28. infectivity of viral strain 681 was stable in pbs plus 3% fbs at 37 c for 3 hr; the titer then decreased by 1.1 log., by 5 hr. at 56 c the infectivity decreased from 10 3 . 8 1mld50 at zero time to trace levels by 5 the size of infectious viral particles. the approximate size of infectious viral particles was determined by the method of atoynatan and hsiung [14] and casals [15] as modified by bhatt et al. [9] . a fresh, 10% suspension of mouse brain was made in pbs plus fbs, clarified by centrifugation at 1,000 g for 20 min, and filtered through miliipore filters (millipore corp., bedford, mass.) of various pore sizes. viral titers obtained were 103. 5, 104.2, 103. 8 , 10 3 . 0, and < lq2 1mld50/0.015 m1 for unfiltered virus and after filtration through pore sizes 1,200 urn, 450/lm, 220/lm, and 100 urn, respectively. results indicate that the size of the virus is less than 220 urn but greater than 100 urn. the particles without membranes measured by electron microscopy were previously reported to be 6<f--70 /lm [1] . detection of hemagglutination. two sources of antigen were used. (1) a 10% suspension of infected salivary gland was tested for its capacity to hemagglutinate red blood cells (rbcs) of rabbits, guinea pigs, and geese by the method of ashe [16] . (2) either a 10% suspension of infected mouse brain in pbs or infected mouse brain extracted with sucrose and acetone was tested for hem agglutinability with rbcs of rats, mice, guinea pigs, and geese. the microtiter method [11] was used for all tests, and incubation was at 4 c, 25 c, and 37 c. rbcs were suspended (0.5%) in pbs. hemagglutination was not detected at 4 c, 25 c, or 37 c using a suspension of infected salivary gland and rabbit, guinea pig, or goose cells. a 10% suspension of infected brain in pbs or sucrose-acetone-extracted antigen from mouse brain gave unsatisfactory results with rbcs of rats, guinea pigs, and mice at 25 c and 37 c, and there was no ha activity after incubation overnight at 4 c. neither was ha detected for goose rbcs at 4 c, 25 c, or 37 c. acridine-orange staining. the cytoplasm of infected cells stained orange-red with acridine orange, indicating that virus replicates in the cytoplasm. antigenic relationship to other viruses. results of these experiments are summarized as follows. (1) serum-neutralization tests were conducted with strain 681 sda virus with immune sera to * names of the individual arboviruses will be furnished upon request. theiler encephalomyelitis virus (strain gd vii), sendai, mvm, and mouse adenovirus were negative. immune sera to toolan h-l virus, kilham rat virus, and simian myxovirus sv 5 (at dilutions of 1: 5) did not neutralize sda virus. (2) sera of mice immune to strain 681 were tested by cf at dilutions of 1: 4, 1: 8, and 1: 16 for reactivity to 118 viral antigens. these agents are listed in table 3. there was no reaction, indicating absence of antigenic relationship between our agent and these 118 agents under the conditions of the cf test. (3) a checkerboard cross-cf test was performed, using two different sera from mice immune to sda agent and commercial mhv antigen and corresponding immune serum. on the table 5 . results of cross-neutralization tests with sialodacryoadenitis virus (sda) and rat coronavirus (rev) and respective immune sera. (4) cross-neutralization tests were performed with parker's rat coronavirus and strain 681, using both homologous and heterologous immune sera. as shown in table 5, there is cross-neutralization, but there are also antigenic differences between these viruses. induction of disease in weanling mice. neither overt illness nor any gross or histologic evidence of lesions was associated with this agent. more specifically, there were no lesions compatible with infection by mhv either in the group treated with cortisone or in the untreated group. production of sda in susceptible rats by mousebrain-adapted strain 681. there was no overt illness in any rat, and at necropsy all organs, including the lacrimal and salivary glands, were grossly normal. histopathologic examination of the harderian, exorbital, parotid, and submaxillary salivary glands showed evidence of sda. there was considerable variation in the severity of the reaction of affected glands. in general, lesions were most numerous and most severe in the parotid and exorbital glands. two important features of this study were the relative severity of lesions in the parotid salivary glands and the failure to isolate an agent pathogenic for mice from these animals. these findings suggest that the mouse-brainadapted virus caused mild but definite lesions compatible with sda. it has been approximately 10 years since the first recognized outbreak of sda in rats. on the basis of the information presented in this report, it is concluded that a viral agent causing this disease in rats has been isolated and adapted to grow in brains of infant mice and in primary rat-kidney cultures. the agent probably has rna as its nucleic acid and is sensitive to lipid solvents. it is antigenically related to the rat coronavirus of parker and to mouse-hepatitis virus. it apparently multiplies in the cytoplasm and forms multinucleated giant cells in tissue culture. some coronaviruses are acid labile, but the sda agent is relatively stable at ph 3.0. the virus of transmissible gastroenteritis is also stable at ph 3.0, yet is considered a coronavirus [17] . thus ph stability may be a variable feature of this group. on the basis of this information, we conclude that this virus belongs to the corona group, even though information on the morphology of the negatively stained viral particle and its mode of replication in cells (as determined by electron microscopy) has yet to be obtained. in the affected tissues, the particles are frequently found in cytoplasmic vesicles that eventually contain lysosomal activity (a. m. jonas, unpublished observation). the titer of infectious virus in mouse brain was not increased by serial passage. this may be a reflection of the facts that only selected cells are involved in viral multiplication and that there may be a low yield of infectious virus per infected cell. (the former supposition has been confirmed by detailed histopathologic study supplemented by fluorescent-antibody tracing of infected cells.) when working with mouse-adapted viruses of rats, it is essential to prove that one has not picked up agents from mice. evidence against this possibility includes the following. (l) sickness in mice is produced only when the animals are inoculated with a suspension of infected salivary gland and not when normal salivary gland is used. (2) agents pathogenic for mice were not recovered when brains from normal mice were passaged consecutively several times by the intracerebral 129 route in mice. (3) dr. r. e. shope tested 118 viral antigens prepared from infected mouse brains with antiserum to sda virus. these mice were from the same colony used for our work. he did not find any reaction in cf tests with immune serum to sda virus. (4) immune sera prepared in rats with virus passaged in salivary glands reacted with antigen from murine brain, while sera taken from the same rats before immunization did not. (5) sda was reproduced in rats by inoculation of mouse-brain-adapted virus. failure to adapt sda virus to tissue-culture systems other than primary rat-kidney cultures confirms the observation that coronaviruses are fastidious in their cultural requirements [18] . absence of viral multiplication in mono1ayers of ratsalivary-gland and mouse-brain tissue may be due to absence in culture of cells that normally support growth of virus in the intact host. complement-fixing and neutralizing antibodies to mhv were detected in the sera of men and rats by hartley et al. [19] and were interpreted as indication of infection by antigenically-related, species-specific viruses. this hypothesis has been confirmed in part by isolation of coronaviruses from man [18] . sda agent and parker's rat corona agent may be responsible for antibodies to mhv found in rats. biologically, sda virus behaves differently from known strains of mhv, since the former does not multiply in the py-al/ n cell line, which is highly susceptible to mhv [6] . in addition, sda virus does not induce lesions compatible with those due to mhv if inoculated ic into infant mice or if inoculated ic or ip into cortisone-treated weanling mice. therefore, our failure to detect antigenic differences between these agents by the complement-fixation test should be interpreted with caution. more information may be obtained for serologic differentiation by use of immune sera prepared by different schedules of immunization and then tested by neutralization, complement-fixation, fluorescent-antibody, and gel-diffusion methods. since there is more than one serotype of human and mouse coronavirus [18, 20] , it is not surprising that, although there is much crossneutralization, there seem to be antigenic differences between our virus and the one isolated by parker et al. [4] . several serotypes of rat coronaviruses may therefore exist. it is necessary to delineate further the serotypes, the disease poten-tials, and the epidemiology of both agents. for example, our agent is known to cause sda, while parker's rat coronavirus causes pulmonary lesions. the ability of mouse-brain-adapted virus to produce the disease in rats indicates that we are dealing with the same agent. an evaluation of the glands involved and the extent of involvement may indicate a shift in tissue tropism and also an apparent reduction in virulence of virus. sda virus was not recovered from submaxillary salivary glands when mouse-adapted-virus was inoculated into rats, but a change in tissue tropism may have accounted for this difficulty. attempts were not made to isolate virus from parotid and exorbital glands, but histopathologically they demonstrated classical lesions. these tissues were only minimally involved in previous studies with the strain passaged in rats. the apparent shift in tissue tropism needs confirmation in both germfree and conventional, susceptible rats of the same strain, particularly since the transmission experiments were performed in susceptible, specific-pathogenfree wistar rats. sialodacryoadenitis in the rat. a light and electron microscopic study studies on the agent of sialodacryoadenitis of rats proc. p rat coronavirus (rcv): a prevalent, naturally occurring pneumotropic virus of rats. arch. gesamte virusforsch fluorescent antibody methods enhanced growth of murine corona viruses in cells transformed by polyoma virus proc. p tissue culture technics for diagnostic virology chandipura, a new arbovirus isolated in india from patients with febrile illness isolation and characterization of a herpeslike (hsiung-kaplow) virus from guinea pigs herpes and pox viruses: laboratory procedures application of a microtechnique to viral serological investigations a simple method of estimating fifty per cent endpoints studies of a myxovirus recovered from patients with infectious hepatitis. i. isolation and characterization. 1 ultrafiltration of simian viruses casals, 1. filtration of arboviruses through "millipore" membranes previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats diseases of swine corona viruses of man antibodies to mouse hepatitis viruses in human sera viruses of laboratory rodents. national cancer institute monograph no key: cord-011722-82qzf8ht authors: keitel, wendy a.; piedra, pedro a. title: influenza a(h5n1) vaccines: are we better prepared for the next pandemic? date: 2014-01-01 journal: j infect dis doi: 10.1093/infdis/jit573 sha: doc_id: 11722 cord_uid: 82qzf8ht nan the ongoing epizootic of influenza a(h5n1) infections has resulted in 641 reported human cases since 2003, 380 (59%) of which resulted in death [1] . these cases have been reported to the world health organization (who) from 15 countries in asia, africa, the pacific, europe, and the near east, and influenza a(h5n1) viruses continue circulate and evolve in poultry in asia and northeast africa. most infections have been the result of exposure to sick or dead poultry; however, limited humanto-human spread has occurred. although these viruses are capable of producing infection and illness in humans and most individuals lack of immunity, to date their pandemic potential has been limited by their inability to transmit readily from an infected person to others. international efforts are ongoing to prepare for a possible influenza a(h5n1) pandemic. four influenza pandemics have occurred since 1918, and the impact of these pandemics varied according to age group. for example, death rates in the 1918 pandemic were highest among persons aged <45 years, whereas death rates during the 1957 and 1986 pandemics were highest among persons aged >65 years. infection rates during the 2009 influenza a(h1n1) pandemic were highest among children. the epidemiology of human influenza a(h5n1) cases varies from country to country, but the median age of patients infected with influenza a(h5n1) viruses is approximately 18 years [2] . hence, a substantial burden of illness occurs in the pediatric population. should an influenza a(h5n1) pandemic occur and the epidemiological pattern of past influenza a(h5n1) infections persist, efforts must be focused on protecting this vulnerable population [3] . this is particularly important because children play an important role in the community-wide spread of influenza [4] . immunization is the cornerstone to the control of seasonal and pandemic influenza. the results of numerous clinical trials of candidate influenza a(h5n1) vaccines in adult populations have yielded several common themes. the avian h5 hemagglutinin (ha) is poorly immunogenic relative to seasonal has, 2 doses of vaccine are necessary to elicit serum antibodies in most subjects, and inclusion of an adjuvant (particularly oil-in-water emulsions) or use of whole-virus (wv) preparations can increase the frequency and magnitude of antibody responses and reduce the amount of vaccine antigen required to achieve antibody levels that have been associated with protection (ie, antigen-sparing approaches). in most cases in which mineral-containing adjuvants such as aluminum hydroxide were evaluated, little additional benefit was observed [5] . limited studies of live attenuated (laiv) influenza a (h5n1) vaccines have shown restricted replication and lower immunogenicity than that seen after immunization with seasonal laiv [6, 7] . the process of influenza a(h5n1) vaccine development is complicated by the ongoing evolution of influenza a(h5n1) viruses [8] . numerous clades now circulate among birds, and which one, if any, will acquire the ability to transmit easily from human to human is unknown. several studies have explored the effect of priming with one clade (1 or 2 doses) and boosting with another clade. these studies have shown that prior priming results in more broadly cross-reactive antibodies and higher levels of antibody than those observed after priming. the results of clinical trials of influenza a(h5n1) vaccines among children are consistent with those seen in adult populations. in this issue of the journal, van der velden et al report that a vero cell culture-derived wv influenza a(h5n1) vaccine derived from wild-type virus was well tolerated in infants and children aged 6 months to 8 years [9] . two doses containing 7.5 μg of a/vietnam(h5n1) ha elicited neutralization antibody titers associated with protection in animal models in 68.8%, 72.9%, and 85.4% in children aged 6-35 months, 3-8 years, and 9-17 years, respectively. booster immunization at 1 year with a/indonesia(h5n1) vaccine elicited the same neutralization antibody levels against both strains in ≥93%, ≥95%, and 100%, respectively. single radial hemolysis antibody assays indicated that 2 priming doses containing 7.5 μg of ha elicited responses that satisfied licensure thresholds established by european authorities (european medicines agency). high rates of seropositivity to the neuraminidase (na) also were observed following immunization. similar to studies conducted in adults with wv and adjuvanted or nonadjuvanted subvirion or purified recombinant ha influenza a(h5n1) vaccines, boosting with vaccine derived from another influenza a(h5n1) clade elicited higher and more broadly cross-reactive antibodies. immunization was reported to be well tolerated. fever was reported among 17%-19% of children in the youngest age stratum and was less frequent after the second and boosting doses. the results of this and other studies conducted among all age groups raise important questions regarding issues related to assessing the immunogenicity of both seasonal and pandemic vaccines. serum hemagglutination inhibiting (hai) antibodies have been regarded as a benchmark for assessing the immunogenicity of seasonal influenza vaccines by the food and drug administration [10] . serum hai antibody responses following immunization with the wv vaccines described in the article were low and not reported, making comparisons between results from this trial to results from other trials difficult. furthermore, neutralization antibody responses were lower than those observed following immunization of children with vaccines containing oil-in-water adjuvants (mf59 and as03). lack of standardization of hai and neutralization antibody assays is well recognized, and repeated calls for international cooperation to develop more reproducible assays have been made [11] . however, these assays will need to be clade specific, which poses a major hurdle. even if assays could be standardized, our understanding of the correlates and determinants of protection against both seasonal and pandemic influenza is far from complete. an hai titer of ≥40 has been associated with a 50% reduction in the risk of infection in adults and is used as a major immunization target. however, the level of antibody may vary with age such that hai levels of ≥110 appear necessary for children [12] . even less is known about the level of neutralization antibody required for protection. na antibody, cd4 + t-cell responses, and mucosal antibody responses all may contribute to protection against influenza a(h5n1) and/or other influenza virus infections [13] [14] [15] . identification of epitopes that stimulate broadly cross-reactive immune responses is a high priority, and vaccines prepared using these epitopes are in development [16] . another important consideration is vaccine safety. the use of an as03-adjuvanted 2009 influenza a(h1n1) vaccine was associated with an increased risk of narcolepsy, an autoimmune disease, particularly among adolescents [17] . other rare adverse events may only be observed when millions of people are exposed, as in a pandemic setting. in 1976, largescale immunization of the us population with another influenza a(h1n1) vaccine was associated with an increased risk of an autoimmune disease known as guillain-barré syndrome [18] . wv vaccines have been associated with an increased risk of fever in young children, but the outcomes have not been serious. depending on the severity of a future pandemic, risk/benefit assessments will need to be made with regard to which vaccines can be used in various age and risk groups. we will need all available manufacturing capacity and accelerated manufacturing approaches to produce and distribute vaccine to the entire population if a pandemic occurs. on the basis of the 2009 influenza a(h1n1) pandemic experience, it took about 6 months from the recognition of a pandemic virus to the start of delivery of pandemic vaccines (live and inactivated) to the population. this was truly a herculean feat by all the partners involved, although not fast enough to have an impact on the major fall wave of the pandemic. finally, several prepandemic influenza a(h5n1) vaccines have been licensed and are being stockpiled. it is time to tackle the difficult issue regarding the use of these vaccines before a pandemic occurs. the benefits include priming for robust responses to the actual pandemic strain, obviation of the need for 2 doses under difficult if not extreme circumstances, and expansion of the safety database. risks include adverse events associated with vaccination and diversion of limited resources in the absence of a pandemic. the strategic advisory groups of experts on immunization, which provides guidance on the work of the who immunization, vaccines, and biologicals department, has developed risk-based recommendations for the use of prepandemic vaccines [19] , as follows. vaccination is strongly recommended for laboratory workers who are at high risk of infection and for first responders to avian outbreaks and healthcare professionals in enzootic areas, and it may be made available to other laboratory workers and other healthcare professionals. notably, vaccination with influenza a(h5n1) vaccine is not recommended for the general population or other essential workers in enzootic or nonenzootic areas. as we learned in 2009, it is not possible to predict with any certainty what virus will cause the next pandemic. influenza a(h7n9) viruses recently caused infections in china [20] , and influenza a(h3n2) variant (swine), influenza a(h7n7), and influenza a(h9n2) viruses have caused human infections in recent years. influenza a(h2n2) was responsible for a pandemic in 1957; thus, some experts consider this subtype to be of major concern [21] . vaccines containing both seasonal and candidate pandemic vaccine antigens are being developed, but which viruses to include, in whom, and when is a matter for serious consideration. although vaccine development is a critical component of a program designed to prepare for the next pandemic, stockpiling of antivirals and antibiotics, development of plans for distribution of vaccines and antimicrobials, and ensuring the availability of adequate supplies of effective countermeasures are but a few of the critical approaches that will be necessary [22] , but are they sufficient to prepare the global community for the next pandemic? let the discussion continue! cumulative number of confirmed human cases of avian influenza a(h5n1) reported to who. www.who.int/influenza/human_ animal_interface/en_gip_20131008cumulative numberh5n1cases writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h5n1) virus. update on avian influenza a (h5n1) virus infection in humans prioritization of influenza pandemic vaccination to minimize years of life lost emerging infections: pandemic influenza pandemic h5n1 influenza vaccine development: an update evaluation of two live attenuated cold-adapted h5n1 influenza virus vaccines in healthy adults safety and immunogenicity of live attenuated influenza reassortant h5 vaccine ( phase i-ii clinical trials) the emergence and diversification of panzootic h5n1 influenza viruses safety and immunogenicity of a vero cell culture-derived whole-virus influenza a(h5n1) influenza vaccine in a pediatric population guidance for industry: clinical data needed to support the licensure of seasonal inactivated influenza vaccines. www.fda.gov/biologics bloodvaccines/guidancecomplianceregulatory information/guidances/vaccines/ucm074794. htm reproducibility of serologic assays for influenza virus a (h5n1) hemagglutination inhibition antibody titers as a correlate of protection for inactivated influenza vaccines in children can immunity induced by the human influenza virus n1 neuraminidase provide some protection from avian influenza h5n1 viruses? correlates of immune protection induced by live, attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine preexisting influenza-specific cd4+ t cells correlate with disease protection against influenza challenge in humans correlates of protection against influenza infection in humans-on the path to a universal vaccine as03's adjuvanted ah1n1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in finland guillain-barre syndrome following vaccination in the national influenza immunization program, united states, 1976-1977 recommendations on the use of licensed human h5n1 influenza vaccines in the interpandemic period emergence of avian influenza a (h7n9) virus causing severe human illness-china vaccinate for the next h2n2 pandemic now planning for an influenza pandemic: thinking beyond the virus potential conflicts of interest. p. a. p. is on the speakers bureau for medimmune. w. a. k. certifies no potential conflicts of interest.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-293579-w5sub348 authors: che, xiao-yan; qiu, li-wen; liao, zhi-yong; wang, ya-di; wen, kun; pan, yu-xian; hao, wei; mei, ya-bo; cheng, vincent c. c.; yuen, kwok-yung title: antigenic cross-reactivity between severe acute respiratory syndrome—associated coronavirus and human coronaviruses 229e and oc43 date: 2005-06-15 journal: j infect dis doi: 10.1086/430355 sha: doc_id: 293579 cord_uid: w5sub348 cross-reactivity between antibodies to different human coronaviruses (hcovs) has not been systematically studied. by use of western blot analysis, indirect immunofluorescence assay (ifa), and enzyme-linked immunosorbent assay (elisa), antigenic cross-reactivity between severe acute respiratory syndrome (sars)—associated coronavirus (sars-cov) and 2 hcovs (229e and oc43) was demonstrated in immunized animals and human serum. in 5 of 11 and 10 of 11 patients with sars, paired serum samples showed a ⩾4-fold increase in antibody titers against hcov-229e and hcov-oc43, respectively, by ifa. overall, serum samples from convalescent patients who had sars had a 1-way cross-reactivity with the 2 known hcovs. antigens of sars-cov and hcov-oc43 were more cross-reactive than were those of sars-cov and hcov-229e. are generally recognized to cause mild upper respiratory tract diseases and, rarely, lower respiratory tract diseases [2] . phylogenetic analyses show that sars-cov is not closely related to any of the previously characterized covs [3] . however, some investigators, using sars-cov-infected vero cells in immunohistochemical antibody tests, have observed cross-reactions between sars-cov and group i covs, but seroepidemiological studies revealed that there were no cross-reactions with sars-cov-infected vero cells in 13 and 14 paired serum samples from patients with hcov-oc43 and hcov-229e, respectively [1] . serum samples from group i cov-infected animals also cross-reacted with the recombinant nucleocapsid protein of sars-cov [4] . results of the recombinant nucleocapsid protein-based elisa were positive in 1.04% of serum samples from healthy blood donors [5] . the nature of this antigenic cross-reactivity is still unknown. in the present study, we cloned the nucleocapsid genes of sars-cov, hcov-229e, and hcov-oc43 and produced specific animal antisera to determine if the nucleocapsid protein is responsible for the observed antigenic cross-reactivity. in addition, the antigenic relationships among sars-cov, hcov-229e, and hcov-oc43 were further studied using serum samples from healthy donors and patients with sars. subjects, materials, and methods. hcov strains 229e (atcc vr740) and oc43 (atcc vr759) were maintained in normal human fetal lung fibroblast cells (mrc-5; atcc ccl-171) and african green monkey kidney cells (bsc-1; atcc ccl-26) in mem (gibco brl) supplemented with 10% fetal bovine serum (gibco brl). a sars-cov (hku-39849) strain isolated from a patient with sars in hong kong was inoculated into vero e6 cells as described elsewhere [6, 7] . all experiments with live viruses were performed in a biosafety level 3 laboratory. murine monoclonal antibodies specific for the nucleocapsid proteins of hcov-229e and hcov-oc43 were obtained from a commercial source (chemicon international). a murine monoclonal antibody specific for the nucleocapsid protein of sars-cov was produced in our laboratory [8] . antiserum to whole virus was prepared in female new zealand white rabbits as described elsewhere [9] . the cdna fragments of the nucleocapsid protein of the 3 covs were cloned into the prokaryotic expression vector pqe30 (qiagen) in frame and upstream of the 6 histidine (his 6 ) residue series, and the his 6 -tagged nucleocapsid proteins were expressed and purified using an ni-nta affinity column (qiagen) in accordance with the manufacturer's instructions. the expressed recombinant nucleocapsid proteins were identified by western blot analysis as described elsewhere [8] . sars-cov-specific igg was identified using a commercially available indirect immunofluorescence assay (ifa) kit (euroimmun) in accordance with the manufacturer's instructions. hcov-229e-and hcov-oc43-specific igg was identified by an inhouse ifa, as described elsewhere [7] , that was modified to use hcov-229e-infected mrc-5 cells and hcov-oc43-infected bsc-1 cells, respectively. igm and igg antibodies to sars-cov were identified using an elisa test kit (huada gbi biotechnology) in accordance with the manufacturer's instructions. the nucleocapsid protein-based elisa was performed as described elsewhere [6] . to ensure biosafety, experiments using serum samples from patients were performed in a biosafety level 2 laboratory. one hundred serum samples were collected randomly from healthy adult donors in october 2003. serum samples were collected from 34 patients with sars 8-81 days after the onset of symptoms. paired serum samples were obtained from 11 of these patients who exhibited seroconversion (table 1) , from whom the acute-and convalescent-phase serum samples were collected on days 1-7 and days 12-159 after the onset of symp-toms. sars was diagnosed in accordance with the world health organization's criteria and was confirmed by assessment of seroconversion or a у4-fold increase in antibody titers against sars-cov by ifa. results. the full lengths of 3 nucleocapsid genes from hcov-229e, hcov-oc43, and sars-cov were amplified with their corresponding primer pairs. the gene sizes were 1182, 1359, and 1281 bp for hcov-229e, hcov-oc43, and sars-cov, respectively. the recombinant plasmids were sequenced, and they were all in frame and had sequences matching those of the nucleocapsid genes of the 3 covs. the expressed recombinant his 6 -tagged n-terminal nucleocapsid proteins were identified by western blot analysis using anti-his monoclonal antibodies. the immunoreactive protein bands with expected sizes are shown in figure 1a . the nucleocapsid proteins of hcov-229e, hcov-oc43, and sars-cov reacted strongly and specifically with the rabbit serum immune to the corresponding nucleocapsid protein as immunoreactive protein bands on the western blot (figure 1b). no cross-reactivity was demonstrated among the nucleocapsid proteins of hcov-229e, hcov-oc43, and sars-cov. these results indicate that no substantial antigenic cross-reactivity occurred among the nucleocapsid proteins of hcov-229e, hcov-oc43, and sars-cov when immune rabbit serum was used. sars-cov-immune rabbit serum reacted very strongly with sars-cov-infected cells, moderately with hcov-229e-infected cells, and weakly with hcov-oc43-infected cells by ifa. conversely, hcov-229e-immune rabbit serum reacted very strongly with hcov-229e-infected cells but did not react with either sars-cov-or hcov-oc43-infected cells. hcov-oc43-immune rabbit serum reacted very strongly with hcov-oc43-infected cells and strongly with hcov-229e-infected cells but did not react with sars-cov-infected cells. furthermore, sars-cov-and hcov-oc43-immune rabbit serum showed weak fluorescent signals from uninfected mrc-5 and bsc-1 cells, compared with the response in nonimmune rabbit serum. to determine the serological response to nucleocapsid proteins of the 3 covs, 100 serum samples collected from healthy donors and 34 serum samples collected from patients with sars were tested with recombinant nucleocapsid proteins of hcov-229e, hcov-oc43, and sars-cov using a western blot analysis. the serum samples from healthy donors showed strong reactivity to the nucleocapsid proteins of hcov-229e and hcov-oc43, with positive results in 97% and 99% of the samples, respectively. only 2 samples (2%) reacted with the sars-cov nucleocapsid protein. in contrast, the serum samples from patients with sars obtained 8-81 days after the onset of symptoms showed strong immunoreactivity to the nucleocapsid proteins of hcov-229e, hcov-oc43, and sars-cov, with positive results in 97%, 100%, and 100% of the samples, respectively. when cov-infected cells were used, results were positive for igg antibodies by ifa in 98% (hcov-229e), 100% (hcov-oc43), and 1% (sars-cov) of the serum samples from healthy donors. two samples from healthy donors had no antibody response to hcov-229e by ifa and no antibody response to the nucleocapsid protein of hcov-229e by western blot analysis. one sample collected in october 2003 had an antibody response to sars-cov by ifa and to the nucleocapsid protein of sars-cov by western blot analysis. however, 100% of the samples from 34 patients with sars had antibody responses to hcov-229e, hcov-oc43, and sars-cov. in healthy donors, the results of the ifa showed the presence of antibodies in response to the nucleocapsid proteins of hcov-229e and hcov-oc43 in association with the presence of igg antibodies to both hcovs, but antibodies to sars-cov were absent in all samples except 1, which had a low antibody titer of 1:10, compared with the usual antibody titer of at least 1:100 in most patients with sars. the serum samples from patients with sars had antibody responses to sars-cov as well as to hcov-229e and hcov-oc43 when nucleocapsid proteins were used in the western blot analysis and when cov-infected cells were used in the ifa. paired serum samples from 11 patients with sars were used to determine antigenic relationships among the 3 covs by ifa and elisa. the antibody titers to hcov-229e, hcov-oc43, and sars-cov are shown in table 1. to determine which viral antigen was responsible for the cross-reactions, the culture filtrate from cells infected with hcov-229e and hcov-oc43 was immunoblotted with the paired serum samples. a strong band at ∼44 kda, the same molecular weight at which there was a reaction with a specific monoclonal antibody to the nucleocapsid protein of hcov-229e, was observed in convalescent-phase samples from 2 patients with sars ( figure 1c) . these serum samples also displayed higher antibody titers in the hcov-229e nucleocapsid protein-based elisa. the paired serum samples from the 9 other patients also had a weak or moderate reactive band at ∼44 kda when they were immunoblotted with hcov-229e. acute-phase or convalescent-phase serum from 11 paired serum samples had a weak reactive band at ∼50 kda, the same molecular weight at which there was a reaction with a specific monoclonal antibody to the nucleocapsid protein of hcov-oc43 when it was immunoblotted with hcov-oc43 (data not shown). our results indicate that no nucleocapsid protein antigenic cross-reactivity was found between sars-cov and rabbit serum immune to either hcov-229e or hcov-oc43. in our previous studies, neither specific monoclonal nor polyclonal antibodies to the nucleocapsid protein of sars-cov crossreacted with hcov-229e or hcov-oc43 [8, 10] . however, a previous study has described cross-reactivity between the nucleocapsid proteins of sars-cov and those of group i covs [4] . this cross-reactivity may depend on the type of serum used. another investigator has demonstrated that hcov-229e-immune animal serum cross-reacted with sars-cov [1] . in the present study, when we used immunofluorescent staining of cov-infected cells, hcov-229e-and hcov-oc43-immune rabbit serum did not cross-react with sars-cov-infected cells, whereas sars-cov-immune rabbit serum had moderate crossreactivity with hcov-229e-infected cells and weak cross-reactivity with hcov-oc43-infected cells. although sars-covimmune rabbit serum that had high antibody titers for sars-cov was slightly contaminated with antibodies to host cell components, it is apparent that the serum had cross-reactivity with hcov-229e and hcov-oc43. in addition, the ifa showed that hcov-oc43-immune rabbit serum had strong cross-reactivity with hcov-229e. this cross-reactivity has been observed by some investigators [11, 12] but not by others [13, 14] . it is possible that some antibodies in the rabbit serum reacted against the host cells or cross-reacted with hcov-229e. because the 2 known hcovs are responsible for ∼30% of all common colds [2] , it is not unexpected that 97% and 99% of serum samples from healthy donors had antibodies to hcov-229e and hcov-oc43, respectively. therefore, it is expected that the antibodies to hcov-229e and hcov-oc43 found in the serum samples from patients with sars either preexisted or were cross-reacting antibodies to hcov-229e and hcov-oc43. further studies of this issue are warranted. the ifa showed that paired serum samples exhibited a у4-fold increase in antibody titers against hcov-229e and hcov-oc43 in 5 of 11 and 10 of 11 patients with sars, respectively. such a high antibody titer response to the known hcovs in patients with sars may represent an anamnestic reaction to previous infections with the 2 known hcovs or other covs or a crossreaction between sars-cov and hcov-229e or hcov-oc43. however, the nucleocapsid protein-based elisa detected increases in antibody titers in only 2 of 11 paired serum samples from patients with sars when the nucleocapsid protein of hcov-229e was used as an antigen, and they had a consistently increased signal reaction to the nucleocapsid protein from hcov-229e-infected cell culture filtrate by western blot analysis ( figure 1c ). paired serum samples from patients with sars showed no consistent increase in antibody titers in the hcov-oc43 nucleocapsid protein-based elisa and no increase in signal reaction to the nucleocapsid protein from hcov-oc43infected cell culture filtrate by western blot analysis. these results confirm the suggestion that the major antigenic crossreactivity with hcov-oc43 in the convalescent-phase serum samples of patients with sars is due not to nucleocapsid proteins but to other viral components. although the paired serum samples from patients with sars showed a partial cross-reaction to hcov-229e, there was no significant close correlation between the ifa titer ratios, which were determined in response to whole virus-infected cells, and the elisa titer ratios, which were determined in response to nucleocapsid proteins. furthermore, antibodies to sars-cov could be detected in only 1 serum sample from a healthy donor by either ifa or nucleocapsid protein-based western blot analysis, even though patients with sars had antibodies to hcov-229e and hcov-oc43. therefore, there is no serological cross-reactivity with sars-cov in healthy donors even though they have a high reactivity to hcovs [15] . in summary, sars-cov had apparent antigenic 1-way crossreactivity to the 2 known hcovs. although it is not clear which antigenic determinants were involved, the overall results suggest that sars-cov and hcov-oc43 are more closely antigenically related than are sars-cov and hcov-229e. studies using a number of purified recombinant viral components from covs as antigens to identify the antibodies produced during infection and to determine the antigenic relationships among covs are in progress. a novel coronavirus associated with severe acute respiratory syndrome human coronavirus infections characterization of a novel coronavirus associated with severe acute respiratory syndrome antigenic cross-reactivity between the nucleocapsid protein of severe acute respiratory syndrome (sars) coronavirus and polyclonal antisera of antigenic group i animal coronaviruses: implication for sars diagnosis evaluation of antibody responses against sars coronaviral nucleocapsid or spike proteins by immunoblotting or elisa relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia detection of sars coronavirus in patients with suspected sars sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome antigenic relationships among proteins of bovine coronavirus, human respiratory coronavirus oc43, and mouse hepatitis coronavirus a59 detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay antigenic relationships amongst coronaviruses antigenic relationships among the coronaviruses of man and between human and animal coronaviruses antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species immunogenicity and antigenicity of human coronaviruses 229e and oc43 coronavirus as a possible cause of severe acute respiratory syndrome we thank biao di (centers for disease control and prevention of guangzhou, people's republic of china), for providing the serological data of patients with severe acute respiratory syndrome for analysis, and san francisco edit, for assistance in editing the manuscript. key: cord-332175-d5suvj8g authors: allen, jawara title: my future in medicine: how covid-19 is inspiring the next generation of infectious disease specialists date: 2020-04-11 journal: j infect dis doi: 10.1093/infdis/jiaa178 sha: doc_id: 332175 cord_uid: d5suvj8g nan on the day that i defended my phd thesis, there were 86 000 confirmed cases of coronavirus disease 2019 (covid-19) globally [1] . i was one of the last students at johns hopkins university with an in-person defense, surrounded by family, friends, colleagues, and mentors. soon after, the centers for disease control and prevention (cdc) released guidelines discouraging the gathering of more than 50 people in one place. three weeks later-on the day i was scheduled to start the clinical portion of my medical training-there were 418 700 confirmed cases of covid-19 globally [1] . by that time, many courses had started remote instruction, most research laboratories had been closed to all nonessential personnel, and medical school core clerkships had been temporarily canceled, all in an effort to decrease student exposure to covid-19 and help "flatten the curve. " stuck between the 2 phases of my training as an md/phd candidate, i could not help but reflect on my future in the healthcare field. as a newly minted scientist specializing in microbiology and genomics, the biology of the virus immediately captured my attention. i contemplated how similarities between coronavirus strains could be exploited to strategically design vaccines, how drugs targeting similar proteins in other viruses could be repurposed, and how unique mutations in this virus's genome could be used to better understand and curb its high rate of transmission. but as a medical student eager to reenter the world of clinical care, the personal stories from the communities that would be most impacted by this relentless pathogen soon consumed my thoughts. these included communities in baltimore with which i had worked over the past 7 years and communities in the virgin islands where i had spent many summers among my extended family. their stories emphasized the effects of an increasingly global society on healthcare: vulnerable populations everywhere are impacted by this virus, and more infectious diseasetrained healthcare workers are needed. in baltimore county, the first case of covid-19 was reported on 11 march 2020. one day later, k-12 schools across the state were ordered to close. hearing this news, i immediately thought of the students i had worked with through several mentoring and outreach programs. i had taught them how to subculture bacteria, edited their college admissions essays, attended their prom send-off parties, and even spoken at their funerals. they live with their mothers, fathers, uncles, grandmothers, and any other combination of direct and extended family. schools had been closed, in part, so that these students would not bring covid-19 back home, so that they would not have to become caretakers should their caregivers get ill. many of their family members have diabetes, hypertension and other comorbidities that put them at increased risk of serious complications if infected with covid-19 [2] . the health of these inner-city communities was already being ravaged by gun violence, police brutality, and institutionalized systems of discrimination that resulted in life expectancies well below the national average [3] . but these students and their families were now facing yet another threat to their health. in the virgin islands, about 1600 miles south of baltimore, the first case of covid-19 was reported on 13 march 2020. this news shifted my thoughts again, this time toward my sprawling caribbean family. as i talked to them about the cdc's new recommendations on social distancing, i remembered the 2 hurricanes that had recently devastated their island, rendering basic necessities scarce, even before covid-19. i thought about how overwhelming an outbreak there would be for a healthcare system that often struggles to meet the daily needs of its people, a healthcare system that, in typical times, must fly its citizens to hospitals in the united states for anything beyond routine care. i worried about what would happen if my 80-yearold grandfather or my 60-year-old aunt with severe asthma contracted covid-19. the hospitals where they typically would have been transferred are already operating at close to full capacity. and the number of ventilators, n95 masks, isolation rooms, and healthcare workers on the island is undoubtedly insufficient to respond to even the slightest increase in demand. in settings as varied as inner-city baltimore and the virgin islands, the consequences of a virus that began in wuhan, china are apparent. few other events before have so poignantly highlighted the global nature of health, the vulnerability of certain populations around the world, and the need for more infectious disease specialists. the spread of covid-19 has shown that rapidly mutating microorganisms are unrestrained by borders. this idea is not novel to the infectious disease field, but unfortunately, it has not risen to the forefront of international conversations. in the united states, this is partly due to the shortage of physicians specializing in infectious diseases. in 2020, only 80% of spots in infectious disease fellowships were filled [4] , highlighting the need to foster more interest among medical students and residents. during this pandemic, the role of infectious disease specialists as leaders has become evident; they are top-tier advisors to policy makers, public health practitioners disseminating health information to society, researchers identifying treatments and vaccines for the virus, and front-line clinicians treating the most vulnerable patients. but the importance of infectious disease specialists will not end once the number of newly diagnosed covid-19 cases starts to decrease. this pandemic will cause a shift in medicine such that when clinical training across the world resumes i will join a healthcare workforce that is markedly different from that of my predecessors. at that time, the demand for physicians who specialize in infectious diseases will be amplified. they will be needed in laboratories not only to perfect the treatments for covid-19, but to initiate research on other pathogens with pandemic potential. they will be needed in hospitals not only to streamline the use of personal protective equipment, but to strengthen our supply channels to prepare for future system surges. they will be needed in government not only to shape and disseminate public health recommendations, but to design policies that preemptively protect those most vulnerable to future pandemics. they will be needed in the clinics not only to treat the complications of covid-19, but to promote healthy preventive habits. and above all, they will be needed as international ambassadors to strengthen scientific ties between countries in the fight against global health threats. this is a decisive moment for students in healthcare fields around the world. the impact of covid-19 on the rising generation of doctors, scientists, and public health professionals is undeniable. we are all part of, or connected to, communities impacted by covid-19 today, but we are also all threatened by the next global pathogen of tomorrow. to protect those communities, we need more infectious disease specialists. i will be one of them. i call on others to join me. potential conflicts of interest. the author: no reported conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. an interactive web-based dashboard to track covid-19 in real time clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study baltimore city health department results and data: specialties matching service; 2020 appointment year key: cord-289745-qtorq2qq authors: esper, frank; weibel, carla; ferguson, david; landry, marie l.; kahn, jeffrey s. title: evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children date: 2005-02-15 journal: j infect dis doi: 10.1086/428138 sha: doc_id: 289745 cord_uid: qtorq2qq background. the etiological agents responsible for a substantial proportion of respiratory tract diseases have not been identified. we sought to determine whether novel human coronaviruses (hcovs) are circulating in new haven, connecticut, and, if so, whether they are associated with respiratory tract disease in infants and young children. methods. we developed a polymerase chain reaction (pcr)-based approach for screening specimens from the respiratory tracts of symptomatic children. pcr probes that target regions of the replicase 1a gene that are conserved among genetically diverse animal covs and hcovs were designed. using these probes, we identified genomic sequences of a novel hcov, designated “new haven coronavirus” (hcov-nh). thereafter, we designed specific probes to screen respiratory specimens from children <5 years old for this novel hcov. clinical features associated with hcov-nh infection were identified. results. seventy-nine (8.8%) of 895 children tested positive for hcov-nh. cough, rhinorrhea, tachypnea, fever, abnormal breath sounds, and hypoxia were the most common findings associated with hcov-nh infection. sequence analysis revealed that hcov-nh is closely related to a novel hcov recently reported in the netherlands. conclusions. the novel hcovs identified in new haven and the netherlands are similar and likely represent the same species. this newly discovered virus may have worldwide distribution and may account for a significant proportion of respiratory tract disease in infants and young children. gence of sars-cov, hcovs were generally thought to cause mild, self-limited infections of the upper respiratory tract [8] . however, it is now known that hcovs can cause severe disease in immunocompromised individuals [9] . the study of hcovs has been hampered by the difficulty in propagating these viruses in vitro and the lack of specific diagnostic tests with which to identify potentially novel viruses. therefore, the possibility exists that unidentified hcovs are the cause of some proportion of the respiratory tract diseases for which etiological agents have not been identified. to determine whether novel hcovs are circulating and, if so, whether they are responsible for respiratory tract disease in children, we developed a strategy to screen for previously unknown hcovs. our initial assumption was that all covs must have conserved functions and that these conserved functions are reflected in the genome. the replicase of covs is an rna-dependent rna polymerase, the function of which is not provided by the host cell and, therefore, must be evolu-tionarily maintained by all covs. therefore, we designed polymerase chain reaction (pcr) probes that target regions of the cov replicase 1a gene that are conserved among hcovs, avian covs, and mammalian covs. using this approach, we identified genetic evidence of a novel hcov circulating in new haven, ct. probes specific for this hcov, designated "new haven coronavirus" (hcov-nh), were then used to screen specimens from the respiratory tracts of symptomatic children. during our screening of respiratory specimens for this hcov, 2 studies from the netherlands reported the identification of a novel hcov [10, 11] . this virus is a group 1 cov and is most closely related to hcov-229e and transmissible gastroenteritis virus (tgev), a virus of pigs. comparisons of the sequences of the hcov identified in the netherlands and hcov-nh revealed that these viruses are closely related and likely represent the same species. here, we describe the seasonal distribution and clinical manifestation of disease associated with hcov-nh infection. primers for the detection of covs were based on conserved regions of the replicase 1a gene of groups 1, 2, and 3 covs and sars-cov. the replicase 1a genes from avian infectious bronchitis virus (genbank accession number aj311317), bovine cov (bcov) strain lun (genbank accession number af391542), bcov strain quebec (genbank accession number af220295), bcov isolate bcov-ent (genbank accession number af391541), hcov-229e (genbank accession number nc_ 002645), tgev (genbank accession number nc_002306), and sars-cov tor2 (genbank accession number ay274119) were aligned by use of clustal w in the lasergene software package (version 5.05; dnastar). two conserved regions were identified, and primers corresponding to these regions were synthesized (yale oligonucleotide laboratory, department of pathology, new haven, ct). the forward primer, 5 -gcgcaa-aataatgaattaatgcc-3 (underscoring indicates a g/c clamp), and the reverse primer, 5 -gacgcaccaccatatga-atcctg-3 , represent consensus sequences of conserved regions within the 3 1000 bases of the replicase 1a gene of all of the covs listed above (relative to sequences 11,781-12,285 of hcov-229e). the predicted length of the amplicons produced by these primers is ∼550 bp. rna from respiratory specimens obtained from the clinical virology laboratory, yale-new haven hospital, was extracted by use of the qiaamp viral rna mini kit (qiagen), in accordance with the manufacturer's instructions. cdna for each respiratory specimen was synthesized with random hexamer primers and mumlv rt (new england biolabs), in accordance with the manufacturer's instructions. cdnas were subsequently screened by pcr with hotstar taq polymerase (qiagen), in accordance with the manufacturer's instructions. the following amplification program was used: 15 min at 95њc; 40 cycles of 1 min at 94њc, 1 min at 50њc, and 1 min at 72њc; and a final extension of 10 min at 72њc. for the initial screening of respiratory specimens, rna was extracted from mrc-5 cells infected with hcov-229e (atcc vr-740) as a positive control. each set of rt-pcr amplifications included appropriate negative controls. pcr products were analyzed by agarose gel electrophoresis. after the initial screening of respiratory specimens, amplicons of the predicted molecular weight were isolated and sequenced. sequencing was performed by use of applied biosystems 377 dna automated sequencers (w. m. keck biotechnology resource lab, yale university school of medicine). sequences that corresponded to a potential novel hcov were identified, and primers specific for this agent were synthesized. the forward primer, 5 -gcgctat-gagggtggttgtaac-3 , and the reverse primer, 5 -cgcg-cagttaaaagtccagaattaac-3 , amplify a 215-bp region of the novel hcov genome. these primers target regions of the novel hcov genome that are distinct relative to the corresponding region of the hcov-229e genome. screening by pcr with these primers was performed by use of the following amplification program: 15 min at 95њc; 40 cycles of 1 min at 94њc, 1 min at 55њc, and 1 min at 72њc; and a final extension of 10 min at 72њc. these primers were used to screen pooled respiratory specimens. respiratory specimens. we chose to screen specimens that were obtained from the respiratory tracts of symptomatic children !5 years old and that tested negative for respiratory syncytial virus (rsv), influenza viruses a and b, human parainfluenza viruses 1-3, and adenovirus by direct fluorescent antibody assay (dfa). these specimens, which were obtained from both ambulatory and hospitalized patients, were screened for presence of human metapneumovirus (hmpv) by use of an rt-pcr-based approach described elsewhere [12, 13] and were submitted to the clinical virology laboratory, yale-new haven hospital, for diagnostic testing. specimens were obtained from 1 january 2002 to 14 february 2003. children could be counted more than once if the specimens were obtained 130 days apart. clinical data from the children who tested positive for hcov-nh were obtained by extracting information from medical records and recording the data on a standardized form. children who had evidence of infection with another viral respiratory pathogen were excluded when the clinical features associated with hcov-nh infection were tabulated. comorbidity was defined as prematurity (gestational age of !35 weeks), underlying pulmonary disease, genetic syndromes, acquired immunosuppression, malignancies, and congenital heart disease. the yale university human investigations committee approved the collection and screening of respiratory specimens. sequencing and phylogenetic analysis. the amplicon of each specimen that tested positive by rt-pcr was sequenced to confirm the presence of hcov-nh. phylogenetic analysis was performed by use of the lasergene software package described above. using the primers that target the conserved regions of the cov replicase 1a gene, we screened 601 respiratory specimens for covs. the screening reaction was performed on pooled rna; each pooled amplification reaction included 5-10 individual specimens. of the 80 pooled amplification reactions, 17 yielded an amplicon of ∼550 bp (data not shown). after these amplicons were sequenced, a nucleotide blast (available at: http://www.ncbi .nlm.nih.gov/blast/) search was performed. hcov-oc43 was identified in 8 pooled reaction products, and hcov-229e was identified in 1 pooled reaction product. six amplicons either were human dna or did not yield an interpretable sequence. the 2 remaining amplicons were similar and represented novel sequences most closely related to group 1 covs. these sequences were ∼69%-71% identical to hcov-229e and tgev on the nucleotide level and ∼68% identical to hcov-229e and tgev on the amino acid level. pcr primers specific for these novel sequences of hcov-nh were then synthesized, and these primers were used to screen all respiratory specimens thereafter. screening of respiratory specimens for hcov-nh. in total, 1265 respiratory specimens from 895 children were screened by rt-pcr with primers specific for hcov-nh (the 601 spec-imens from the initial screening were rescreened by rt-pcr with these primers). screening reactions were again pooled, with 5-10 individual specimens in each pool. if a pooled reaction produced an appropriately sized amplicon, the specimens in that pool were individually tested. seventy-nine (8.8%) of these individual specimens were found to be positive for hcov-nh. two children had 2 positive specimens each; the specimens were obtained 5 days apart for 1 child and 7 days apart for the other child. in both instances, the 2 positive specimens were considered to be the result of a single episode of hcov-nh infection. the median age of the 79 hcov-nhpositive children was 6.5 months. fifty (63.3%) were !1 year old, and 27 (34.2%) were 0-3 months old. forty-nine (62.0%) were male. eleven of the hcov-nh-positive children had been hospitalized since birth at the newborn intensive care unit (nicu), yale-new haven hospital. the age distribution of the hcov-nh-positive children is shown in figure 1 . clinical data were available for 76 of the 79 hcov-nh-positive children. of these 76 children, 9 (11.8%) had evidence of recent infection with another respiratory virus-2 were coinfected with hmpv, and 7 were found to be coinfected with another respiratory virus by dfa on other respiratory specimens that had been collected during the same hospitalization or illness (1 had a parainfluenza infection, and 6 had an rsv infection). among the 67 children who tested positive for hcov-nh only, cough, rhinorrhea, tachypnea, fever, abnormal breath sounds, and hypoxia were the most common findings (table 1) . thirtyfive (52.2%) of these 67 children had an underlying comortwo of the 79 hcov-nh-positive children died; both had been hospitalized since birth at the nicu. one child had been diagnosed with hydrops fetalis in utero and died on day 2 of life, 1 day after a respiratory specimen had been collected that tested positive for hcov-nh. the second child, who had been born at 28 weeks gestation, died on day 170 of life; a respiratory specimen collected on day 151 of life tested positive for hcov-nh. this child also had a history of necrotizing enterocolitis and liver failure before day 151 of life and ultimately died of multiorgan system failure. phylogenetic analysis of hcov-nh. sequence analysis was performed on the amplicon derived from each positive specimen (genbank accession numbers ay870943-ay871008). phylogenetic analysis of a 126-bp portion of the hcov-nh-specific primer region, which contains representative sequences of hcov-nh, is shown in figure 3 . overall, the amplified region of the putative replicase 1a gene of hcov-nh was highly conserved. these sequences closely matched the sequences of the replicase 1a gene of the novel hcov recently identified in the netherlands [10, 11] . several distinct polymorphisms were identified among the hcov-nh isolates; these polymorphisms were not present in the hcov identified in the netherlands. most of the polymorphisms in the replicase 1a gene among hcov-nh isolates did not change the predicted amino acid sequence (data not shown). using a pcr-based approach to screen for previously unknown hcovs, we identified evidence of the existence of a novel hcov, and we have described the clinical features associated with infection with this agent. our approach consisted of identifying conserved regions of the replicase 1a gene of a variety of animal covs and hcovs and then exploiting the conserved genomic domains to identify a previously unknown hcov. this approach is based entirely on knowledge of cov genomes, and so identification of a cov does not require the ability of the virus to propagate in vitro or in animal models. this type of genome-based screening has been used previously to identify human bacterial pathogens by targeting conserved elements of rrna [14] . after the observation of cov-like particles in respiratory secretions from individuals with sars, broadly reactive pcr was used to identify the pathogen; unlike in the present study, in which the replicase 1a gene was targeted, the replicase 1b gene was targeted in the identification of the agent that causes sars [5] . in the present study, the initial set of primers, used to screen pooled specimens, were less sensitive than the hcov-nh-specific primers. many of the respiratory specimens in the initial pool of 601 specimens that tested negative by pcr with the replicase 1a gene primers tested positive by pcr with the hcov-nh-specific primers. subsequent sequence analysis of the target sites for the replicase 1a gene of hcov-nh (data not shown) revealed several mismatches between the genome and primer sequence. this finding likely accounts for the relative decreased sensitivity of the replicase 1a gene primers. among the 601 specimens initially screened, the 2 pooled reactions that tested positive for hcov-nh contained multiple positive specimens. this may account for why these pooled specimens tested positive. it is also unclear why primers constructed for the detection of the 2 common hcovs, hcov-229e and hcov-oc43, would not have detected hcov-nh. presumably, the genomic sequence targeted for the detection of hcov-229e and hcov-oc43 is specific for these viruses. the sensitivity of the cov consensus primers and the hcov-nh-specific primers is unknown. determination of the sensitivity will require propagation of the virus and development of a method for quantitating the number of viral particles (such as an infectivity assay or a method based on electron microscopy, neither of which have been completely developed for hcov-nh). as further genetic data become available, moresensitive primers may be developed. our approach complements that of traditional virology, in which viruses are identified by their effects on cells in tissue culture. however, the identification of previously unknown viruses on the basis of in vitro findings has significant shortcomings, not the least of which is the inability to amplify and characterize unique genetic sequences. this obstacle was overcome by fouchier et al. [10] and van der hoek et al. [11] , who used the tools of molecular biology to identify and sequence the genome of a novel hcov in the netherlands after propagation in cell culture. hmpv was discovered by use of a similar approach that was based on in vitro virus propagation and genomic amplification techniques [15] . like the 2 groups from the netherlands, we have evidence that this novel hcov can be propagated in cell culture. the results of rt-pcr assays of passaged cell culture supernatants suggested the growth of hcov-nh in vitro (data not shown). there were early hints that hcov-229e and hcov-oc43 were not the only common hcovs. the first hcov isolated, b814, was not serologically related to either hcov-229e or hcov-oc43 [16] . mcintosh et al. demonstrated that 3 of the 6 oc strains isolated from adults with colds appeared to be serologically unrelated, or at least distantly related, to hcov-oc43 and hcov-229e [17] . experimental infection of human volunteers with these 6 oc strains confirmed this finding [18] . it was, therefore, unlikely that hcov-229e and hcov-oc43 were the only hcovs. the viral pathogen newly identified in new haven and the netherlands may represent the first of many antigenically distinct groups of hcovs to be characterized. in the present study, hcov-nh was associated with both upper and lower respiratory tract disease in infants and young children. because ours was not a population-based study and because a control group was not included, it is impossible to determine the spectrum of disease caused by hcov-nh infection. our study screened specimens submitted to a diagnostic laboratory only; therefore, the proportion of children with clinical illness reported here likely does not reflect that of the general population. likewise, the issue of causality remains unresolved. prospective population-based studies are required. nonetheless, the identification of genetic sequences of this hcov from a large number of children with respiratory tract illnesses strongly suggests that hcov-nh infection plays a role in disease. our data suggest that hcov-nh, like other hcovs, may have a seasonal distribution [8] . animal covs cause disease in organ systems other than the respiratory tract, such as the gastrointestinal system and the central nervous system [19] . sars-cov is excreted in the stool of infected individuals [6] . therefore, it is necessary to determine whether hcov-nh is present in other body fluids and whether it is associated with other clinical syndromes. the percentage of children who tested positive for this novel hcov in our study (8.8%) was higher than the percentages of patients who tested positive in the studies by van der hoek et al. (1.6%) and fouchier et al. (2.9%) [10, 11] . the reasons for this are unclear. both our study and those in the netherlands screened respiratory specimens submitted to a diagnostic virology laboratory and used a pcr-based approach for screening. the characteristics of the study populations and the time of the acquisition of the specimens screened may explain the differences in the observed percentages of positive subjects between our study and the 2 studies in the netherlands. fouchier et al. limited screening for the novel hcov to 3 months during late autumn and early winter [10] and, therefore, may have missed the peak circulation of the virus in the population. also, we chose to screen only children !5 years old, whereas van der hoek et al. chose to screen individuals of all ages [11] . similar to other respiratory viruses, symptomatic infection with this newly discovered hcov may be more common during childhood; therefore, we may have been more likely to detect a higher percentage of infected subjects in the present study. hcovs can cause severe disease in infants [20] . in the present study, a majority (50/79 [63.3%]) of hcov-nh-positive children were, in fact, !1 year old, and 34.2% were р3 months old. eleven (13.9%) of the 79 hcov-nh-positive children in the present study had been hospitalized since birth at the nicu. there were 2 apparent temporal clusters of hcov-nh infection at the nicu, during february 2002 and during january and february 2003. although these may represent nosocomial outbreaks, other modes of infection, such as perinatal transmission, cannot be discounted. hcov-nh was detected in a child diagnosed with hydrops fetalis. whether this represents congenital infection with hcov-nh remains to be determined. in conclusion, the present results demonstrate the power of the tools of molecular biology to define and characterize potential infectious agents associated with human disease. the novel hcov identified in new haven and the netherlands was associated with both upper and lower respiratory tract disease in infants and young children. whether this virus is associated with other clinical syndromes remains to be determined. population-based studies are required to define the burden of disease caused by this novel hcov, and such studies could provide information on causality. the global burden of disease 2000 project: aims, methods and data sources. in: global programme on evidence for health policy. geneva: world health organization prospective comparative study of viral, bacterial and atypical organisms identified in pneumonia and bronchiolitis in hospitalized canadian infants. pediatr etiology of community-acquired pneumonia: impact of age, comorbidity, and severity the tucson children's respiratory study. ii. lower respiratory tract illness in the first year of life a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome coronavirus 229e-related pneumonia in immunocompromised patients a previously undescribed coronavirus associated with respiratory disease in humans identification of a new human coronavirus a 1-year experience with human metapneumovirus in children aged !5 years human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children identification of the uncultured bacillus of whipple's disease a newly discovered human pneumovirus isolated from young children with respiratory tract disease antigenic relationships amongst coronaviruses antigenic relationships among the coronaviruses of man and between human and animal coronaviruses coronative antibody tires in sera of healthy adults and experimentally infected volunteers coronaviridae: the viruses and their replication coronavirus infection in acute lower respiratory tract disease of infants we are indebted to george miller, john f. enders professor of pediatric infectious diseases (yale university school of medicine), for his continued support, intellectual and scientific input and exchange, and critical review of the data and manuscript. we thank the staff of the clinical virology laboratory, yale-new haven hospital (ct), for their assistance. we are also indebted to eugene d. shapiro (yale university school of medicine), for his thoughtful review of the manuscript. key: cord-333429-bq7kfpby authors: shi, ding; wu, wenrui; wang, qing; xu, kaijin; xie, jiaojiao; wu, jingjing; lv, longxian; sheng, jifang; guo, jing; wang, kaicen; fang, daiqiong; li, yating; li, lanjuan title: clinical characteristics and factors associated with long-term viral excretion in patients with sars-cov-2 infection: a single center 28-day study date: 2020-07-02 journal: j infect dis doi: 10.1093/infdis/jiaa388 sha: doc_id: 333429 cord_uid: bq7kfpby background: despite the ongoing spread of covid-19, knowledge about factors affecting prolonged viral excretion is limited. methods: in this study, we retrospectively collected data from 99 hospitalized patients with covid-19 between january 19 and february 17 in zhejiang province, china. we classified them into two groups based on whether the virus test results eventually became negative. cox proportional hazards regression was used to evaluate factors associated with sars-cov-2 shedding. results: among 99 patients, 61 patients had sars-cov-2 clearance (virus-negative group), but 38 patients had sustained positive results (virus-positive group). the median duration of sars-cov-2 excretion was 15 days (iqr 12-19) among the virus-negative patients. the shedding time was significantly increased if fecal sars-cov-2 rna test results was positive. male sex (hr, 0.58 [95% ci, 0.35-0.98]), immunoglobulin use (hr, 0.42 [95% ci, 0.24-0.76]), apache ii score (hr, 0.89 [95% ci, 0.84-0.96]), and lymphocyte count (hr, 1.81 [95% ci, 1.05-3.1]) were independent factors associated with a prolonged duration of sars-cov-2 shedding. antiviral therapy and corticosteroid treatment were not independent factors. conclusions: sars-cov-2 rna clearance time was associated with sex, disease severity and lymphocyte function. the current antiviral protocol and low-to-moderate dosage of corticosteroid had little effect on the duration of viral excretion. a c c e p t e d m a n u s c r i p t for asymptomatic patients. shedding cessation was defined as the occurrence of two consecutive rt-pcr negative results of respiratory specimens in a 24-hour interval. for most variables, descriptive statistics such as the median with interquartile range (iqr; for data with skewed distribution) and proportion (%) were calculated. the t test, mann-whitney u test, and kruskal-wallis test were used for continuous variables. the χ 2 test and fisher's exact test were used for categorical variables. to identify risk factors associated with a prolonged duration of sars-cov-2 rna shedding, we performed a time-dependent cox proportional hazards model adjusted for baseline covariates. outcomes were defined as time to sars-cov-2 rna negativity, and we censored patients if they never cleared sars-cov-2 rna during hospitalization. kaplan-meier curves were used to estimate the cumulative sars-cov-2 rna negativity rate and the stratified log-rank statistic to compare the differences in viral clearance between the two groups. statistical analyses were performed using spss software, version 20.0. for all analyses, probabilities were two-tailed, and a two-tailed p value of < 0.05 was considered significant. ; p < 0.01). the number of elderly patients (>65 years old) in the virus-positive group was significantly higher than that in the virus-negative group (p < 0.01). a total of 61.6% of patients were male, but no significant difference in sex was found between the two groups. hypertension (n=36, 36.4%) and diabetes mellitus (n=16,16.2%) were the most common comorbidities among the covid-19 patients. additionally, a few patients had chronic lung disease, cardiac disease, immunosuppressive disease and pregnancy, but there were no significant differences between the two groups (table 1) . among laboratory indicators tested at admission, inflammatory indexes such as crp (table 1) . other laboratory indicators, such as leukocyte count, hemoglobin, platelet count, coagulation profile, creatine, liver function, and a c c e p t e d m a n u s c r i p t myocardial enzyme showed no difference between the two groups (supplementary table 1 ). from the results, persistent virus-positive patients showed lower immune cell counts and higher inflammation levels. only 1 patient (1.6%) tested sars-cov-2 negative within 5 days, 9 patients (14.7%) tested negative within 10 days, and 49 (80.3%) tested negative within 20 days from illness onset. in addition, a small subset of 12 patients with sars-cov-2 had detectable levels of the virus up to 30 days from symptom onset. the median time of persistent viral shedding was 16 days (iqr [13] [14] [15] [16] [17] [18] [19] [20] [21] . the virus-positive group showed a median viral shedding time of 19 days, which was significantly longer than that in the virusnegative group (median 15, iqr 12-19, p =0.002, table 2 ). no apparent difference in time from illness onset to covid-19 diagnosis was found between the two groups. sars-cov-2 rna was positively detected in the stool specimens of 21 patients table 2 ). darunavir (800 mg once daily) was prescribed to replace lpv/rtv if patients experienced significant drug side effects. the duration from illness onset to arv start was 6 days (iqr 4-8.5). there was no significant difference in arv start time between the virus-positive and virus-negative groups. a total of 77 patients (77.8%) received corticosteroid treatment with an initial dosage of 60 mg/d (iqr 40-80), and the time from illness onset to corticosteroid treatment was 8 days (iqr 6-10). there was no significant difference in the application, initial dosage or start time of corticosteroid treatment between the two groups ( table 2) . forty-nine patients (49.5%) were treated with antibiotics, and the median time from illness onset to antibiotic use was 9 days (iqr 6-13). there was no difference in antibiotic use proportion or antibiotic start time between the two groups (p>0.05). in addition, 43 patients (43.4%) received intravenous immunoglobulin treatment. the proportion of patients receiving immunoglobulin therapy in the virus-positive group was much higher than that in the virus-negative group (63.2% vs 31.1%, p=0.002). the patients with persistent sars-cov-2 rna positivity showed greater disease severity ( table 2) . among 99 covid-19 patients, a total of 30 (30.3%) were transferred to the intensive care unit (icu), and the patients in the virus-positive group had a higher rate of icu admission than the virus-negative group (52.6% vs. 16 .4%, a c c e p t e d m a n u s c r i p t p<0.01). the median length of icu stay for the 30 patients was 7.5 days (iqr 4-11), and in the virus-positive group, the length of icu stay was 8.5 days (iqr 6.3-11), which was longer than that in the virus-negative group (4 days, iqr 3-5.8, p <0.01). the acute physiology and chronic health evaluation ii (apache ii) score was used to evaluate the severity of hospitalized patients as well as to predict mortality [10] . the apache ii score was 9.5 (iqr 5-15) in virus-positive group, which was much higher than that of virus-negative patients, with a score of 5 (iqr 2-8). mechanical ventilation was performed in a minority of patients (12.1%). compared with the virus-negative group, the virus-positive group had a higher rate of mechanical ventilation (26.3% vs. 3.3%, p<0.01). moreover, all patients receiving extracorporeal membrane oxygenation (ecmo) treatment were from the virus-positive group (15.8% vs. 0%, p<0.01), indicating more severe lung injury and oxygenation. we conducted a multivariable time-dependent cox proportional hazards model to identify risk factors associated with the duration of sars-cov-2 rna shedding ( (table 3) . interestingly, the use of immunoglobulin may prolong the duration of viral shedding. we found that the disease tended to be more severe in patients prescribed immunoglobulins, and these patients were older with higher apache ii scores and worse immune function than those who did not use immunoglobulins (supplementary table 2 ). using kaplan-meier survival analysis, we found sars-cov-2 rna clearance was associated with disease severity, which was significantly delayed in patients who were transferred to icu and whose apache ii scores >10 compared with that of patients in the general ward and whose apache ii scores ≤ 10 (log-rank test, p<0.001; figure 1a , 1b). compared with patients with a high lymphocyte count (> 0.5×10 9 /l), patients with low immunity (lymphocyte count ≤ 0.5×10 9 /l) showed a significant delay in virus clearance (log-rank test, p=0.002; figure 1c ). it took longer for fecal viral rnapositive patients to clear sars-cov-2 rna than fecal viral rna-negative patients (log-rank test, p=0.003; figure 1d ). despite the ongoing spread of covid-19, knowledge of the factors affecting prolonged viral shedding is still limited. thus, we conducted a retrospective, single-center study of 99 hospitalized patients with confirmed sars-cov-2 infection identified between january 19 and february 17, 2020 in zhejiang province, china. we identified that male sex, immunoglobulin use, apache ii score, and lymphopenia were independent risk factors associated with the duration of sars-cov-2 rna shedding, whereas arv a c c e p t e d m a n u s c r i p t combination therapy and corticosteroid treatment were not independent factors. in addition, patients with fecal viral rna tested positive needed more time to clear sarsconsistent with previous findings, median age of hospitalized patients with sars-cov-2 infection was 54 years, and 22.2% patients were aged > 65 years [11] . patients with persistent sars-cov-2 rna positivity were older. although age >65 years was considered as a risk factor associated with disease severity [2] . however, our study did not find age to be a risk factor for viral clearance. the key difference in age between the two groups may be related to weak immune response as well as coexisting illnesses. a majority of covid-19 patients had hypertension and diabetes, and a small proportion had chronic lung disease, cardiac disease, immunosuppression or pregnancy. covid-19 patients with underlying comorbidities, including hypertension, diabetes, cardiovascular disease, were more likely to be transferred to icu [12] . underlying diseases has been shown to be related to prolonged viral shedding in sars patients [6] . however, we found no significant differences between virus-positive patients and virusnegative patients with regard to comorbidity. this result suggests that underlying diseases such as hypertension and diabetes may be related to the patient's disease prognosis but have little to do with the duration of viral shedding. male sex was found to be an independent risk factor for prolonged sars-cov-2 viral shedding. in sars-cov infection, males experience higher mortality than females [13] . a c c e p t e d m a n u s c r i p t imbalanced levels of angiotensin-converting enzyme 2 (ace2) between males and females may play an important role in the sex-based differences in the response to the disease [14] . ace2 has been identified as the host receptor of sars-cov, which regulates both cross-species and human-to-human transmissions in sars-cov infection [15] . sars-cov-2 also use the ace2 receptor to facilitate viral entry into target cells [16] . consistent with other reports, the most common laboratory abnormalities among the covid-19 patients were depressed total lymphocyte and lymphocyte subset counts, as well as an elevated crp level [2, 3, 17] . compared with the virus-negative patients, patients with persistent sars-cov-2 positivity had lower lymphocyte count and t-cell subset count but higher crp and pct level. this suggested more severe cellular immune deficiency and inflammatory activation in sars-cov-2 virus-positive patients. decreases in t cell counts were strongly associated with the severity and progression of sars and mers in patients [18, 19] . the underlying reasons for lymphopenia may be the direct infection of lymphocytes by sars-cov-2, lymphocyte sequestration in the lung or cytokine-mediated lymphocyte trafficking [20, 21] . all of which may lead to extend the virus clearance time. although various drugs have been tested for covid-19, there is still an urgent need for a clinically proven, effective antiviral treatment for sars-cov-2 infection. in this study, the antiviral therapy mainly included lpv/rtv and arbidol. nevertheless, we a c c e p t e d m a n u s c r i p t observed that there was no apparent effect on viral shedding from the use of theses antiviral drugs or their combined use. previous researches on the efficacy of lpv/rtv in sars and mers showed disparate results. compared with those using ribavirin alone, improved outcomes for sars and decreased viral loads were reported for patients using a combination of lpv/rtv and ribavirin [22] , whereas no obvious antiviral effect against mers-cov with lopinavir was observed in vitro [23] . another study revealed that lpv/rtv could improve pulmonary function without inhibiting virus replication in mers-cov infection [24] . current evidence regarding the effect of arbidol against sars-cov-2 was rare. in vitro experiment have indicated that arbidol showsed some antiviral activity against sars [25] . it was previously demonstrated that remdesivir had excellent antiviral activity against mers-cov in vitro and in vivo [24] . moreover, remdesivir exhibited a beneficial effect in the first case in america [26] . this novel nucleotide analogue had not been put into use in our center yet, while its therapeutic effect in illness improvement is worth attention. overall, further studies are warranted to uncover the exact efficacy of these different antiviral agents on viral shedding. corticosteroids have been used frequently for the treatment of severe patients infected with coronavirus by reducing inflammatory-induced lung injury [3] . several studies have reported that the use of corticosteroids is associated with delayed viral rna clearance in mers or sars patients and even with higher mortality in influenza pneumonia [27] [28] [29] . in this study, corticosteroid usage did not independently increase a c c e p t e d m a n u s c r i p t the risks for viral shedding regardless of the initial dosage or an earlier implementation. however, the percentage of corticosteroid use in our center reached up to 77%, and the dosages prescribed were relatively lower for these covid-19 patients than for mers and sars patients in previous studies. it was reported that high dosages of corticosteroids (>150 mg/d) showed a similar influence on h7n9 infection with prolonged viral shedding time, whereas proper use of corticosteroids was associated with clinical improvement in sars [30, 31] . thus, more clinical data are urgently needed to indicate the effects of corticosteroids and the optimal scheme of their usage. in addition, the beneficial effect of immunoglobulin has also been observed in viral infection [32] . our study found immunoglobulin application was associated with delayed viral clearance. in fact, patients using immunoglobulin were more likely to be in the more severe condition. thus, more studies should be done to confirm the association between immunoglobulin treatment and viral shedding. we identified the predictive value of the apache ii score in sars-cov-2 infection. moreover, patients transferred to the icu showed delayed sars-cov-2 rna clearance. the result indicated that sars-cov-2 viral shedding was associated with disease severity. similarly, mers-cov sub-genomic mrna was detected more frequently in the severe group than in the mild group [5] . severe patients had more prolonged mers-cov shedding, up to 18-27 days following the onset of symptoms [33] . sars patients with apache ii scores ≥ 20 were associated with an increased risk of transmission of sars-cov [34] . it can be explained by the higher a c c e p t e d m a n u s c r i p t apache ii score, more severe immunosuppression and higher viral load among patients in the icu. fecal-oral transmission was considered as an additional route for sars-cov-2 spread. in this study, we found a high percentage of covid-19 patients with positive fecal virus results, who needed more time to clear sars-cov-2 than those with negative fecal virus results. fecal viral rna can remain positive even after respiratory specimens test results return negative. consistent with our results, stool specimens tested positive in up to 53% of covid-19 patients, and the positive staining of ace2 and sars-cov-2 nucleocapsid protein was observed in gastrointestinal epithelium as well as isolated infectious sars-cov-2 from feces [4] . the duration of sars fecal viral excretion could last for more than 100 days [6] . positive fecal viral rna was highly suggestive of virus intestinal infection; thus, a longer isolated observation time should be considered in these covid-19 patients. our study has some notable limitations. first, we only observed the hospitalization of covid-19 patients for 28 days, and many patients remained in hospital with sars-cov-2 rna positivity. the duration of viral shedding may be longer than this cut-off time point. second, some patients were transferred to our hospital after the disease had progressed and become worse; hence, our cohort might represent a more severe range of covid-19. a c c e p t e d m a n u s c r i p t in conclusion, we found that male sex, immunoglobulin use, apache ii score, and lymphopenia were independent risk factors associated with the duration of sars-cov-2 rna shedding, whereas arv combination therapy and corticosteroid treatment were not. a prolonged sars-cov-2 rna clearance time was associated with disease severity and fecal viral rna positivity. our findings suggest that more suitable antiviral agents and low-to-moderate corticosteroids rather than high dosages should be considered in the treatment of severe covid-19, while seeking solutions to enhance immune function and to reduce the inflammatory response were also of great importance for viral clearance. in addition, more data based on randomized and controlled multicenter studies are needed to rigorously assess the clinical relevance of our proposed indicators. m a n u s c r i p t among patients who were transferred to icu and those who were in general ward by day after illness onset. b, cumulative proportion of patients with detectable sars-cov-2 rna among those whose apache ii score >10 and those whose apache ii≤10 by day after illness onset. c, cumulative proportion of patients with detectable sars-cov-2 rna among those whose lymphocyte count ≤0.5ⅹ10 9 /l and those whose lymphocyte count > 0. a c c e p t e d m a n u s c r i p t m a n u s c r i p t a novel coronavirus from patients with pneumonia in china clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan evidence for gastrointestinal infection of sars-cov-2 replicative virus shedding in the respiratory tract of patients with middle east respiratory syndrome coronavirus infection long-term sars coronavirus excretion from patient cohort viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection antiviral therapy and outcomes of influenza requiring hospitalization in ontario clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: 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development and progression to respiratory failure in mers-cov infected patients a clinicopathological study of three cases of severe acute respiratory syndrome (sars) in vitro detection of apoptosis in monocytes/macrophages infected with human coronavirus role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov first case of 2019 novel coronavirus in the united states corticosteroid therapy for critically ill patients with middle east respiratory syndrome effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients treatment of severe acute respiratory syndrome with glucosteroids: the guangzhou experience severe acute respiratory syndrome: report of treatment and outcome after a major outbreak l or more. effect on viral, opportunistic, and bacterial infections. the national institute of child health and human development intravenous immunoglobulin clinical trial study group comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity risk factors for sars transmission from patients requiring intubation: a multicentre investigation in toronto this study was funded by science and technology department of zhejiang province a c c e p t e d m a n u s c r i p t key: cord-324001-m7ys95z7 authors: kobinger, gary p.; meunier, isabelle; patel, ami; pillet, stéphane; gren, jason; stebner, shane; leung, anders; neufeld, james l.; kobasa, darwyn; von messling, veronika title: assessment of the efficacy of commercially available and candidate vaccines against a pandemic h1n1 2009 virus date: 2010-04-01 journal: j infect dis doi: 10.1086/651171 sha: doc_id: 324001 cord_uid: m7ys95z7 background. the emergence and global spread of the pandemic h1n1 2009 influenza virus have raised questions regarding the protective effect of available seasonal vaccines and the efficacy of a newly produced matched vaccine. methods. ferrets were immunized with the 2008–2009 formulations of commercially available live attenuated (flumist; medimmune) or split-inactivated (fluviral; glaxosmithkline) vaccines, a commercial swine vaccine (flusure; pfizer), or a laboratory-produced matched inactivated whole-virus vaccine (a/mexico/indre4487/2009). adaptive immune responses were monitored, and the animals were challenged with a/mexico/indre4487/2009 after 5 weeks. results. only animals that received the swine or matched vaccines developed detectable hemagglutinationinhibiting antibodies against the challenge virus, whereas a t cell response was exclusively detected in animals vaccinated with flumist. after challenge, all animals had high levels of virus replication in the upper respiratory tract. however, preexisting anti—pandemic h1n1 2009 antibodies resulted in reduced clinical signs and improved survival. surprisingly, flumist was associated with a slight increase in mortality and greater lung damage, which correlated with early up-regulation of interleukin-10. conclusions. the present study demonstrates that a single dose of matched inactivated vaccine confers partial protection against a pandemic h1n1 2009 virus, and it suggests that a higher dose or prime-boost regimen may be required. the consequences of mismatched immunity to influenza merit further investigation. 1414,000 confirmed cases and ∼5000 deaths worldwide, and the real numbers are likely to be considerably higher, because countries are now only required to confirm severe cases by laboratory diagnosis [1] . even though most patients experience a disease similar to seasonal influenza, reports of severe cases are increasing [2] [3] [4] . studies in different animal models reveal more efficient spread of the pandemic h1n1 2009 viruses to the lower respiratory tract and demonstrate increased virulence of some field isolates, suggesting that the genetic makeup of the respective strain may significantly contribute toward disease outcome [5, 6] . this observation, in combination with reports of more frequent incidents of severe disease in the southern hemisphere [7] , also increases concerns about the fall, which is typically the period of the most severe influenza activity in the northern hemisphere [8, 9] . the rapid spread of the virus in countries with high seasonal influenza vaccine coverage suggests that there is little to no cross-protection conferred by these vaccines [10] . at the same time, the presence of neutralizing antibodies and the generally milder course of dis-ease observed in individuals 160 years of age are indicative of a protective effect of prior infection with antigenetically related viruses [6] . as this pandemic unfolds, and especially in light of the emerging resistance to available antivirals [11] , assessment of the safety and efficacy of available seasonal vaccines as well as matched candidate vaccines is becoming increasingly urgent. the current study evaluates in ferrets-the preferred preclinical animal model for influenza vaccine testing-3 commercially available influenza vaccines from the 2008-2009 season and 1 fully matched laboratory-produced inactivated whole pandemic h1n1 2009 virus vaccine. immune responses were monitored, and the animals were challenged 5 weeks after vaccination with a pandemic h1n1 2009 influenza isolate that exhibits moderate to high virulence in ferrets. viral loads, morbidity, mortality, and postchallenge immune responses were documented for 2 weeks. groups of five 16-to 20-weekold ferrets without antibodies against circulating influenza strains were immunized with one of the 2008 seasonal inactivated split vaccines (fluviral; glaxosmithkline) or the cold-adapted live attenuated vaccine (flumist; medimmune), a swine influenza vaccine (flusure; pfizer), or a matched laboratory-produced inactivated vaccine (ph1n1inact). the latter vaccine consisted of a madin-darby canine kidney (mdck) cell-produced whole-virus preparation of a/mexico/ indre4487/2009 (mx10; h1n1) that was isolated during the ongoing h1n1 influenza outbreak, purified by ultracentrifugation, subsequently inactivated by addition of formalin to a final concentration of 0.1%, and incubated for 3 days at 4њc (fisher scientific). the animals received the recommended dose of the respective commercial vaccines or a dose containing 15 mg of hemagglutinin (ha) of the experimental vaccine. with the exception of flumist, which was inoculated intranasally, all vaccines were injected in the gluteal muscle at the recommended dose for humans or pigs, respectively. five weeks later, the animals were challenged intranasally with 50% tissue 5 10 culture infectious doses (tcid 50 ) of mx10. clinical signs, body temperature, and weight were assessed daily, and animals were euthanized based on clinical evaluation or at the end of the study on day 16. virus quantification and pathology. nasal washes were collected on days 1, 3, 6, 9, and 16 after challenge, and virus titers were quantified by limiting dilution. in brief, 10-fold serial dilutions were incubated on mdck cells with 6 replicates per dilution. at 72-96 h after infection, the plates were scored for cytopathic effect, and the tcid 50 virus titers were calculated using the method of reed and muench [12] . rna was isolated, and viral copy numbers were quantified using real-time reverse-tran-scription polymerase chain reaction (rt-pcr). tissues preserved in rnalater were homogenized using a bead mill homogenizer for extraction of total rna. rna was isolated from nasal washes and swabs, by use of the qiaamp viral rna mini kit (qiagen), and from tissues, by use of the rneasy mini kit (qiagen). the h1n1 virus was detected by quantitative real-time rt-pcr performed using the lightcycler 480 rna master hydrolysis probes (roche) assay targeting the ha gene (nucleotide position 714-815; genbank accession number gq160606). reaction conditions were as follows: at 63њc for 3 min; at 95њc for 30 s; and 45 cycles at 95њc for 15 s and at 60њc for 30 s with the use of a lightcycler 480 (roche). the low detection limit for this h1n1 assay is 0.1 pfu/ml. the primer sequences are as follows: haf, ggatcaagaagggagaatgaactatt; har, aatgcata-tctcggtaccactagattt; and hap, ccgggagacaaa-ataacattcgaagcaac. after euthanasia, necropsy was performed for all animals, and photographs of their lungs were taken before the lungs were harvested for histopathologic analysis. lungs were inflated by slow injection of ∼5 ml of phosphate-buffered saline (pbs; invitrogen) in the trachea, and formalin-fixed and paraffinembedded tissue sections were stained with hematoxylin-eosin. immune response assessment. serum samples were collected on days 3, 7, 10, 14, and 21 after vaccination and were analyzed for the presence of hemagglutination-inhibiting (hai) antibodies against mx10 and the seasonal h1n1 strain a/brisbane/59/2007. hai antibody titers are expressed as the reciprocal of the highest serum dilution that inhibits hemagglutination of turkey red blood cells. on day 10 after vaccination, heparinized blood was collected for proliferation assays. in brief, peripheral blood mononuclear cells were isolated by ficoll-hypaque (ge healthcare) gradient purification and cultivated in the presence of overlapping peptide pools covering the nucleocapsid (np), neuraminidase (na), and ha proteins of the related h1n1 strain a/brevig mission/1918. the proliferation response was measured by adding 5-bromo-2-deoxyuridine (brdu) to the peptide-exposed peripheral blood mononuclear cells after 72 h. the next day, cells were fixed, and brdu incorporation was quantified by immunostaining performed using a chemoluminescent substrate (roche). the proliferation index is expressed as the ratio of brdu incorporation measured for the respective influenza peptide pool and for an ebola virus peptide as negative control. messenger rna profiles of cytokines, including interferon (ifn)-a, ifn-g, interleukin (il)-6, and il-10, were generated from nasal wash rnas isolated on days 1, 3, 6, and 9 after infection and from rna isolated from the right and left lung, respectively, of animals euthanized on day 9. the assays were performed using the primers and method outlined elsewhere [13] . hai antibody titer kinetics were monitored for 21 days after immunization, because titers of 140 reciprocal dilutions remain the reference standard that is predictive of protective immunity elicited by influenza vaccine candidates [14] . hai antibody titers against the pandemic h1n1 2009 mx10 isolate were detected at day 7 in ferrets receiving the laboratory-produced matched vaccine ph1n1inact or the swine influenza vaccine flusure, reaching titers 140 on day 7 or 10, respectively. flusure or ph1n1inact did not generate detectable hai antibody titers against the seasonal h1n1 strain a/brisbane/59/2007 included in conventional seasonal influenza vaccines, such as fluviral or flumist. in contrast, between day 7 and day 10, both seasonal vaccines elicited hai antibody titers against a/brisbane/59/2007 that were 140, whereas no cross-reactive response to mx10 was detected before challenge ( figure 1b) . none of the vaccines elicited an ifn-g enzyme-linked immunospot response upon stimulation with overlapping peptides covering the np, na, and ha proteins of h1n1 strain a/brevig mission/1918. these proteins share 94%, 87%, and 86% amino acid identity with the respective mx10 proteins, which may have contributed to the weak response observed. however, increased proliferation activity in response to np peptide pools was detected in animals immunized with flumist, indicating a cross-reactive t cell response ( figure 1c) . correlation of presence of hai antibodies with milder disease and improved survival. upon intranasal challenge with mx10, all vaccinated animals displayed a 1-day delay in the onset of fever, and they then followed a course comparable to that of nonvaccinated controls (figure 2a) . clinically, animals immunized with the swine vaccine flusure demonstrated more complete protection with mild and transient signs of disease, less weight loss in the first week than in the other groups, and 100% survival. the matched ph1n1inact vaccine also resulted in reduced weight loss, clinical signs of disease, and improvement of the survival rate from 50% to 80%, compared with observations in naive controls ( figure 2b-d) . in contrast, there were no statistically significant differences in recorded clinical signs of disease, weight loss, or survival rate between the animals given fluviral and the control groups, over the course of the experiment after challenge ( ). the group of ferrets vac-p 1 .05 cinated with flumist showed a slight improvement in average body weight at days 5 and 6 after challenge. however, weight loss increased from day 6 to day 9, at which point clinical signs of disease, including nasal seromucous exudates, shallow and labored breathing, and reduced activity forced euthanasia for 4 of the 5 animals given flumist, resulting in a small increase in the mortality rate, compared with that noted for the unvaccinated control group. this increase in the mortality rate associated with mismatched flumist immunization was also observed in a second experiment with 4 animals, although the increase was not statistically significant (data not shown). at the respective times of euthanasia, gross pathological evaluation of lungs demonstrated minimal lesions in all 5 animals vaccinated with flusure, including 1 animal euthanized on day 9, without reaching experimental end points to obtain, on a timely basis, tissue samples matched to those obtained from the other groups ( figure 3 ). animals vaccinated with ph1n1inact showed a slight improvement, with smaller lesions noted in 3 of the 5 ferrets. three of 4 control animals had severe lesions with hepatization, hemorrhages, and widespread alveolitis and bronchiolitis, which were comparable to lesions observed in 4/5 or 5/5 ferrets vaccinated with fluviral or flumist, respectively. all groups reached nasal infectious titers of ∼ tcid 50 at day 1 after challenge, with the exception 6 10 of the animals immunized with the swine vaccine flusure, which had a 10-fold lower titer ( figure 4a ). the group vaccinated with flumist maintained relatively high levels of virus replication through day 3, whereas the other groups experi-enced titer decreases of у20-fold. with the exception of one animal in the control group, no infectious viral particles were detectable by titration in the nasal washes of animals on day 6 or later, although viral rna could be detected by real-time rt-pcr until the end of the experiment ( figure 4b ). animals immunized with ph1n1inact or flusure experienced the lowest ifn-a levels and up-regulation of ifn-g during the early stage of the infection. moderately elevated levels of il-6 were detected later in the course of disease-at day 9 exclusively in these 2 groups, which showed evidence of protection ( figure 5 ). in contrast, the group vaccinated with flumist exhibited the highest average of mrna transcripts for ifn-a, up to day 6, and for ifn-g, at day 3, possibly reflecting a better cellular response. of interest, at day 3, levels of il-10 transcripts were significantly higher in the upper airway of animals vaccinated with flumist, and at day 9 after infection, they were slightly increased in the lower airway, compared with observations in control animals and animals vaccinated with ph1n1inact or flusure ( figure 5 ). no differences in tumor necrosis factor-a and il-8 levels were observed between the groups (data not shown). the availability of an efficient vaccine is essential to alleviate the effect of the ongoing influenza pandemic. a possible intervention strategy to mitigate the 2009 fall influenza season in the northern hemisphere was to initially perform mass immunization with the seasonal vaccine, followed by mass immunization with the fully matched pandemic h1n1 2009 vaccine as soon as would become available. to direct a concerted public health response to control the spread of the virus, the efficacy of a newly produced matched inactivated vaccine, as well as that of already available inactivated and live attenuated vaccines, has to be assessed. toward this end, we compared the antibody and cellular responses elicited by 2 seasonal vaccines (fluviral and flumist), the commercial swine vaccine flusure, and a laboratory-produced matched inactivated whole-virus preparation. we found that only the swine and matched vaccines resulted in production of hai antibodies against the pandemic h1n1 2009 virus, whereas only flumist triggered a crossreactive cellular response. intranasal challenge with the virulent mexican isolate mx10, similar to mx/4482, which also leads to a 50% mortality rate among naive animals [6] , revealed that none of the vaccines was able to confer complete protection after only one immunization. however, flusure was associated with the best reduction in morbidity and complete protection from mortality, whereas the matched inactivated vaccine resulted in moderate clinical improvement and reduced mortality. as was expected from undetectable hai antibody titers, animals vaccinated with fluviral or flumist did not experience a beneficial effect, compared with unvaccinated control animals. the partial protection observed in animals vaccinated with one dose of the matched inactivated vaccine, despite the detection of an hai antibody response within the protective range, indicates that protection from aggressive isolates may require more than a single immunization, which would put an additional strain on vaccine availability. the use of a more virulent challenge strain enables assessment of vaccine efficacy in a worst-case scenario. however, the disease severity associated with currently circulating pandemic h1n1 2009 strains in most patients is more similar to that associated with seasonal influenza [3] . it is thus possible that a single 15-mg dose of a matched inactivated vaccine will be sufficient to confer protection against most pandemic h1n1 2009 strains, especially in individuals with some levels of cross-protection due to previous influenza infection. the efficiency of a commercially available swine vaccine indicates that this product would be adequate to protect animals, including pig herds, which could minimize interspecies transmission and maybe limit evolution of the virus. the flusure vaccine consists of an inactivated h1n1 and h3n2 type a field isolate formulated with amphigen (pfizer) as an adjuvant [15] , indicating that the use of this and other adjuvants merits a more in-depth evaluation in the context of the development of improved human influenza vaccines. a curious observation is the more severe cases of disease and the higher mortality rate noted for animals vaccinated with flumist, which correlated with slightly more infectious virus in the nasal washes of this group at day 3. this correlation figure 5 . relative quantification of cytokine messenger rna (mrna) induction. changes in cytokine mrna levels were determined by semiquantitative real-time reverse-transcription polymerase chain reaction in nasal wash rna or rna isolated from lung tissue harvested on day 9. ten nanograms of rna were used for each reaction, and the fold change was calculated using the comparative cycle threshold (ddct) method. columns denote the mean of all values obtained for the respective group, and error bars denote the standard error. ctl, nonimmunized control group; dpi, days post infection; ifn, interferon; il, interleukin. between the infectious viral load in the upper respiratory tract and the clinical outcome was in fact observed for all groups in the present study, confirming previous reports that the infectious viral load in the airway is predictive of levels of protection in vaccinated ferrets after challenge with respiratory viruses, including severe acute respiratory syndrome-associated coronavirus and influenza [16, 17] . on the other hand, data from this study also indicate that weight loss was, on average, more severe for the unvaccinated control animals than for any other vaccinated group of animals. the present study was designed to evaluate protective efficacy after vaccination and not the possible subtle negative effects caused by immunization with mismatched influenza antigens. larger study group sizes will be necessary to conclusively address this question with an appropriate degree of statistical confidence. antibody-mediated enhancement of influenza infection, including subtype-crossreactive, nonneutralizing antibodies, has been previously described in cultured cells [18] [19] [20] . this mechanism has never been directly associated with a worsened clinical condition in animal models of influenza infection, although a recent study reported that maternally derived antibodies possibly enhanced swine influenza virus-induced pneumonia in pigs [21] . these observations further support the need for a more detailed evaluation of the efficacy of influenza vaccine in controlled exper-imental conditions where various levels of preexisting immunity to mismatched influenza antigens could be studied. all animals demonstrated a strong induction of the proinflammatory cytokine il-6 at day 6; this level remained elevated at day 9 only in the 2 groups of ferrets showing noticeable protection. animals vaccinated with flumist, the only group that mounted a strong cross-reactive cellular response, had the highest ifn-g response. however, this response was not sufficient to control the disease. in fact, the strong expression of il-10, an anti-inflammatory cytokine, detected in that group on day 3 may have suppressed an appropriate inflammatory response, including il-6 expression, and temporarily favored virus replication, as previously demonstrated in pigs infected with foot-and-mouth disease [22] . there are reports showing the negative effect of il-10 on influenza virus-infected mice and pigs [23, 24] , and increased il-10 production correlated with a low antibody response in elderly individuals after influenza vaccination [25] . evaluating the response of cytokines, including il-6 and il-10, at early time points in patients may help predict unfavorable outcome and allow for better allocation of resources to individuals requiring more intensive clinical intervention. the present study reports the immune responses and protective efficacy of commercially available vaccines and one lab-oratory-produced matched vaccine with regard to prevention of pandemic h1n1 2009 infection in ferrets. the findings of this study may help to guide ongoing preparations for the influenza season in the northern hemisphere. world health organization (who) pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico swine influenza a (h1n1) strikes a potential for global disaster severe respiratory disease concurrent with the circulation of h1n1 influenza emergence and pandemic potential of swine-origin h1n1 influenza virus transmission and pathogenesis of swine-origin 2009 a(h1n1) influenza viruses in ferrets and mice influenza a(h1n1)v in the southern hemisphere-lessons to learn for europe? influenza activity in europe during eight seasons (1999-2007): an evaluation of the indicators used to measure activity and an assessment of the timing, length and course of peak activity (spread) across europe surveillance for influenza-united states, 1997-98, 1998-99, and 1999-00 seasons emergence of a novel swine-origin influenza a (h1n1) virus in humans oseltamivir-resistant novel influenza a (h1n1) virus infection in two immunosuppressed patients a simple method of estimating fifty percent endpoints severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction a trivalent virus-like particle vaccine elicits protective immune responses against seasonal influenza strains in mice and ferrets adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques investigation of the biological indicator for vaccine efficacy against highly pathogenic avian influenza (hpai) h5n1 virus challenge in mice and ferrets antibody-mediated growth of influenza a nws virus in macrophagelike cell line p388d1 infection enhancement of influenza a nws virus in primary murine macrophages by anti-hemagglutinin monoclonal antibody subtype cross-reactive, infectionenhancing antibody responses to influenza a viruses the immune response and maternal antibody interference to a heterologous h1n1 swine influenza virus infection following vaccination immunosuppression during acute infection with foot-and-mouth disease virus in swine is mediated by il-10 alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs il-10 deficiency unleashes an influenza-specific th17 response and enhances survival against high-dose challenge high interleukin-10 production is associated with low antibody response to influenza vaccination in the elderly we would like to thank eric poeschla, for providing the flumist doses, and naveed zafar janjua and nicholas svitek, for help with literature review or the cytokine real-time reverse-transcription polymerase chain reactions, respectively. key: cord-344271-5aynmdsk authors: de souza luna, luciano kleber; panning, marcus; grywna, klaus; pfefferle, susanne; drosten, christian title: spectrum of viruses and atypical bacteria in intercontinental air travelers with symptoms of acute respiratory infection date: 2007-03-01 journal: j infect dis doi: 10.1086/511432 sha: doc_id: 344271 cord_uid: 5aynmdsk respiratory infections after air travel are frequent, but epidemiological data are incomplete. using sensitive polymerase chain reactions, we studied the spectrum of atypical bacteria and respiratory viruses in travelers fulfilling the case definition of severe acute respiratory syndrome. a pathogen was identified in 67 travelers (43.2%). influenza and parainfluenza viruses were most prevalent, at 14.2% and 15.5%, respectively. prevalences of adenoviruses, human metapneumovirus, coronaviruses, and rhinoviruses ranged between 2.6% and 4.8%. human bocavirus, respiratory syncytial virus, and legionella, mycoplasma, and chlamydophila species were absent or appeared at frequencies of <1%. to our knowledge, these are the first specific baseline data for the mentioned agents in the context of air travel. istered, laboratory data on the spectrum of causative agents are actually not available [1, 4, 5] . the epidemic of severe acute respiratory syndrome (sars) in 2003 involved a period of heightened awareness of respiratory infections after air travel. samples for laboratory testing were routinely obtained from patients, which provided a unique opportunity for studying their disease etiologies. after initial characterization of the causative agent of sars, we acted as a diagnostic reference laboratory for the world health organization (who) [6] . during the epidemic, we accepted samples for initial and confirmatory testing exclusively from those patients who fulfilled the who case definition of suspected or probable sars. the definition was designed to be sensitive and thus to prevent any possible transmission, but its specificity was low. it covered most respiratory illnesses compatible with viral or atypical bacterial infection. respiratory agents are best diagnosed by direct assays, the most sensitive of which is polymerase chain reaction (pcr). because of the rapid pace at which the technique evolves, we searched the literature for the most up-to-date pcr assays that cover the broadest possible range of genetic diversity for each respective agent. high sensitivities had to be clearly proved in studies. where assays fulfilling these criteria were not available, we established sensitive real-time pcrs de novo. these assays were used to determine a point prevalence of the full spectrum of respiratory viruses and atypical bacteria in sars-compatible patients. patients and methods. respiratory samples ( ) from n p 214 172 patients were available. all patients fulfilled the who case definition of suspected or probable sars, which required a combination of fever and lower-respiratory-tract symptoms (e.g., cough or difficulty breathing) plus either a stay in an affected area during the preceding 10 days or close contact with suspected patients. probable cases additionally had radiological evidence of respiratory distress syndrome without other reason. samples were prepared as described elsewhere [6] . assays included adenovirus (adv) [7] ; rhinovirus (rv) [8] ; human bocavirus (hbov) [9] ; human coronaviruses (hcov) 229e, nl63, and oc43 (table 1) ; influenza virus (inf) a (table 1) ; and a combined assay for inf a and b, parainfluenzaviruses (pivs) 1-3, respiratory syncytial virus (rsv), and human metapneumovirus (hmpv) (hexaplex plus; prodesse). the commercial system was chosen because several published respiratory multiplex assays gave instable results in our laboratory. we also tested for the presence of atypical bacteria, including mycoplasma, chlamydophila, and legionella species [10] . fortable 1 . formulations of reverse-transcription (rt)-polymerase chain reaction (pcr) assays designed for the study. sense primer probe antisense primer formulation hcov 229e and oc43 tactatgactggcagaatgtttca and tactatgactactagacagtttca, mulations of new assays established for the present study are provided in table 1. their limits of detection (lods) were determined using in vitro-transcribed rna [6] . lods for hcovs nl63 and inf a were !5 copies of synthetic rna genomes per reaction, corresponding to ∼50 rna copies/swab sample. lods for hcov 229e and oc43 were !20 copies/ reaction, corresponding to ∼200 copies/swab sample. results. travel histories were reconstructed by telephone interviews with hospitals, family physicians, or patients themselves. for 164 patients (hereafter called "flight patients"), additional information was retrieved. this included 124 for whom the exact dates and destinations of departure to germany were known and 22 for whom only destinations could be reconstructed. seventeen patients had not traveled as initially reported but had been in contact with patients who had suspected sars. for 8 patients, no additional information could be re-trieved. they were included in the cohort because a travel history had been confirmed before samples were received. only those patients who had reportedly not traveled were evaluated separately. they are hereafter called "contact patients." the average age of flight patients was 42.2 years (range, 1-79 years). the average age of contact patients was 41.8 years (range, 4-79 years). neither the average or median ages nor the sds in both groups were significantly different. figure 1 shows the age distributions in both groups. some 71% of flight patients were between 19 and 60 years old, as were 76% of contact patients ( ). only 12 patients were !19 years old, 11 of p p .57 whom were flight patients. they contributed 13.4% of all positive findings in flight patients, which was significantly more than the mean positivity rate in all age groups ( , stu-p ! .012 dent's t test). at least 1 pathogen was detected in 67 flight patients (43.2%) and in 8 contact patients (47%) ( , student's t test). table p p .7 2 shows the global pathogen detection rates by age group, as well as the relative and absolute prevalences of pathogens in flight patients. in contact patients ( ), adv, human piv, n p 17 inf b, and hmpv were detected in 2 patients each and inf a in 1 patient. double infections occurred in only 2 patients: rv/ piv in a flight patient and adv/piv in a contact patient. inf and piv were clearly the most prevalent agents in flight patients, at 14.2% and 15.5%, respectively, without significant differences between age groups (1-way analysis of variance [anova], 95% significance level). equal distribution of these viruses was also seen in contact patients. covs were more frequent than one would expect in a mostly adult cohort. detection rates did not differ between age groups at the 95% significance level. rsv and hmpv appeared to be significantly more frequent in flight patients !18 years old than in the other age groups ( and .01, f test). for hmpv, this was also p p .006 seen in contact patients ( , f test). the novel hbov was p p .02 not detected in any patient, which indicates that this agent may be restricted to children. indeed, all data available so far about bov have been derived from cohorts of young infants [9, 11, 12] . a complete absence was also observed of chlamydophila species, but it cannot be derived from our data what fraction of patients had already received preemptive antibiotic treatment at the time of sampling. there was no association between the airport of departure and the detection of pathogens in general ( , 1-way anova) p ! .3 or of any pathogen in particular. to identify any possible influence of sampling on detection rates, we analyzed common categories of clinical samples separately for both flight and contact patients. samples were categorized as follows: upper-respiratory-tract samples (category 1), throat-wash fluids (category 2), and lower-respiratory-tract samples (sputum and bronchoalveolar lavage [bal] fluids; category 3). a comparison of age versus sample type yielded a significant lack of lower-respiratory-tract samples in patients !19 years old (1-way anova, 95% significance level). because a significantly higher detection rate of viruses had been identified in these patients, they were eliminated from the analysis, to avoid a bias. after elimination, no significant age differences in the 3 categories of samples remained ( , f test). the p p .3 average ages for categories 1, 2, and 3 were 42.1, 43.9, and 47.0 years, respectively. upper-respiratory-tract samples analyzed included 39 pharyngeal, nasal, and nasopharyngeal swabs and 99 throat-wash fluids. lower-respiratory-tract samples included 50 sputum specimens and 4 bal fluids. the detection frequencies of every pathogen in the 3 categories were compared by separate anovas. global detection frequencies did not differ significantly between categories (any pathogen: 51.3%, 34.3%, and 46.3% in category 1, 2, and 3, respectively). only for inf was the detection frequency in swabs was significantly higher than that for other samples (category 1, 30.1%; category 2, 10.1%; category 3, 14.8%; , f test). the same was observed for p p .01 inf a only (21%, 5%, and 9%, respectively; ) but not p p .01 for inf b alone. discussion. baseline data on the prevalence of respiratory viruses and atypical bacteria after air travel are not currently available. these patients constitute a specific subcohort of patients with community-acquired respiratory disease. unlike general cohorts, children and elderly persons are underrepresented, and adults of working age constitute the majority of patients (71% in our study). normally, respiratory infections in adults are mild and infrequent, but this may change in the context of travel. there is evidence that air travel increases the incidence of respiratory disease in general and that significant transmission in modern aircraft has occurred of sars, inf, and other agents of respiratory infection [2, 4] . because morbidity during working life is economically relevant and new thera-peutic options are at hand, investigation into the etiology of travel-associated respiratory disease seems to be well justified. the criteria of the who sars case definition, including fever, possibly caused an underrepresentation of milder infections in our cohort (rsv and hmpv). however, this made our patients representative of those who are likely to be absent from work. we used up-to-date diagnostic assays to determine the spectrum of viral or atypical agents in these patients. the rate of resolved etiologies was markedly higher than that in studies of community-acquired respiratory infections, including recent collective analyses and some of the latest original studies [5, [13] [14] [15] . this may be due to the extended spectrum of highly sensitive assays applied. our main finding is that the spectrum of agents in returning travelers is broad. almost one-half of all patients were infected with respiratory viruses. agents with treatment options, such as influenza or atypical bacteria, were present in only 15.4% of flight patients and 15.7% of all patients, making even upto-date diagnostics unrewarding. in this context, it is interesting that lower-respiratory-tract samples did not yield increased detection rates. the general assumption that nasopharyngeal washes are more sensitive than swabs in patients with community-acquired respiratory disease could not be confirmed in our study [5] . good detection rates and a low risk of aerosolization suggest that swabs should be the preferred in this type of patients. it cannot be told whether the high prevalence and diversity of respiratory viruses seen in our study is specific to patients with recent intercontinental air travel. because otherwise healthy adults of working age are usually not tested for respiratory viruses, comparison data from similar cohorts are not available. our contact patients provided a small control group, and the observed prevalence of viruses confirms the findings seen in flight patients. however, it should be noted that all contact patients fulfilled the case definition of sars, requiring recent contact with a suspected patient [6] . in germany, this means that all of them most likely had contact with a recent intercontinental traveler. it is unclear why and how patients acquire viral respiratory disease in the context of air travel [2, 4] . in our study, it was interesting that significant clusters of patients with the same diagnosis, who could have been on the same flight, did not exist. similarly, no association was detected between any pathogen and a particular airport. it would thus be likely that viruses are picked up during prior travel rather than being acquired in flight. in any case, data from this unique cohort suggest that the prevalence of known, emerging, and potentially novel respiratory viruses in adults must be carefully studied. understanding the spectrum and the etiological contribution of viruses in adult respiratory disease will be of growing importance in the future. with new antiviral drugs at hand, more consultations will occur for respiratory diseases. targeted therapy will require broad-spectrum pathogen detection with rapid results, most likely by pcr. given the large range of assays required for this study and considering current reagent costs, it is obvious that developing more efficient diagnostic assays is as essential as developing drugs themselves. today, diagnostic technology is far from attaining this aim. respiratory infections during air travel aircraft cabin air recirculation and symptoms of the common cold prevalence of respiratory symptoms among female flight attendants and teachers transmission of infectious diseases during commercial air travel community-acquired pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome internally controlled real-time pcr monitoring of adenovirus dna load in serum or plasma of transplant recipients amplicon sequencing and improved detection of human rhinovirus in respiratory samples cloning of a human parvovirus by molecular screening of respiratory tract samples single-run, parallel detection of dna from three pneumonia-producing bacteria by real-time polymerase chain reaction evidence of human coronavirus hku1 and human bocavirus in australian children detection of human bocavirus in japanese children with lower respiratory tract infections enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests surveillance of respiratory virus infections in adult hospital admissions using rapid methods characterization of viral agents causing acute respiratory infection in a san francisco university medical center clinic during the influenza season we are grateful to laurent kaiser, eric claas, and harald kessler for providing detailed bench protocols for the published polymerase chain reaction assays used in the study. key: cord-329392-fufattj8 authors: den hartog, gerco; schepp, rutger m; kuijer, marjan; geurtsvankessel, corine; van beek, josine; rots, nynke; koopmans, marion p g; van der klis, fiona r m; van binnendijk, robert s title: sars-cov-2–specific antibody detection for seroepidemiology: a multiplex analysis approach accounting for accurate seroprevalence date: 2020-08-08 journal: j infect dis doi: 10.1093/infdis/jiaa479 sha: doc_id: 329392 cord_uid: fufattj8 background: the covid-19 pandemic necessitates better understanding of the kinetics of antibody production induced by infection with sars-cov-2. we aimed to develop a high-throughput multiplex assay to detect antibodies to sars-cov-2 to assess immunity to the virus in the general population. methods: spike protein subunits s1 and receptor binding domain, and nucleoprotein were coupled to microspheres. sera collected before emergence of sars-cov-2 (n = 224) and of non-sars-cov-2 influenza-like illness (n = 184), and laboratory-confirmed cases of sars-cov-2 infection (n = 115) with various severities of covid-19 were tested for sars-cov-2–specific igg concentrations. results: our assay discriminated sars-cov-2–induced antibodies and those induced by other viruses. the assay specificity was 95.1%–99.0% with sensitivity 83.6%–95.7%. by merging the test results for all 3 antigens a specificity of 100% was achieved with a sensitivity of at least 90%. hospitalized covid-19 patients developed higher igg concentrations and the rate of igg production increased faster compared to nonhospitalized cases. conclusions: the bead-based serological assay for quantitation of sars-cov-2–specific antibodies proved to be robust and can be conducted in many laboratories. we demonstrated that testing of antibodies against multiple antigens increases sensitivity and specificity compared to single-antigen–specific igg determination. coronavirus disease 2019 (covid-19) caused by the newly emerged severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has resulted in a pandemic in a largely immune-naive population. the presence of specific antibodies is currently being investigated to assess the induction of an immune response in patients and to assess the degree of exposure and immunity in the general population [1] [2] [3] . as it is a recently emerged coronavirus variant, the kinetics and degree of immunity induced following contact with the virus and covid-19 disease are largely unknown. sars-cov-2 expresses a spike protein, highly similar to spike of sars-cov, which binds to angiotensin converting enzyme 2 (ace2) [4, 5] . binding of antibodies to the receptor binding domain (rbd) of spike neutralizes the ability of the virus to infect cells [6] . in addition, antibodies are detected against other viral proteins, including nucleoprotein (n) [7] . n is shielded within the virion and therefore n-specific antibodies are probably unable to neutralize the virus. although n may not be involved in neutralization of the virus, antibodies to n could provide an indicator of exposure to the virus. antibodies to n induced by sars-cov reportedly recognize n of sars-cov-2 but not of seasonal coronaviruses [8] . estimates of the prevalence of seroconversion as proxy for protection of the general population may support health decision making, including the decision to lift lockdown measures. to appropriately apply an assay for serosurveys we need to know the precision of the assay, that is the sensitivity and specificity, which are variable between currently available tests [9, 10] . performing and sustaining large studies to assess changing population immunity requires high-throughput screening assays that are robust and accurate [11] . many countries now aim to assess the protective status of the general population for covid-19 using antibody assays. to guarantee high specificity, the assay should be validated with a representative number of sera from patients infected with other coronaviruses and other pathogens causing influenza-like illness (ili), but this is often lacking [11] [12] [13] . to date, covid-19 prevalence of seroconverted individuals is relatively low and there is a risk of significant overestimation if an assay has insufficient specificity (supplementary table 1 ). thus, high specificity is important at this stage [11, 12] . our laboratory has extensive experience in developing multiplex assays to quantify antibodies to many bacterial and viral pathogens in the general population, of which most are part of the national immunization program [1, [14] [15] [16] [17] . we developed a high-throughput and highly quantitative bead-based multiplex immunoassay to assess the prevalence of seropositivity in the general population, and also anticipating the introduction of future sars-cov-2 vaccines. by multiplexing a broader range of sars-cov-2 antigens in a single assay we may generate a better understanding of the proportion of persons that have seroconverted. moreover, in a multiplex assay positivity can be compared among antigens to provide a more detailed evaluation of the antibody levels and to enhance assay performance [17] . the developed assay was tested on samples from covid-19 patients with various severities of disease collected at multiple timepoints to determine the kinetics of seroconversion. serum samples were obtained from the following cohorts: (1) a random selection of individuals (n = 224) from a national (dutch) cohort representing all age groups and obtained 3 years prior to sars-cov-2 emergence (pienter3 study, netherlands trial register number nl5467); (2) individuals (supplementary table 2 ) with proven non-sars-cov-2 ili caused by human coronaviruses (n = 110, hcov ili) or other viruses (n = 74, non-hcov ili) obtained from the national institute for public health and the environment (rivm), bilthoven, the netherlands (trial register number nl4666) [18] , and from erasmus medical center, rotterdam, collected prior to the sars-cov-2 outbreak and at least 2 weeks after polymerase chain reaction (pcr) detection of the virus; and (3) the steps in assay validation were similar to recently developed bead-based multiplex immunoassays for cmv, ebv, and rsv, with minor modifications as described below [16, 17] . for the multiplex bead-based immune assay the following antigens obtained from sino biological were used: sars-cov-2 monomeric spike s1 (40591-v08h), rbd (40592-v08b), and nucleoprotein (n) (40588-v08b). microplex fluorescent beads were activated in 50 mm 2-(n-morpholino)ethanesulfonic acid (mes) ph 5.5. the proteins were diluted to a concentration of 0.2 mg/ml in phosphate-buffered saline (pbs) ph 7.4 and added at 5 µg per 75 µl of activated beads. an internal reference sample was created by pooling 13 sera of covid-19 patients with varying immunoglobulin g (igg) concentrations. an arbitrary antibody concentration unit of 100 was assigned on the basis of the mean fluorescence intensity (mfi) signal in the upper limit of linearity of a 3-fold serial dilution of the reference sample. sera (25 µl) diluted 1:400 and 1:8000 in sm01 buffer (surmodics) plus 2% fetal calf serum were incubated with antigen-coated beads for 45 minutes at room temperature at 750 rpm in the dark. following incubation, samples were washed 3 times with pbs, incubated with phycoerythrin-conjugated goat anti-human igg for 30 minutes and washed. samples were acquired on an lx200 or fm3d (luminex). mfi was converted to arbitrary units (au/ml) by interpolation from a 5-parameter logistic standard curve, using bioplex manager 6.2 (bio-rad laboratories) software and exported to microsoft excel. different batches of antigen-conjugated beads were incubated with serially diluted sera to test linearity and parallelism between bead conjugations, reference, and serum samples. assay robustness was tested by analyzing a serum panel by 3 different operators on independent days using 2 different bead and 2 reference batches. the ability to discriminate igg concentrations between covid-19 patients and controls was evaluated by receiver operator characteristic (roc) analyses. to select the optimal assay defaults, both the youden j statistic, which balances between sensitivity and specificity, and a specificity-optimized cutoff (specificity of at least 98.5% for low-prevalence settings of 5%-10%) were selected. data were entered into graphpad prism 8.4.1 to generate graphs and perform statistical analyses. reproducibility of the assay was evaluated using r 2 and coefficient of variation (% cv) calculated by standard deviation divided by average × 100. for the roc analyses antibody concentrations of cross-sectional pienter3 participants (n = 224), ili patients with coronavirus (n = 74), or other viral infection (n = 110) were used as the negative control group and pcr-confirmed covid-19 samples (n = 115) with various clinical severities were used in the positive group. we selected for serum samples that were obtained more than 10 days post onset of disease symptoms to meet a reasonable degree of seroconversion, as shown in recent reports [8, 19] . both the youden j statistic-determined cutoff and the specificity-optimized cutoff (specificity of at least 98.5%) were determined. to compare differences in concentrations, data were logtransformed and tested with either a t test between 2 groups, or 1-way anova and tukey's multiple comparison test to compare multiple groups and adjusted p values reported. antibody kinetics was fitted using a nonlinear 4-parameter least square fit in graphpad prism 8.4.1. we prepared a reference serum by pooling 13 pcr-confirmed covid-19 sera and tested serial dilutions in the multiplex assay consisting of distinct fluorescent beads coupled to sars-cov-2 nucleoprotein (n), s1, and the s1 subunit rbd ( figure 1a ). this was repeated for varying batches of beads to assess consistency of performance. the assay was able to quantify concentrations in a 1000 to 10 000-fold concentration range, using a single dilution of the serum. to reliably quantify antibody concentrations between the reference serum and test samples, we confirmed that the reference and a selection of samples display the same rate of decline of fluorescence signal with increasing dilutions, which is referred to as parallelism ( figure 1b) . these data show that the triplex assay is a highly quantitative assay to detect antibodies to sars-cov-2. applying an assay in large population and longitudinal studies requires reproducibility of assay results. therefore, antibody concentrations were determined in independent experiments performed on 6 different days, using a selection of 214 samples for rbd and 268 samples for n and s1 with different concentrations of sars-cov-2 antibodies ( figure 1c ). in addition, the reproducibility test was performed by 3 different technicians using different bead batches and references to reflect the expected maximum variability of the assay over time. comparison of sample data determined on 2 independent assays runs resulted in an r 2 of 0.982, 0.985, and 0.988 for n, s1, and rbd, respectively ( figure 1c ). the obtained % cvs were 19.1, 25.5, and 14.6 for n, s1, and rbd, respectively, showing that the assay results were reproducible. sera of 115 pcr-confirmed covid-19 patients after 10 days of symptoms were tested in the assay and the results compared to a control panel of 408 sera collected prior to the outbreak of sars-cov-2. in covid-19 patients, high concentrations of igg were observed to all 3 antigens (figure 2a ). despite clear discrimination of igg concentrations between groups of control and covid-19 patients, some samples overlapped between the 2 groups. therefore, the specificity and sensitivity of the assay to discriminate between covid-19 patients and controls using igg concentrations was evaluated by an established statistical standard to analyze assay performance, the roc analyses. for the roc analyses, concentration data of hospitalized and nonhospitalized covid-19 disease cases were included to provide a realistic evaluation of the performance of the assay (figure 2a ). the area under the curves ranged from 0.9839 to 0.9859 ( figure 2b ). the roc generated cutoff concentrations of 14.8, 0.85, and 8.21 au/ml using the roc youden j statistic. to gain a higher specificity of the assay optimized for a low population seroprevalence, the cutoff concentrations were 19.7, 2.37, and 19.1 for n, s1, and rbd, respectively ( figure 2c ). the latter cutoffs resulted in a specificity of 98.5%, 99.0%, and 98.5% at a sensitivity of 89.4%, 84.4%, and 83.6% for n, s1, and rbd, respectively. to study how our assay discriminates between antibodies of individuals with different laboratory-confirmed viral infections, antibodies were measured in a cross-sectional population panel (n = 224), a panel of noncorona ili patients (non-hcov ili, n = 74), and non-sars-cov-2 corona ili patients (hcov ili, n = 110) and compared to pcr-confirmed covid-19 patients' samples. some of the covid-19 patients were admitted to hospital (n = 50) because of severe covid-19 and these were compared to nonhospitalized covid-19 cases (n = 65). for each of the 3 negative control groups the majority of the samples had concentrations below the cutoff for all 3 antigens ( figure 3a ). the number of falsepositive samples ranged from 5 to 6 out of 404 or 408 samples tested for the different antigens. the non-hcov and hcov ili panels were from persons infected with multiple different non-sars viruses including 4 different endemic coronavirus (supplementary table 2 ). the proportion of false positives did not increase by testing the convalescent sera from patients with a laboratory (pcr)-confirmed infection with either of the 4 seasonal coronaviruses ( figure 3b , and data not shown), indicating that the antigens used in the assay are selective for sars-cov-2-induced antibodies. comparison of pcrconfirmed sars-cov-2 patients samples shows that all hospitalized patients induced antibodies to n and the majority of hospitalized patients induced antibodies to s1 and rbd. the majority of the nonhospitalized cases showed antibody concentrations above the cutoff for n, whereas around 10% of the nonhospitalized patients did not produce antibodies above the cutoffs for s1 and rbd. overall, the concentrations of antibodies in serum samples from patients that were hospitalized were significantly higher compared to patients that were not hospitalized. following infection, an immune response is initiated, resulting in the production of serum antibodies. to study the time between onset of disease symptoms and the development of antibodies, paired serum samples were collected from the majority of patients. data were separated for patients that were either admitted to the hospital or not ( figure 4a and 4b) . apart from the paired samples from 2 patients that were obtained before 7 days after onset of disease, all other hospitalized cases showed seroconversion for all 3 antigens tested ( figure 4a ). in line with other reports, hospitalized covid-19 patients seroconverted around day 10 of disease onset. of 53 nonhospitalized cases, 48 seroconverted, whereas 5 showed slight increases in concentrations but failed to formally cross the cutoff value for any of the 3 analytes to be regarded a specific seroconversion. hospitalized patients reached a plateau of antibody production shortly after 2 weeks from onset of symptoms, which took at least 25 days for the nonhospitalized cases (4-10 fold lower slope; figure 4c and 4d). as a consequence of the slower increase of antibody concentrations the time to detectable antibodies was delayed, especially with respect to antibodies reacting to s1 and rbd. the variance in the nonhospitalized cases was high compared to the hospitalized cases, which is illustrated by the lower r 2 of the nonlinear least square fit of the 2 patient groups. cutoff (au/ml) figure 2 . ability of the assay to identify covid-19 patients. a, control sera (n = 408) and covid-19 sera (n = 115) collected after day 10 of symptoms were tested and compared for concentrations of igg. median concentration and 95% confidence intervals are shown. b, the sera tested in (a) were analyzed by roc. c, the roc data were used to determine youden j statistic cutoff (lower cutoff) and a specificity-optimized cutoff of at least 98.5% specificity (higher cutoff). abbreviations: au, arbitrary unit; igg, immunoglobulin g; n, nucleoprotein; rbd, receptor binding domain; roc, receiver operator characteristic; s1, spike protein subunit 1. table 2 ) and concentration data of ili patients are shown to confirm that the assay discriminates sars-cov-2-specific antibodies from antibodies induced by various laboratory-confirmed viral infections. abbreviations: au, arbitrary unit; covid-19, coronavirus disease 2019; hcov, human coronavirus; mers-cov, middle east respiratory syndrome coronavirus; n, nucleoprotein; non-hcov, noncoronavirus; rbd, receptor binding domain; rsv, respiratory syncytial virus; s1, spike protein subunit 1. the engagement of different structural sars-cov-2 proteins in 1 serological determination (multiplex testing) instead of 1 protein could improve the sensitivity and the specificity. if only 1 analyte is analyzed, the sensitivities for hospitalized cases were 94.1%, 94.3%, and 100% for rbd, s1, and n, respectively, using the specificity-optimized cutoff (table 1) . using the roc youden j statistic cutoff the sensitivities were 97.1% for both s1 and rbd and 100% for n. nonhospitalized cases typically had lower concentrations of igg, which reduced the sensitivity: 76.3% for s1 and up to 82.7 % for n using the specificity-optimized cutoff. using the youden j statistic cutoff, the sensitivity increased to 91.3% for s1. in this multiplex approach an increased sensitivity can be obtained by evaluating a sample as positive when either 1 of the antibody concentrations determined is higher than the set cutoff (logical or analysis in table 1 ). any combination of antigen reached a sensitivity of 100% when n was used in hospitalized cases and ranged from 90.4% (s1 or rbd) up to 95.1% (n or s1 or rbd) using the specificity-optimized cutoff. applying the youden j statistic cutoff resulted in a sensitivity for nonhospitalized cases of at least 92.8% (n or s1) up to 98.8% (n or s1 or rbd). the specificity of the youden j analyses using n or s1 or rbd dropped to 90.9%. this specificity is far too low for serosurveillance purposes in areas of low prevalence. the specificity-optimized cutoff (95.8%-97.8%) is clearly better, which may be considered adequate if the true prevalence in the population is above 20%. because in most countries the overall covid-19 seroprevalence is currently under 20%, high specificity is required to provide reliable seroprevalence estimates (illustrated in supplementary table 1 ). this could be achieved by defining a sample positive when at least 2 antibody test results in multiplex are above the cutoff. this resulted in a specificity of 100% for any of the combinations and both the specificityoptimized and the youden j statistic-determined cutoffs (logical and; table 1 ). as expected, this increased specificity comes at the expense of the sensitivity. here, if only s1 and rbd are taken into consideration, this combination resulted in the highest possible sensitivity of 87.3% and 97.1% for nonhospitalized and hospitalized patients, respectively. we aimed to develop a high-throughput quantitative assay to measure true concentrations of antibodies to spike s1, spike rbd, and n of sars-cov-2. the assay presented here uses a very small sample volume, which can be obtained from, for example, fingerstick blood, while retaining highly quantitative output. this bead-based multiplex immunoassay generates robust results and is able to discriminate covid-19 with different degrees of disease severity, especially from day 10 of disease onward. the results of the assay presented here provide detailed insight into the performance of the assay in terms of parallelism between the references and sera containing different concentrations of antibodies. in addition, we show consistency of assay results when the same samples are measured on independent days, by different investigators using different batches of reagents, basically incorporating all potential variability. large population studies are in high demand to provide insight into the spread of the virus and the protective status of the population, which can be used for policy makers to manage the pandemic or lift the lockdown measures [2, 3, 11, 12] . assays results have to be accurate to generate reliable seroprevalence data of the general population. in addition to knowing the performance of an assay, we need to understand how the majority of infections in the general population relate to the induction of detectable antibodies. our data comparing hospitalized and nonhospitalized cases revealed that milder disease results in both lower levels of antibodies and later seroconversion, which is in line with previous reports [19, 20] . also, comorbidities may play a role in the production of specific serum antibodies following infection, which warrants further study. approximately 10% of the nonhospitalized cases in our selection did not show any seroconversion at all, indicating that such mild infections may not be detected by serological assays. however, assay performance could be improved by adding other sars-cov-2 proteins or subunits of these to further improve the sensitivity of the assay to detect low seroconversion in some cases. essential performance characteristics of assays aiming to identify seroprevalence in the population are the specificity and sensitivity. the specificity and sensitivity determine the positive and negative predictive value (ppv and npv) of the assay given the prevalence of seropositivity in the population [21] . in current low-prevalence settings insufficient specificity will generate a low ppv, resulting in a significant overestimation of the proportion of seropositive individuals (illustrated in supplementary table 1 ). however, the accuracy of the reported sensitivity and specificity of an assay also highly depends on the patient selection used for this evaluation, for example, using sera of severe covid-19 patients will result in beneficial statistics of an assay because of the acknowledged higher antibody concentration and seroconversion rate [22] . these statistics will not apply in a population serosurvey where the majority of persons will not develop severe covid-19. for this reason, we included a heterogeneous group of covid-19 patients' samples, consequently reducing sensitivity. scoring samples positive if at least 2 of the analytes generated positive results improved the specificity of the assay to 100% at a sensitivity > 90%. at a true seroprevalence of 5%, this would provide a seroprevalence estimate of 4.5% and therefore would be much more accurate than using a single analyte. we recommend transparent reporting of underlying assay performance using heterogeneous panels of controls and covid-19 patients. furthermore, implementation of international reference materials as being distributed by, for example, the national institute for biological standards and control, to facilitate comparison of seroepidemiological data between studies and countries is greatly recommended [1, 23] . from an immunological point of view, it needs to be established which sars-cov-2-specific antibodies correlate with protection. antibodies to rbd of s1 have been shown to associate with neutralization of the virus in vitro, and preliminary data indicate that the antibodies reported in our assay correlate quantitatively with virus neutralization in vitro as well [6] . the data presented here show detection of total igg. another study has shown that igg subclasses are not equally induced by sars-cov-2 infection, with a bias towards the production of igg3, at least in the first weeks after infection [24] . infection with sars-cov-2 also induces the production of iga and igm, which can contribute to protection and in vitro neutralization of the virus, but these isotypes are currently not captured by our assay [7, 8, 25] . follow-up studies are needed to establish the longevity of the production of antibodies, the degree of protection antibodies confer through various fc receptor-mediated and other mechanisms, and how b-cell memory is induced. such studies should also consider different viral loads detected in a patient and degree of severity of covid-19. in conclusion, we developed a robust multiplex assay to detect antibodies to sars-cov-2 in small blood volumes. our study is unique in validating the assay against hcov and non-hcov ili panels. because of the differences in seroconversion rates and quantitative antibody concentrations among nonhospitalized covid-19 cases, which represents the majority of patients in the general population, further investigation is required to improve assay performance for serosurveys in general. we show the advantages of multiplexed analysis in determining seroconversion and provide a framework for reliable seroprevalence estimates in different settings. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. immune surveillance for vaccine-preventable diseases developing antibody tests for sars-cov-2 towards the next phase: evaluation of serological assays for diagnostics and exposure assessment ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor • jid 2020:xx (xx xxxx) • den hartog et al neutralizing antibodies against sars-cov-2 and other human coronaviruses temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study profiling early humoral response to diagnose novel coronavirus disease (covid-19) evaluation of nine commercial sars-cov-2 immunoassays. medrxiv test performance evaluation of sars-cov-2 serological assays. medrxiv the role of antibody testing for sars-cov-2: is there one? world health organization a serological assay to detect sars-cov-2 seroconversion in humans third national biobank for population-based seroprevalence studies in the netherlands, including the caribbean netherlands immunogenicity of 13-valent pneumococcal conjugate vaccine administered according to 4 different primary immunization schedules in infants: a randomized clinical trial the development of a bead-based multiplex immunoassay for the detection of igg antibodies to cmv and ebv development and standardization of a high-throughput multiplex immunoassay for the simultaneous quantification of specific antibodies to five respiratory syncytial virus proteins influenza-like illness incidence is not reduced by influenza vaccination in a cohort of older adults, despite effectively reducing laboratory-confirmed influenza virus infections severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients antibody responses to sars-cov-2 in patients with covid-19 diagnostic tests: how to estimate the positive predictive value antibody responses to sars-cov-2 in patients of novel coronavirus disease national institute for biological standards and control. coronavirus (covid-19)-related research reagents available from the nibsc detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals longitudinal change of severe acute respiratory syndrome coronavirus 2 antibodies in patients with coronavirus disease 2019 acknowledgments. the authors acknowledge jorgen de jonge and puck van kasteren for critically reviewing our manuscript and gert-jan godeke for providing technical assistance.financial support. this work was supported by the national institute for public health and the environment, the netherlands.potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-315457-w1nx9g91 authors: siedner, mark j; gandhi, rajesh t; kim, arthur y title: desperate times call for temperate measures: practicing infectious diseases during a novel pandemic date: 2020-04-21 journal: j infect dis doi: 10.1093/infdis/jiaa209 sha: doc_id: 315457 cord_uid: w1nx9g91 nan m a n u s c r i p t the covid-19 pandemic will likely be the defining public health event of this generation. like no other event in recent memory, it has demonstrated the resolve and commitment of so many in healthcare. colleagues in nursing, emergency medicine and critical care (among many others) deployed to the frontlines, and those in infection control quickly intervened to protect our staff and patients. we, as infectious disease clinicians, have been asked to play our part, as the trusted purveyors of knowledge on the evaluation and management of communicable diseases; from loved ones inquiring about antiviral prophylaxis to calls from colleagues for salvage therapies for worsening pneumonia. for a field of medicine that takes pride on its reliance on evidence and experience, these have been desperate times. in normal times, we make every effort to respond to such requests with evidence-based decision making. in the case of covid-19, our struggles to reply have not been for a lack of information. between january 1 and april 12, 2020, there have been 3,300 manuscripts indexed in pubmed including the terms "covid-19" or "sars cov-2", and another 1,552 in medrxiv or biorxiv (with some duplications between them). however, to date, there has been scant evidence to guide evidence-based clinical decision making. of the thousands of peer-reviewed articles indexed in pubmed, exactly one has reported the results of a randomized controlled trial; a single-centered study with approximately 200 covid-19 infected individuals, investigating a drug developed for another virus, and resulting in a null finding. 1 as of this writing, there are 52 registered trials for covid-19 in the united states, but many of these have not yet launched. this abundance of creativity based on strong foundational science has translated so far to only a handful of clinical trials that are currently enrolling, mostly at tertiary care hospitals. we need to do more . . . and faster. but this phenomenon is not new to infectious disease clinicians. we are often asked to solve diagnostic and therapeutic dilemmas without the benefit of compelling clinical trial data. we take pride in our clinical acumen (and epic documentation); treating non-tuberculous mycobacterial infections and fevers of unknown origin with dizzying cocktails of antebellum drugs or nothing more than the tincture of time. experience and wisdom matter. but here again, we are knocked off-kilter, by a disease that has been in existence for less than six months, and with its closest equivalents being a blip in the annals of epidemic time in 2003, that only a handful of clinicians saw in person, and a contagion that only some of our grandparents were old enough to experience. so we find ourselves without either of our two most reliable supports. to keep ourselves propped up, we have been desperate for information. we have scoured the literature, and the online forums, and the town halls from our own institutions and others. we have all been drinking from the covid-19 firehose. we are soaked, but still thirsty for reliable knowledge. to fill this void we have sought information in places that, as medical academics, we have not been wont to go previously. and although we agree with ethical obligations that compel medical researchers to make their data publicly available so it can inform the epidemic response, we also remain acutely aware that there has been a corresponding pandemic of covid-19 misinformation. indeed, this epidemic has brought an unprecedented supply of rapidly disseminated data that are frequently not peer-reviewed, of variable quality, and, in some m a n u s c r i p t cases, retracted altogether. many of the therapeutic agents proposed in this barrage are toxic, and others are being repurposed for covid-19 on grand scales, creating shortages for patient populations who depend on them for chronic disease control. despite all the unknowns, one thing is clear at this time: none are proven to be beneficial for covid-19. this is where our test has come and our judgment will follow. do we reach for this arsenal of unproven medications before we know how to aim? can we resist the temptation to put our desire to offer guidance to desperate colleagues over our perspicacity about the lack of data in support of these medicines? we confess to failing this challenge more than once over the past few weeks. but if we can do so, we do have a third strategy in our pockets. one well known to veteran clinicians who have been wrong before and know they will be wrong again: temperance. it is something we do as well as anyone: withdrawing antibiotics, waiting another day or week or month to decide how long their duration needs to be, or perhaps, never starting them in the first place. in the coming months, there will be results from well-designed and peer-reviewed trials that we hope will reveal therapeutic options for the treatment and prevention of covid-19. even more likely, there will be at least as many that do not work. in the meantime, we will be asked countless times to help decide which ones are which, often by trusted colleagues in search of a miracle for patients in extremis. in the face of this uncertainty, we can hope that preliminary data from a preprint can provide that miracle, or we can return to first principles and ensure we are reflecting on what the data tell us when asked, simply by responding with the painfully honest truth, "i do not know. ask me again tomorrow." rajesh t. gandhi has served as an advisory board member for gilead sciences and merck & co. pharmaceuticals. arthur y. kim has served as an advisory board member for biomarin pharmaceuticals. a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 mark j. siedner receives research support from the national institutes of health (nih r01 ai124718). key: cord-341548-gazsszs6 authors: buscho, r. o.; saxtan, d.; shultz, p. s.; finch, e.; mufson, m. a. title: infections with viruses and mycoplasma pneumoniae during exacerbations of chronic bronchitis date: 1978-04-17 journal: j infect dis doi: 10.1093/infdis/137.4.377 sha: doc_id: 341548 cord_uid: gazsszs6 the association of viral and mycoplasma pneumoniae infections with acute exacerbations of chronic bronchitis was studied by serologic or isolation techniques in 46 adult men during the five years from 1964 through 1968. serologic evidence of viral or m. pneumoniae infection was detected in 25% of 166 episodes of exacerbation and 14% of 138 remission periods (p = 0.02). influenza a virus, parainfluenza virus type 3, and coronavirus oc43 predominated; infections with other viruses were infrequent. infection with m. pneumoniae was detected serologically in four patients, but this organism was never isolated from sputum specimens. rhinoviruses were isolated from frozen-stored sputum specimens in 2.7% of the episodes of exacerbation and from 0.55% of the remission intervals (p not significant). these data suggest that although exacerbations of chronic bronchitis may be accompanied by viral and m. pneumoniae infections, patients with chronic bronchitis also acquire such infections without a worsening of their respiratory status. most of the microorganisms that infect the respiratory tract can be recovered from individuals undergoing exacerbations of chronic bronchitis [1, 2] . however, the role of viral and mycoplasmal infections in exacerbations of chronic bronchitis has not been fully documented. moreover, the observations regarding infections in chronic bronchitics in a designated patient population during any specified period may not be comparable to those in other populations. because of the high prevalence and morbidity of chronic bronchitis among patients of veterans administration hospitals, we have conducted surveillance of one of these groups to assess the impact of respiratory tract infections on the natural course of this disease and to investigate further the occurrence and relative importance of viruses and mycoplasmas in exacerbations [3] . evidence of viral and mycoplasma pneumoniae infections (obtained mainly by serologic testing and to a lesser extent by isolation of organisms) was correlated with the pattern of clinical disease in patients with chronic bronchitis. design of study. forty-six male adults with chronic bronchitis, outpatients at the veterans administration hospital, hines, ill., constituted the study group. these patients all fulfilled the criteria for the diagnosis of chronic bronchitis, defined as a chronic or recurrent increase in the volume of mucoid or mucopurulent bronchial secretions sufficient to cause expectoration, during at least three months of each of the two years prior to entry into the study group. their clinical and microbiological status was studied longitudinally during the five years from 1964 through 1968. monthly or in alternate months and during periods of exacerbation, each patient reported to the outpatient clinic for examination. at each visit a symptom questionnaire was completed by the examiner, a physical examination was performed, pulmonary function tests were done, a sputum specimen was collected for isolation of virus and mycoplasma, and a serum specimen was obtained for serologic evaluation. the questionnaires and definitions of remissions and exacerbations were modeled after the guidelines reported by the medical research council [4] . exacerbations were defined as an increase in cough or sputum production since the patient's previous visit. symptoms of upper airway infection were not included among these criteria. an exacerbation was considered satisfactorily tested for viral and m. pneumoniae infections when serum pairs were obtained before and after this episode and a sputum specimen was collect-edior mycoplasmal and viral isolation. one hundred sixty-six (62%) of 270 exacerbations were tested in this manner. for comparison, 138 intervals when patients did not experience an exacerbation or were otherwise in remission were similarly tested. population characteristics. all patients were veterans who attended the pulmonary outpatient clinic on a regular basis. they visited the clinic for monthly follow-up an average of 10.3 months per year and made an average of 11.0 total visits per year. persons who were habitual poor attenders were not included in the study population. the ages of the 46 patients at the beginning of the study period ranged from 31 to 79 years; the distribution of age was as follows: 30-39 years, two patients; 40-49 years, 13; 50-59 years, nine; 60-69 years, 13; and 70-79 years, nine. the median age decade was 50-59 years. all patients were or had been cigarette smokers. the range of pack-years (one pack per man per year) was five to 125 (median, 49 years). pulmonary function tests on entry into the study disclosed a marked degree of obstructive pulmonary disease. the mean values of forced vital capacity (fvc) , forced expiratory volume (fev 1)' and the ratio fev 1/fvc were 2.45 liters, 1.31 liters, and 52%. expressed as a percentage of normal, the fev 1 for study patients ranged from 10% to 80%, but two-thirds of the group had values of 40%. similarly, the fev 1/fvc ratio ranged .from 30% to 80% of normal, and two-thirds of the group had values of~50%. procedures for isolation of virus. sputum specimens for viral isolation were stored at -70 c for five to eight years. when ready for use, speci-mens were rapidly thawed, and 0.4 ml was suspended in 2.0 ml of chilled veal infusion broth treated with 500 units of penicillin rrnl, soo p,g of streptomycin rml, and 0.02 mg of amphotericin b /ml of broth for 1 hr at 4 c. a volume of 0.2 ml of the treated sputum suspension was inoculated into each of two roller-tube cultures of human diploid fibroblasts (vvi-38 strain) for recovery of picornaviruses, adenoviruses, herpesviruses, and coronavirus strain 229e. because of the long period of storage of the sputum specimens, no attempt was made to isolate myxoviruses. wi-38 roller-tube cultures were obtained from flow laboratories, rockville, md. maintenance medium for these cell cultures consisted of eagle's minimal essential medium supplemented with 2% inactivated fetal calf serum; the complete medium was adjusted to ph 7.0 with 7.sc;o nahc0 3 [s] . cultures were incubated on a rotating drum at 33 c for at least 21 days and often for as long as 28 days. subpassages of negative cultures were not made. cell cultures were examined three times a week for cpe, and the maintenance medium was changed at these times. viral isolates \.vere identified by procedures previously described [5] . mycoplasma. a fresh agar medium consisting of pleuropneumonia-like organism agar (difco, detroit, mich.), 20% horse serum, 10% yeast extract, penicillin g (2,000 unitsj ml). amphotericin b (5.6 jlg/ml), and thallium acetate (0.05%) was used for the isolation of all mycoplasmas. duplicate plates were inoculated with sputum. one plate was incubated aerobically and the other anerobically at 37 c for four weeks. mycoplasma isolates were identified by growth inhibition procedures using specific hyperirnmune antiserum [6] . included in the test were antisera to m. pneumoniae, mycoplasma orale) mycoplasma hominis, mycoplasma [errnentans, mycoplasma arthriditis, and mycoplasma salioarium. approximately one-half of the cultures were passaged on agar a second time from an area selected adjacent to the zone of inhibition in an attempt to identify mixed cultures of mycoplasmas. none were detected. serologic procedures. serial serum specimens were tested by cf for antibodies to influenza a and b viruses, respiratory syncytial virus (rsv), parainfluenza virus types 1, 2, and 3, adenoviruses, and coronavirus types 229e and oc43 and for m. pneumoniae. microtiter cf tests containing 1.7-1.8 units of complement were employed after fixation overnight at 4 c. four units of each viral and mycoplasmal antigen were used. serologic evidence of infection was defined as a fourfold or greater rise in antibody titer between the acuteand convalescent-phase sera. occurrence of acute respiratory illness in patients with chronic bronchitis. the total number of exacerbations experienced by each patient during their participation in this study ranged from one to 14 (mean, 5.9). the number of exacerbations of anyone patient usually was directly proportional to the number of years of participation in the surveillance program. the mean number of years of patient participation was 3.7. only one patient reported no exacerbations during the four-year period of the study. exacerbations of chronic bronchi tis tended to occur more often in the winter months than in the summer months, although they occurred frequently in all months of the year (table 1) . serologic evidence -of viral and m. pneumoniae infections. infections with respiratory virus or m. pneumoniae were detected at least once in 40 of the 46 patients studied. the mean num-379 ber of infections was 1.9 (range, 1-6). although patients included in the study for a longer time experienced somewhat more virus and m. pneumoniae infections, the relationship was not linear, a finding suggesting that individual differences in rates of infection could not be attributed exclusively to the duration of participation in the study. fourfold or greater rises in titer of antibody to virus or m. pneumoniae were detected in 50 (30.1%) of 166 exacerbations of chronic bronchitis in the 46 adult patients studied (table 2) . by comparison, viral and m. pneumoniae infections were detected in 38 (27.5%) of 138 remissions. these rates were not significantly different and suggest that patients with chronic bronchitis can undergo infections with these agents apparently without an acute worsening of their respiratory status. when rises in titers of antibody to multiple agents which occurred simultaneously during a single episode were excluded from this analysis, the distribution of single-agent infections could be assessed (table 3) . infections defined as a rise in titer of antibody to a single agent (a virus or m. pneumoniae) were detected in 41 (24.7%) of 166 exacerbations of chronic bronchitis and only 19 (13.8%) of 138 remissions, a difference which was significant (p = 0.02). the frequency of individual viral and m. pneumoniae infections was analyzed using only these single-agent antibody titer rises (table 4) . fortyone (82%) of 50 rises in antibody titer during exacerbation were single-agent antibody titer rises, and during remission 19 (50%) of the 38 rises were single-agent antibody titer rises. the higher incidence of multiple-agent antibody titer rises in remission intervals may be a reflection of the study design. more attention was paid to specimen collection during exacerbations with the result that the average intervals defined by available serum pairs were 1.5 months for exacerbation and 3.2 months for remission. myxovirus and coronavirus infections predominated (table 4) . influenza a virus, parainfluenza virus type 3, and coronavirus oc43 accounted for nearly two-thirds of all infections. in each instance these infections occurred more often in exacerbation than in remission, a finding that suggests their association with the occurrence of exacerbations of chronic bronchitis; however, the difference was significant only for influenza a virus and coronavirus oc43 (p < 0.05). all other viral infections occurred as often in exacerbation as in remission. m. pneumoniae infection alone occurred in 2.4~o of exacerbations but not in any remission. fifteen additional rises in titer of antibody to m. pneumoniae occurred simultaneously with respiratory virus infections. these rises were equally distributed between periods of exacerbation and remission. viral infections among patients with chronic bronchitis occurred as defined outbreaks ( figure 1) in this study, viral and m. pneumoniae infections were common in patients during exacerbation, but such infections occurred also in these patients without worsening of their respiratory status. we reported our findings in two ways: as rises in titer of antibody to a single agent and as rises in titer of antibody to two or more agents within the same interval. rises in titers of antibody to multiple agents predominated in remission intervals, which tended to be longer than exacerbation intervals. this difference may have accounted for some variation in the antibody titer which was not indicative of specific viral infection. antibody titer rises to multiple agents also may reflect heterotypic responses and overestimate the number of infections. by analyzing antibody titer rises to single agents in this report, we estimated the importance of infection with each specific virus, but the approach precluded assessment of the role of dual infection with two viruses or a virus and m. pneumoniae in exacerbations. rises in titer of antibody to m. pneumoniae, however, were detected most often in association with a titer rise to one or more respiratory viruses. the possibility that m. pneumoniae can function as a secondary or synergistic pathogen in respiratory tract infections cannot be excluded and requires further investigation. that viruses may playa role in exacerbations of chronic bronchitis is suggested by the finding that one-fourth of exacerbations were associated with viral infections. this rate was twice that of viral infection in remission. infections with influenza a virus and coronavirus oc43 occurred significantly more often in exacerbations than in remissions. coronaviruses have been recognized recently as etiologic agents of respiratory illness in children and adults [8] [9] [10] . gump and coworkers also reported association of these agents with exacerbations of chronic bronchitis [11] . they found that four of seven infections with coronavirus oc43 and two of seven infections with coronavirus 229e were associated with an exacerbation. evidence of this relationship has accumulated very slowly because of the requirement for recovery of strain oc43 in human fetal trachea organ cul-tures, a difficult procedure. infection with coronavirus 229e was not detected either by isola tion or rise in antibody titer in the patients in our study. the finding in this study of rhinovirus infections in only 2.7% of exacerbations is considerably lower than the 23% recovery rate for rhinoviruses in exacerbations reported by eadie [14, 15] . carilli et al., however, failed to isolate these agents [16] . in his controlled study, stenhouse found that rhinovirus infection was not more common in' subjects with bronchitis than in the control population, but that rhinovirus infection was more likely to be associated with the development of acute lower respiratory tract symptoms [14j. the extended period of frozen storage of specimens before tissue culture inoculation may have contributed to our low recovery rate of rhinoviruses. sommerville first detected rsv infection in 50% of 82 exacerbations [17j. carilli et al. [16j and mcnamara et al. [13j also found rsv associated with a significant number of exacerbations, 17% and 12%, respectively. unlike these investigators, we detected rsv infection by serologic procedures in only one remission interval of only one of our patients. the paucity of m. pneumoniae infections detected in exacerbations in this study agrees with the results of another recent report [18] . in a number of prospective studies, m. pneumoniae infections have been associated with 8.7% and 9.5% of exacerbations [12, 13j and with no exacerbations [19] . one consistent finding, however, has been the failure to recover the organism from sputum, even from patients with rising antibody titers. these results question the significance of serologic responses alone only in this infection and should be further investigated. m. saliuarium seems ubiquitous in samples of expectorated sputum as well as in specimens obtained during bronchoscopy [2oj . our findings are similar to those reported in a number of other studies on viral and m. pneumoniae infections in exacerbations of chronic bronchitis (table 5) . in various studies the rates of infection in exacerbations ranged widely (4%-64%). these data have been interpreted as constituting strong, but not conclusive, evidence for an etiologic role of viruses or m. pneumoniae in the pathogenesis of acute exacerbations of chronic bronchitis. however, the available data cannot be easily compared because different sam-piing procedures and methods were employed. in addition, individual authors often did not specify the number of remission intervals examined but reported only infections detected during exacerbations and compared patients with chronic bronchitis with healthy (control) populations. furthermore, the definition of an exacerbation is a subjective judgment, and different interpretations of the criteria will affect the percentage of association with infection. two groups of investigators have derived a more striking correlation of infection with exacerbation by interpreting their findings in a timeweighted fashion. thus gump et al. found that the incidence of infection was 32% per patient week of exacerbation but only 0.9% per patient week spent in remission [11j. similarly, lamy et al. compared patient months spent in exacerbation and remission with an incidence of infection of 52% and 3.7%, respectively [24] . our data did not permit a time-weighted analysis since the duration of exacerbation was not recorded. when the duration of exacerbation was estimated using the interval between the collection of two samples of serum bracketing an exacerbation, we tested 257 months of exacerbation and 442 months of remission. the rate of viral infection was 15.4% per exacerbation month and 4.3% per remission month. this trend is similar to that in the data of gump et al. [11] . a special problem in interpretation is posed by the detection of infection in patients with chronic bronchitis without an associated acute compromise in their respiratory status. in our study approximately one-third of all viral and m. pneumoniae infections detected belonged in this category. whether subclinical infections contribute to continuing clinical deterioration remains unexplored, and long-term epidemiologic and clinical surveillance of patients with chronic bronchitis will be required to answer this question. role of infection in the cause and course of chronic bronchitis and emphysema role of infection in chronic bronchitis virus and mycoplasma infections in exacerbations of chronic bronchitis definition and classification of chronic bronchitis for clinical and epidemiological purposes. a report to the medical research council the role of viruses, mycoplasmas and bacteria in acute pneumonia in civilian adults mycoplasma species identification based upon growth 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for mycoplasma infections in patients with chronic bronchitis infection with influenza and parainfluenza viruses in chronic bronchitis persistence of viral antibodies in patients with chronic bronchitis pilot study of factors associated with exacerbations in chronic bronchitis debacker-willame, e. respiratory viral infections in hospital patients with chronic bronchitis: observations during periods of exacerbation and quiescence key: cord-315448-bosazmlm authors: crawford, katharine h d; dingens, adam s; eguia, rachel; wolf, caitlin r; wilcox, naomi; logue, jennifer k; shuey, kiel; casto, amanda m; fiala, brooke; wrenn, samuel; pettie, deleah; king, neil p; greninger, alexander l; chu, helen y; bloom, jesse d title: dynamics of neutralizing antibody titers in the months after sars-cov-2 infection date: 2020-09-30 journal: j infect dis doi: 10.1093/infdis/jiaa618 sha: doc_id: 315448 cord_uid: bosazmlm most individuals infected with sars-cov-2 develop neutralizing antibodies that target the viral spike protein. here we quantify how levels of these antibodies change in the months following sars-cov-2 infection by examining longitudinal samples collected between ~30 and 152 days post symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. neutralizing antibody titers declined an average of about four-fold from one to four months post symptom onset. this decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike or its receptor-binding domain. importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. additional studies of long-lived b-cells and antibody titers over longer time frames are necessary to determine the durability of immunity to sars-cov-2. onset, sera from most infected individuals can bind to the viral spike and neutralize infection in vitro [5, 7, 9] . the reciprocal dilution of sera required to inhibit viral infection by 50% (nt 50 ) is typically in the range of 100 to 200 at 3-4 weeks post symptom onset [10] , although neutralizing titers range from undetectable to >10,000 [2, 5, 9] . there are currently limited data on the dynamics of neutralizing antibodies in the months following recovery from sars-cov-2. for most acute viral infections, neutralizing antibodies rapidly rise after infection due to a burst of short-lived antibody-secreting cells, and then decline from this peak before reaching a stable plateau that can be maintained for years to decades by long-lived plasma and memory b cells [11, 12] . these dynamics have been observed for many viruses, including influenza [13] , rsv [14] , mers-cov [15] , sars-cov-1 [16, 17] , and the seasonal human coronavirus 229e [18] . several recent studies have tracked antibody levels in individuals who have recovered from infection with sars-cov-2 for the first few months post symptom onset [5, 7, 8, [19] [20] [21] [22] . most of these studies have reported that over the first three months, antibodies targeting spike decline several fold from a peak reached a few weeks post symptom onset [5, 7, 19] , suggesting that the early dynamics of the antibody response to sars-cov-2 are similar to those for other acute viral infections. a c c e p t e d m a n u s c r i p t 5 here we build on these studies by measuring both the neutralizing and binding antibody levels in serial plasma samples from 32 sars-cov-2-infected individuals across a range of disease severity with follow-up as long as 152 days post symptom onset. on average, neutralizing titers decreased ~4-fold from ~30 to >90 days post symptom onset. this decline in neutralizing titers was paralleled by a decrease in levels of antibodies that bind spike and its receptor-binding domain (rbd). nonetheless, most recovered individuals still had substantial neutralizing titers at three to four months post symptom onset. plasma samples were collected as part of a prospective longitudinal cohort study of individuals with sars-cov-2 infection. individuals 18 years or older with laboratory confirmed sars-cov-2 infection were eligible for inclusion. individuals who were hiv+ were excluded from this study due to concerns that antiretroviral treatment may affect our pseudotyped lentivirus neutralization assay. individuals were recruited from three groups: inpatients, outpatients, and asymptomatics. inpatients were hospitalized at harborview medical center, university of washington medical center, or northwest hospital in seattle, wa and were enrolled while hospitalized. outpatients were identified through a laboratory alert system, email and flyer advertising, and through identification of positive covid-19 cases reported by the seattle flu study [23] . asymptomatic individuals in this study were recruited through outpatient testing and identified when they answered "none" on their symptom questionnaire. they were confirmed to be symptom-free for the first 30 days after diagnosis. a c c e p t e d m a n u s c r i p t 6 we initially enrolled 34 individuals following rt-qpcr-confirmed sars-cov-2 infection. two individuals (participant ids (pids) 19c and 196c) were seronegative at all timepoints in the neutralization assay and all rbd and spike elisas (supplementary figure 1) . we then tested these individuals in the abbott architect anti-nucleoprotein assay where they were also seronegative, with index values of 0.01 (for both samples from pid 196c) or 0.02 (for both samples from pid 19c), which are far below the threshold for seropositivity of 1.40 [24] . because these individuals had only a single positive rt-qpcr test (and pid 196c tested negative in 6 subsequent rt-qpcr tests conducted within 15 days of their initial test), and because the abbott architect assay has been validated to have very high (95.1-100%) sensitivity by day 17 post symptom onset [24] , we assessed these two individuals were likely not truly infected but rather false positives in a single rt-qpcr viral test. therefore, they were excluded from all further analyses, resulting in a final cohort of 32 individuals. participants or their legally authorized representatives completed electronic informed consent. sociodemographic and clinical data were collected from electronic chart review and from participants via a data collection questionnaire (project redcap [25] ) at the time of enrollment. the questionnaire collected data on the nature and duration of symptoms, medical comorbidities, and care-seeking behavior (supplementary table 1 ). based on these data, individuals were classified by disease severity as asymptomatic, symptomatic non-hospitalized, and symptomatic hospitalized. individuals who were recruited as inpatients were enrolled during their hospital admission and had samples collected during their hospitalization. after hospital discharge, these participants subsequently returned to an outpatient clinical research site approximately 30 days after symptom a c c e p t e d m a n u s c r i p t 7 onset for follow-up. in person follow-up only occurred if participants were asymptomatic as per cdc guidelines. outpatients and asymptomatic individuals completed their enrollment, data collection questionnaire, and first blood draw at an outpatient visit approximately 30 days after symptom onset (or positive test for asymptomatic individuals). all participants subsequently were asked to return at day 60 and then at day 90 or 120 for follow-up. the majority of samples collected from participants were from outpatient visits after recovery. however, the first sample from pid 13, the first three samples from pid 23, and the first six samples from pid 25 were collected during their hospitalizations. table 1 . for analyses of fold-change, we required individuals to have a sample collected at the 30-day timepoint. the numbers of individuals included in the foldchange analyses are indicated in table 1. a c c e p t e d m a n u s c r i p t 8 this study was approved by the university of washington human subjects institutional review board. whole blood was collected in acid citrate dextrose tubes then spun down, aliquoted, and frozen at -20ºc within 6 hours of collection. prior to use in this study, plasma samples were heat inactivated at 56ºc for 60 min and stored at 4ºc. some samples from the early timepoints were stored at -80ºc after heat inactivation and underwent no more than two freeze/thaw cycles. plasma samples were spun at 2000xg for 15 min at 4ºc immediately prior to use to pellet platelets. protein expression and purification sars-cov-2 rbd and spike (s-2p trimer [26] ) proteins were produced in mammalian cells as previously described [26] [27] [28] . proteins were purified from clarified supernatants as described in [28] . sds-page was used to assess purity prior to flash freezing and storage at -80°c. testing of serum samples with the abbott architect sars-cov-2 igg assay was performed according to the manufacturer's instructions for use and as described in [24] . index values associated with the igg enzyme-linked immunosorbent assays (elisas) to spike and rbd were conducted as described previously [27] , and were based on a published protocol that recently received emergency use authorization from new york state and the fda [29, 30] . plasma samples were diluted with five serial auc was calculated as the area under the titration curve after putting the serial dilutions on a logscale. neutralization assays were conducted using pseudotyped lentiviral particles as described in [31] , with a few modifications. first, we used a spike with a cytoplasmic tail truncation that removes the a c c e p t e d m a n u s c r i p t 10 last 21 amino acids (spike-∆21). the map for this plasmid, hdm-sars2-spike-delta21, is in supplementary file 1 and the plasmid is available from addgene (plasmid #155130). we used a spike with a c-terminal deletion because, since publishing our original protocol [31] , other groups have reported that deleting spike's cytoplasmic tail improves titers of spike-pseudotyped viruses [32] [33] [34] [35] . indeed, we found that the c-terminal deletion increased the titers of our pseudotyped lentiviral particles without affecting neutralization sensitivity (supplementary figure 2) . at 50-52 hours post-infection, luciferase activity was measured using the bright-glo luciferase assay system (promega, e2610) as described in [31] , except luciferase activity was measured directly in the assay plates. two "no plasma" wells were included in each row of the neutralization plate and fraction infectivity was calculated by dividing the luciferase readings from the wells with plasma by the average of the "no plasma" wells in the same row. after calculating fraction infectivities, we used the neutcurve python package (https://jbloomlab.github.io/neutcurve/) to calculate the a c c e p t e d m a n u s c r i p t 11 plasma dilution that inhibited infection by 50% (ic50) by fitting a hill curve with the bottom fixed at 0 and the top fixed at 1. nt 50 s for each plasma sample were calculated as the reciprocal of the ic50. individuals whose plasma was not sufficiently neutralizing to interpolate an ic50 using the hill curve fit were assigned an nt 50 of 20 (the limit of our dilution series) for plotting in figures 1a, 1c, and 2b and for fold-change analyses in figure 1b . results from sars-cov-2 spike-pseudotyped lentivirus neutralization assays have been shown to correlate well with full virus sars-cov-2 neutralization assays [36, 37] . nonetheless, in an effort to help standardize comparisons between neutralization assays, we also ran our assay with a standard serum sample from nibsc (research reagent for anti-sars-cov-2 ab, nibsc code: 20/130). this sample had an nt 50 of ~3050 (supplementary figure 3) . raw data for each sample, including ic50, nt 50 , auc and relevant demographic data (age, sex, we used spike-pseudotyped lentiviral particles [31] to measure neutralizing antibody titers in the longitudinal plasma samples from all 32 infected individuals ( figure 1a) . all individuals had detectable neutralizing antibody titers (nt 50 >20) at their first convalescent plasma sample, which was generally collected roughly one month post symptom onset. these data are consistent with prior studies showing that most sars-cov-2 infected individuals develop neutralizing antibodies [5, 7, 9] . qualitative inspection of figure 1a shows that these titers modestly decreased for most a c c e p t e d m a n u s c r i p t 13 individuals over the next few months, although the dynamics were highly heterogeneous across individuals. to quantify the dynamics of neutralizing antibody titers over time, we calculated the fold change at ~60 and >90 days post symptom onset relative to the ~30 day timepoint, excluding any individuals who lacked a 30-day sample. taken across all individuals, neutralizing titers significantly declined from 30 to 60 days, and again from 60 to 90 days (see legend of figure 1b for details). at >90 days, the median neutralizing titer was reduced 3.8-fold relative to the 30-day value ( figure 1b) . however, most individuals (27/32) still had detectable neutralizing titers at the last timepoint. we compared the dynamics of neutralizing antibody titers between individuals with different disease severity (figure 1c) . individuals with more severe disease tended to have higher neutralizing antibody titers during early convalescence, consistent with prior studies [5, 38, 39] . specifically, at both ~30 and ~60 days post symptom onset, individuals who required hospitalization had significantly higher neutralizing antibody titers than individuals who did not ( figure 1c) . from ~30 to >90 days post symptom onset, the nt 50 for symptomatic hospitalized individuals decreased ~18-fold, which is significantly more than the ~3-fold decrease in the nt 50 for non-hospitalized individuals (p=0.03, wilcoxon rank-sum test) (supplementary figure 4) . by >90 days post symptom onset, neutralization titers were not significantly different between disease severity groups (figure 1c) . at all timepoints, asymptomatic individuals had neutralization titers similar to those of symptomatic non-hospitalized individuals. for all plasma samples, we also used elisas to measure iga, igm, and igg binding to the rbd of spike, and igg binding to the full spike ectodomain [29] . figure 2a shows each individual's iga, igm, and igg binding antibody titers as quantified by area under the curve (auc) of the elisa readings (see methods for detailed description). like neutralizing antibody titers, these antibody binding titers tended to decrease over time, although there was substantial variation among individuals. all the elisa-measured antibody-binding titers are clearly correlated with neutralizing antibody titers ( figure 2b) . individuals with severe disease had higher antibody binding titers at early timepoints. specifically, individuals who were hospitalized as part of their care had higher igg, iga, and igm binding responses than asymptomatic or symptomatic non-hospitalized individuals at ~30 days post symptom onset (figure 2c) . by ~60 days post symptom onset, anti-rbd igm levels were no longer significantly different between severity groups, and by >90 days post symptom onset, binding responses did not differ between severity groups for any antibody subtype. this trend is consistent with data in figure 1c showing that neutralizing antibody responses were higher for individuals with more severe disease early during convalescence, but reached similar levels across all diseaseseverity groups by >90 days post symptom onset. among all patients, regardless of disease severity, iga and igm levels decreased more than igg levels from ~30 to >90 days post symptom onset, consistent with other studies [7, 8, 19, 22] . a c c e p t e d m a n u s c r i p t 15 we have measured the dynamics of neutralizing antibody titers over the first three to four months following infection with sars-cov-2 in a well-characterized prospective longitudinal cohort of individuals across a range of disease severities. the titers of neutralizing antibodies declined modestly, with the titers at three to four months post symptom onset generally about four-fold lower than those at one month. this decline in neutralizing antibodies was paralleled by a decline in antibodies binding to the viral spike and its rbd. this decline is generally similar in magnitude to that reported in several other recent studies of antibody dynamics in the months immediately following sars-cov-2 infection [5, 7, 19] . individuals with more severe disease tended to have higher peak antibody responses at one to two months post symptom onset, consistent with many other studies reporting higher early titers in severely ill sars-cov-2 infected individuals [5, 6, 38, 39] . however, by three to four months post symptom onset, neutralizing antibody titers among individuals with severe disease were no longer significantly higher than those of individuals with mild symptoms or even asymptomatic infections. therefore, it seems possible that the large peak in antibody production in severely ill individuals wanes more dramatically than in milder cases, consistent with severe disease often leading to an exaggerated burst of short-lived antibody-secreting cells [40, 41] . importantly, most individuals in our study still had substantial neutralizing antibody titers at three to four months post symptom onset. while some recent studies have interpreted a modest drop in titers in the first few months after infection as alarming, it is entirely consistent with antibody responses to other respiratory viruses. acute infection is always associated with an initial peak in antibody titers due to a burst of short-lived antibody-secreting cells [42] . for many other infections, titers decline from this initial peak but then reach a stable plateau that is maintained for years or a c c e p t e d m a n u s c r i p t 16 even decades by long-lived plasma cells and memory b cells that can be recalled during subsequent infections [12] [13] [14] [15] [16] 18, 43, 44] . the modest declines in antibody titers that we observe over time do have implications for efforts to collect convalescent patient plasma for use in treatment of sick individuals [45] . fda guidelines suggest minimum cutoffs for the antibody activity in such convalescent plasma (e.g., nt 50 > 160; [39] ). our results suggest that plasma from convalescent donors collected in the first few months post symptom onset will be more likely to meet these cutoffs; others have made a similar observation [46] . additionally, our results indicate that if an individual is donating convalescent plasma over time, each plasma sample should be tested for antibody titers to ensure that they remain above the fda cutoff. the limitations of this study include the small number of samples, particularly in the asymptomatic and symptomatic hospitalized groups, and recruitment of participants from a single study site, which potentially limits the generalizability of these results. furthermore, since symptom-onset date relies on individual recollections, it is difficult to precisely match the timing of blood draws across all participants. additionally, we only had follow-up to about four months post symptom onset and only measured plasma antibody responses. further studies over longer time frames and with direct interrogation of plasma and memory b cells will be necessary to determine longer term durability of immunity to sars-cov-2, as well as its relationship to protection against re-infection [47] . despite these limitations, our study shows that titers of neutralizing and binding antibodies targeting sars-cov-2 spike remain detectable in most individuals out to >90 days 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covid-19 through a citywide pandemic surveillance platform performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support structure, function, and antigenicity of the sars-cov-2 spike glycoprotein serological identification of sars-cov-2 infections among children visiting a hospital during the initial seattle outbreak elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for sars-cov-2. biorxiv a serological assay to detect sars-cov-2 seroconversion in humans sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup protocol and reagents for pseudotyping lentiviral particles with sars-cov-2 spike protein for neutralization assays a replication-competent vesicular stomatitis virus for studies of sars-cov-2 spike-mediated cell entry and its inhibition neutralizing antibody and soluble ace2 inhibition of a replication-competent vsv-sars-cov-2 and a clinical isolate of sars-cov-2. cell host microbe rapid isolation of potent sars-cov-2 neutralizing antibodies and protection in a small animal model characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov human neutralizing antibodies elicited by sars-cov-2 infection a novel receptor-binding domain (rbd)-based mrna vaccine against sars-cov-2 sars-cov-2 neutralizing antibody responses are more robust in patients with severe disease relationship between anti-spike protein antibody titers and sars-cov-2 in vitro virus neutralization in convalescent plasma immunologic perturbations in severe covid-19/sars-cov-2 infection. biorxiv [internet iga dominates the early neutralizing antibody response to sars-cov-2. medrxiv somatically hypermutated plasmodium-specific igm+ memory b cells are rapid, plastic, early responders upon malaria rechallenge original antigenic sin priming of influenza virus hemagglutinin stalk antibodies others. investigational covid-19 convalescent plasma-emergency inds. accessed on april longitudinal analysis of the humoral response to sars-cov-2 spike rbd in convalescent plasma donors neutralizing antibodies correlate with protection from sars-cov-2 in humans during a fishery vessel outbreak with high attack rate we thank marion pepper for helpful input, drs. david koelle and anna wald for sharing reagents, and andrea loes and meei-li huang for experimental assistance. we thank ariana magedson, dylanmcdonald and nicholas franko for assistance with enrollment. we additionally thank all our research participants in the haarvi study for their generosity in participation. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-321580-3ru92tra authors: hadler, james l title: will sars-cov-2 prevention efforts affect the coming influenza season in the united states and northern hemisphere? date: 2020-09-07 journal: j infect dis doi: 10.1093/infdis/jiaa571 sha: doc_id: 321580 cord_uid: 3ru92tra nan a c c e p t e d m a n u s c r i p t studies have examined the effects of community-wide non-pharmaceutical interventions (npis) on the influenza pandemic of 1918-20, and speculated on the effect of deploying them in a modern pandemic [1] . the initial country-level responses to sars-cov-2 have provided substantial evidence of their collective impact on the covid-19 pandemic and, incidentally, on seasonal influenza epidemics. in this issue of the journal of infectious diseases, lei et al present an analysis of the impact of china's -lockdown‖ response to covid-19 on influenza as measured by china's outpatient hospital-based sentinel surveillance system during the anticipated peak of the influenza season in late january 2020 [2] . the chinese national influenza surveillance system includes collection of number of visits, number that were for influenza-like illness (ili), number of influenza laboratory tests and test results from 554 hospitals scattered around china. the system is large, including up to 4 million visits and 10-25,000 laboratory tests for influenza each week during the peak winter influenza season. outcomes presented in graphic form by week from october 2019mid-may 2020 include total number of visits, percentage ili, total number of tests for influenza, percentage positive, and estimated influenza rate among those with hospital visits (number of ili visits times percentage of flu tests positive for influenza). there were several very importantfindings. prior to implementation of npi nationally, the 2019-20 influenza season was not substantially different from the two preceding seasons except that the number of samples tested for influenza was substantially higher in the month before npi implementation (late decemberlate january), the positivity rate was lower, and the estimated influenza rate was lower one week before. following npi implementation compared to previous seasons, the number of visits decreased dramatically over the next two weeks, the percentage for ili increased, the percentage positive for influenza decreased and the influenza incidence rate began plummeting, reaching nearly undetectable levels within 5 weeks and remaining so through the mid-may mark when the a c c e p t e d m a n u s c r i p t data presented in the analysis ended. the authors concluded that the marked decline in influenza they documented was associated in time with the implementation of npi, suggesting that the npis implemented had collateral benefit in preventing influenza. the findings from china are particularly important. as a country, it has perhaps the largest influenza sentinel surveillance system in the world, including active testing for influenza, and it was the first country to implement highly successful npis against sars-cov-2, giving it the best opportunity to see a possible change in influenza before the expected seasonal decline. second, the outcomes of percentage influenza tests positive and calculated influenza rate per hospital visit are optimal outpatient outcomes to use, given changing dynamics in hospital visits brought about by npis and a sharp decline in all outpatient hospital visits. however, china's response to covid-19 was the most comprehensive and effective of any large country. it consisted of stopping all travel within the country, closing all schools, entertainment venues and non-essential work, eliminating public gatherings, isolating outside the home all documented cases and persons needing quarantine in special facilities, conducting massive fever screening, using artificial intelligence to detect movement and contacts, and mandating and enforcing universal face-mask use in public places, among other things. the bottom line is that if you can virtually eliminate covid-19, you can eliminate influenza transmission using the same strategy. in other northern hemisphere countries with less stringent, but still largely effective, measures to control covid-19, influenza activity also appeared to decline rapidly, shortening the expected spring 2020 influenza season by as much as six weeks [3, 4] . to know what might happen with influenza this coming fall and winter, we need to know what has happened prospectively in southern hemisphere countries, especially those that like the us have used fewer npi's and used them with less rigor and, correspondingly, been less successful in controlling sars-cov-2 transmission. the one comprehensive source of data while we focus on every infection with sars-cov-2, we have not historically nor are we likely to pay as much attention to influenza infection. testing for influenza will not be as rigorous, contact investigations followed by quarantine recommendations for contacts of m a n u s c r i p t those testing positive are unlikely to be made, and those with mild symptoms who test negative for sars-cov-2 will be less likely to isolate. countries that have opened the most after their coronavirus -lockdowns‖ including, ironically, those such as china that have had the most success at controlling sars-cov-2, are the ones with the highest potential to have a distinct seasonal epidemic and the largest related healthcare burden. despite the potential for a greatly diminished influenza season (sars-cov-2 is not going away, nor will the need diminish for social distancing efforts), vaccination against influenza remains important given the possibility of an influenza epidemic superimposed on the covid-19 pandemic. it will be especially important for those who are most vulnerable to complications from influenza (young children, older adults, the elderly, those with high risk underlying medical conditions), and those in congregate settings that facilitate amplification of spread (e.g., schools, hospitals, correctional facilities, long term care facilities, crowded workplaces, homeless shelters) to get vaccinated, both for their personal benefit and to further reduce the potential for epidemic spread [8] . in this context and for this school year only, massachusetts is requiring vaccination against influenza by december 31 of children attending massachusetts child care, pre-school, kindergarten, k-12, and colleges and universities [9] . in the disease control context, fall 2020 and winter 2021 will be a challenging but fascinating time. a c c e p t e d m a n u s c r i p t nonpharmaceutical interventions implemented by us cities during the 1918-1919 influenza pandemic nonpharmaceutical interventions used to control covid-19 reduced seasonal influenza transmission in china how coronavirus lockdowns stopped flu in its tracks australian government department of health prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on flu vaccine now required for all massachusetts school students enrolled in child care, pre-school, k-12, and post-secondary institutions a c c e p t e d m a n u s c r i p t immunization practices -united states, 2020-21 influenza season. mmwr key: cord-332537-rtdu4jae authors: tong, tommy r. title: airborne severe acute respiratory syndrome coronavirus and its implications date: 2005-05-01 journal: j infect dis doi: 10.1086/429637 sha: doc_id: 332537 cord_uid: rtdu4jae nan airborne transmission of the severe acute respiratory syndrome (sars) coronavirus (cov) has been the favored explanation for its transmission on an aircraft [1] and appeared to explain a large community outbreak of sars in the amoy gardens in hong kong [2] . the article by booth et al. in this issue of the journal of infectious diseases [3] suggests that airborne dissemination of sars-cov may also occur in the health-care setting. a patient with sars who was breathing quietly but coughing occasionally in a hospital room contaminated the surrounding air with sars-cov, as shown by experiments conducted during the sars outbreak in canada in early 2003. several viruses and other pathogens, such as mycobacterium tuberculosis, have been shown to be transmitted by airborne dissemination [4] [5] [6] [7] [8] . however, the possibility of airborne dissemination of sars-cov has been controversial. the important work by booth et al. has shown beyond doubt that sars-cov aerosol generation can occur from a patient with sars. the study was well conceived and designed and employed nucleic acid amplification and state-of-the-art air slit-sampling technology. to ensure the accuracy of their results, the authors followed stringent control measures in their studies. for example, empty specimen containers made the same trip from outside to the hospital ward and then to the laboratory, in the same way as the real specimen containers. all samples were tested similarly, and the technologists were blinded to their true nature. these procedures helped to control for possible contamination of the outside of the specimen containers, a little-thought-of possible cause of false-positive test results. another, even more stringent measure was the dedication of special rooms for these experiments. these researchers anticipated laboratory contamination as a possible cause of false-positive results long before news broke of sars-cov escaping microbiology laboratories through infection of workers [9, 10] . other measures, such as use of dummy controls (with water only), confirming the identity of sars-cov by testing more than one region of the viral genome, and sequencing the amplified products, add to the credibility of their results. the authors use their findings to make several valid recommendations regarding proper ventilation, air filtration, and aerosol prevention. because none of the sars-cov cultures were found to be positive and host infection was not involved, the authors rightly avoided drawing a conclusion of airborne transmission of sars-cov. definitive proof of transmission will need to come from experiments similar to those performed by riley et al. in the 1950s, which involved exposure of guinea pigs to air shared by patients with active pulmonary tuberculosis [11] . in vitro viral culture tests may not be sensitive enough for this purpose. however, if sars-cov is naturally airborne (produced by breathing and coughing), as was shown by booth et al., then there is sufficient concern that it can be transmitted successfully by air. a number of factors may affect the ability of a virus to establish infection after successful transmission. the lack of proofreading [12, 13] during sars-cov replication suggests that some assembled viral genomes are defective and not packaged within viral capsids to form infectious viral particles. viability may also be compromised, even for nondefective viral particles, after release into the environment. considerable airborne viral dilution may also occur, adding another challenge to a pathogen that employs air for dissemination. finally, the number of viral particles needed to cause an infection differs among viral pathogens, with influenza virus requiring as few as 3 particles to cause infection [14] . it is not clear how many sars-cov particles are required to cause infection. circumstances limited the booth et al. their detection of the virus on frequently touched surfaces, including a bed table, a television remote control, and even a medication refrigerator at a nurses' station, emphasizes the need for even stricter infection control precautions than are usually applied. as the authors point out, electronic equipment, because of its moisture sensitivity, may need particular attention. this work by booth et al. can be looked at from multiple perspectives. the first is from that of patients: the study's results justify their concern about health-care facilities as places in which infectious organisms may be encountered. however, with knowledge of transmission mechanisms should come a better understanding of how to prevent transmission. improving the indoor air quality of health-care facilities, including not just isolation wards but also common areas, will help to pre-vent the notion of them being potential "centers of contagion." the second perspective follows from the first-namely, that of the caregivers, clinical microbiologists, and health-care policy makers. acknowledgment of the fact that sars-cov can be aerosolized justifies the actions of those who have already committed resources for providing a safer environment in terms of preventing airborne transmission of infectious diseases and might provide the needed pressure for others to follow suit. public health officials will also be more likely to recommend "smart" quarantine [16] and to provide point-of-care diagnostics. avoiding crowding in the clinic is important in the prevention of nosocomial transmission of any infectious diseases, especially those spread by air. engineers and architects interested in designing safer institutional and other public environments should read the article by booth et al. with interest and be provided with additional momentum to advance novel concepts [17] [18] [19] . architectural advances in the design of safer hospital facilities, particularly isolation rooms for patients with airborne communicable diseases, are needed. hopefully, the work of booth et al. will spur these efforts. transmission of the severe acute respiratory syndrome on aircraft evidence of airborne transmission of the severe acute respiratory syndrome virus detection of airborne severe acute respiratory system (sars) coronavirus and environmental contamination in sars outbreak units airborne transmission of communicable infection-the elusive pathway detection of varicellazoster virus dna in air samples from hospital rooms a school outbreak of norwalk-like virus: evidence for airborne transmission airborne dispersal as a novel transmission route of coagulase-negative staphylococci: interaction between coagulase-negative staphylococci and rhinovirus infection experimental airborne transmission of prrs virus laboratory-acquired sars raises worries on biosafety world health organization. western pacific region. summary of china's investigation into the april outbreak aerial dissemination of pulmonary tuberculosis: a twoyear study of contagion in a tuberculosis ward lack of evidence for proofreading mechanisms associated with an rna virus polymerase the population genetics and evolutionary epidemiology of rna viruses how contagious are common respiratory tract infections? inhaling to mitigate exhaled bioaerosols efficiency of quarantine during an epidemic of severe acute respiratory syndrome-beijing, china ventilation of wards and nosocomial outbreak of severe acute respiratory syndrome among healthcare workers hospital preparedness and sars severe acute respiratory syndrome and biology, air quality, physics, and mechanical engineering key: cord-324280-e8mj6ecl authors: shaman, jeffrey; morita, haruka; birger, ruthie; boyle, mary; comito, devon; lane, benjamin; ligon, chanel; smith, hannah; desalle, rob; planet, paul title: asymptomatic summertime shedding of respiratory viruses date: 2018-04-01 journal: j infect dis doi: 10.1093/infdis/jix685 sha: doc_id: 324280 cord_uid: e8mj6ecl to determine rates of both symptomatic and asymptomatic infection among ambulatory adults, we collected nasopharyngeal swab specimens, demographic characteristics, and survey information from 1477 adult visitors to a new york city tourist attraction during april–july 2016. multiplex polymerase chain reaction analysis was used to identify specimens positive for common respiratory viruses. a total of 7.2% of samples tested positive for respiratory viruses; among positive samples, 71.0% contained rhinovirus, and 21.5% contained coronavirus. influenza virus, respiratory syncytial virus, and parainfluenza virus were also detected. depending on symptomatologic definition, 57.7%–93.3% of positive samples were asymptomatic. these findings indicate that significant levels of asymptomatic respiratory viral shedding exist during summer among the ambulatory adult population. much of the surveillance for respiratory virus infections in humans is conducted through networks of clinics and hospitals performing patient services. specifically, patients presenting with influenza-like illness or another nonspecific illness are recorded at medical clinics and emergency departments, and these numbers are reported to county and state health agencies [1, 2] . specimens are also obtained from some individuals presenting with influenza-like illness or similar symptoms and tested for the presence of respiratory virus, using a rapid diagnostic or laboratory assay. these data provide an estimate of respiratory virus infection rates among those seeking medical attention. however, these surveillance systems, owing to their passive form, do not capture infection rates among the population of individuals who do not seek medical attention. these latter, unrepresented individuals-persons not seeking medical attention-constitute a generally undocumented population experiencing asymptomatic or symptomatic respiratory virus infection. such persons may be wholly unaware of their infection, experience mild symptoms, or become iller but choose not to seek medical attention. here, we explore respiratory virus infection rates in a segment of this population through a convenience survey and sampling study of adult visitors to a new york city tourist attraction during spring and summer 2016. we solicited participants from among visitors to the new york city tourist attraction during 29 april-31 july 2016. the location, a collection point for both tourists and locals, provides a cross-section of potential participants who are broadly representative of the local and visiting populations of new york city. all activities, including participant solicitation, consenting, surveying, and sampling for respiratory viruses, were performed on weekends at the attraction. adults aged ≥18 years who were interested in participating were provided a detailed study description and consent form (columbia university medical center institutional review board [irb] approval aaaq4358; american museum of natural history irb approval fwa00006768). consenting adults were then administered a baseline survey and 2 nasopharyngeal swab samples, one from each nostril, were collected. participants were asked to provide information on their age, race, sex, recent travel, and preexisting medical conditions, including seasonal allergies, as well as a rating of 9 current symptoms commonly related to respiratory tract infection (see the supplementary materials for the full survey) per the common cold questionnaire [3, 4] . these symptoms-fever, chills, muscle pain, watery eyes, runny nose, sneezing, sore throat, cough, and chest pain-were recorded on a likert scale (ie, none, mild, moderate, or severe); each individual symptom was then quantified on the basis of these designations (ie, 0 for none, 1 for mild, 2 for moderate, and 3 for severe), and a total symptom score was tallied by summing all 9 symptoms values (range, 0-27). the nasopharyngeal samples were collected using minitip flock swabs (vwr catalog no. 10755-196; copan diagnostics). both samples were stored jointly in 2 ml of dna/rna shield (product no. r1100-250; zymo research) at 4°c-25°c for up to 30 days and then were aliquoted into two 2-ml cryovials and stored at -80°c. nucleic acids were extracted from 200 μl of thawed sample, using the easymag nuclisens automated system (biomerieux). reverse transcription-polymerase chain reaction analysis amplification was performed using the veriti 96-well thermal cycler (applied biosystems) per the genmark package instructions. samples were then subjected to genmark's rvp exonuclease polymerase chain reaction program, transferred to a genmark rvp cartridge, and loaded into the esensor for measurement of signal intensity, per manufacturer protocols. the genmark esensor rvp system separately detects influenza a virus of any subtype, influenza a(h1n1), influenza a(h3n2), 2009 pandemic influenza a(h1n1), influenza b virus, respiratory syncytial virus a and b, parainfluenza virus 1-4, human metapneumovirus, human rhinovirus (hrv), adenovirus b/e and c, coronavirus (cov) 229e, cov nl63, cov oc43, and cov hku1. the esensor system measures electrical signal intensity in nanoamps per millimeter squared. per manufacturer specifications, samples positive for a particular virus were identified by an intensity of ≥3 na/mm 2 . to test the sensitivity of our findings, because no standard definition of symptomatic infection exists, we used multiple definitions to delineate symptomatic from asymptomatic participants. the first symptomatic classification (definition 1) required self-report of ≥2 symptoms, with at least 1 being moderate or severe [5] . definition 2 relaxed this standard and required that only ≥1 symptom was moderate or severe. only fever, cough, and sore throat, the symptoms used to diagnose influenza-like illness [1] , were used to delineate the remaining 3 symptomatic definitions. for definition 3, a symptomatic participant had to self-report fever, cough, or sore throat as moderate or severe. a mild or worse fever and a mild or worse cough or sore throat was needed to be symptomatic according to definition 4. definition 5 required a mild or worse fever and a moderate or worse cough or sore throat. while the research protocol was exploratory, we hypothesized that shedding participants would more likely be symptomatic and that travel and age would be associated with shedding. statistical differences in symptom scores among categorical groupings (eg, age and race) were assessed using analysis of variance (anova) and the tukey test, whereas differences across pairs of categorical variables (eg, being symptomatic and positive for infection) were assessed using χ 2 analysis and the fisher exact test. associations between signal intensity among positive samples (all positive samples, hrv-positive samples, and cov-positive samples) and race, sex, age category (18-29, 30-39, 40-49, 50-64, and ≥ 65 years), allergies, travel, residence, and hispanic self-identification were described using univariate and multivariate regression. best-fit models were identified using the akaike information criterion. similarly, logistic regression was used to identify factors associated with a virus-positive sample (ie, positive vs negative). we consented, surveyed, and swabbed 1477 adults between 29 april and 31 july 2016. a total of 57.4% of participants were female, 41.8% were male, and 0.8% responded either "transgender, " "gender nonconforming, " or "don't know. " there were 67.7% of participants who self-identified as white, 14.8% as asian, 3.6% as black/african american, 1.5% as american indian/alaska native, 0.3% as native hawaiian/pacific islander, and 5.8% as another race, and 6.1% gave no response. a total of 22.3% self-identified as hispanic. the age distribution was as follows: 38.5% were aged 18-29 years, 20.9% were aged 30-39 years, 20.4% were aged 40-49 years, 13.9% were aged 50-64 years, and 4.5% were aged ≥65 years. more than half (53.6%) of participants received an influenza vaccine in the preceding year. a total of 7.2% of samples (107 of 1477) tested positive for respiratory virus. of the positive samples, 71.0% (76) were hrv positive, 21.5% (23) were cov positive, and 7.4% (8) were positive for human metapneumovirus, influenza virus, respiratory syncytial virus a, and parainfluenza virus 2-4 ( table 1) . viruspositive participants were identified throughout the study period; however, rates of positivity were lower in july than during april, may, and june. no coinfections were detected. across all participants, women reported significantly higher total symptom scores than men (p < .024 by anova), and participants aged 30-39 years, 40-49 years, and 50-64 years reported significantly lower total symptom scores than participants aged 18-29 years (p < .05 by the tukey test). in addition, consumption of cold and influenza medicines was significantly positively associated with higher total symptom scores (p < .0001 by anova), and this association held when the analysis was restricted to those testing positive for respiratory virus (p = .0001 by anova). among all participants there was a statistically significant positive association between reporting a greater tendency to get sick and total self-reported symptom score (p < .0001 by the tukey test); however, there was no significant association between reporting a greater tendency to get sick and actual detection of respiratory virus shedding (p = .10 by χ 2 analysis). testing positive for respiratory virus (≥3-na/mm 2 signal intensity) was positively associated with consumption of cold and influenza medicine (p < .0001 by the fisher exact test). there was no association between testing positive and having received influenza vaccine during the previous year, recent travel, or location of residence. among participants testing positive, 6.7%-42.3% qualified as symptomatic, depending on the definition used ( table 2 ). rates of being symptomatic differed significantly among virus-positive and virus-negative participants (p < .002 for all comparisons by χ 2 analysis and the fisher exact test) with positive participants more likely to qualify as symptomatic. the best-fit logistic regression model supported an association between an increased likelihood of testing positive for respiratory virus infection and a higher total symptom score (p < .0001) and being hispanic (p < .005). a similar association was found for the likelihood of testing positive for hrv. only a higher total symptom score (p = .001) was associated with an increased likelihood of testing positive for cov. among those testing positive for respiratory virus, the best-fit model revealed a statistically significant negative association between genmark esensor rvp signal intensity and the age categories 40-49 years and 50-64 years (p = .036 and p = .003, respectively) relative to the age category 18-29 years, and a positive association with seasonal allergies (p = .034). when only the hrv signal intensity was regressed, a statistically significant negative association with the age category 50-64 years (p = .038) relative to the age category 18-29 years emerged. regression of the cov signal intensity revealed a more complex positive association with total symptom score (p = .007) and negative associations with self-identification as black and american indian/alaska native (p = .003 for both), relative to white, and with the age category 50-64 years (p = .002), relative to the age category 18-29 years. here, we found that 7.2% of adult participants visiting a new york city tourist attraction during late spring and summer tested positive for a common respiratory virus. depending on the definition used, 57.7%-93.3% of those testing positive qualified as asymptomatic. the asymptomatic percentages derived using definitions 1 and 2 (59.6% and 57.7%, respectively), which had laxer criteria, fell within the broad range (9%-80%) found in prior studies [6] [7] [8] [9] . definitions 3-5, which used more-stringent influenza-like illness symptom criteria, yielded higher asymptomatic percentages. twenty-six percent of participants testing positive reported a total symptom score of 0. in contrast, 83.0%-98.7% of participants testing negative for a common respiratory virus qualified as asymptomatic, depending on the definition used. regardless of symptom definition, all participants testing positive were ambulatory and taking the time to visit the tourist attraction. however, the differences suggest that people experiencing influenza-like illness symptoms, in particular fever, may be more apt to stay home. indeed, fever was the least commonly reported of the 9 surveyed symptoms. among those testing positive, for either all viruses or hrv alone, we found an association between esensor signal intensity and participant age. in addition, signal intensity was positively associated with self-reported total symptom score for cov. previous studies, as well as manufacturer specifications, have indicated little association between esensor signal intensity and the amount of virus present [10, 11] . the finding here that symptom severity and shedding of cov are positively associated warrants further investigation. the current findings, based on a large, diverse sample of ambulatory adults, provide a baseline prevalence of respiratory virus shedding among this subpopulation during the northern hemisphere summer in a major city, where tourists abound. it is unclear how this shedding among approximately 1 in 14 adults contributes to the transmission of these pathogens. indeed, while the nasopharyngeal specimens document shedding, the contagiousness of the participants is unclear. rhinovirus and coronavirus were most prevalent. while their greater abundance might indicate that these 2 viruses are more communicable, it is possible that their higher prevalence is linked with immune escape or innate transmissibility. our sampling scheme introduces some biases that may make the findings not reflective of shedding across the entire population; specifically, very ill individuals stay home, and those feeling symptoms might have been more willing to participate in this study. participants self-reported fever, whereas a thermometer measure might have provided more-definitive data. further, participants only reported symptoms over the last 48 hours; however, for some respiratory viruses, rna can be detected weeks following acute illness. thus, we cannot fully determine whether asymptomatic positive samples were due to prior illness, represented an incubating infection, or were truly asymptomatic. the findings clearly indicate that substantive levels of asymptomatic shedding exist among the sampled adult ambulatory population. it will be important to repeat this study during the winter cold and influenza season and determine how overall infection rates, infection rates by virus, and asymptomatic infection rates vary from summer to winter. the findings presented here can be used to complement findings from household surveys, to improve estimates of virus infection incidence, and to inform model simulation, forecast, and control of infections due to these pathogens. in particular, medical countermeasures might be deployed more effectively if the true scope of respiratory virus infection incidence in the population were better understood. disclaimer. the views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the department of defense or the us government. financial support. this work was supported by the defense advanced research projects agency (grant w911nf-16-2-0035). potential conflicts of interest. j. s. and columbia university disclose equity in sk analytics. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. weekly us influenza surveillance report world health organization (who) validity of the common cold questionnaire (ccq) in asthma exacerbations a prospective study of respiratory viral infection in pregnant women with and without asthma prophylactic intranasal alpha 2 interferon and viral exacerbations of chronic respiratory disease household transmission of the 2009 pandemic a/h1n1 influenza virus: elevated laboratory-confirmed secondary attack rates and evidence of asymptomatic infections cider research team. asymptomatic ratio for seasonal h1n1 influenza infection among schoolchildren in taiwan role of rhinovirus c respiratory infections in sick and healthy children in spain prospective evaluation of rhinovirus infection in healthy young children comparison of the genmark diagnostics esensor respiratory viral panel to real-time pcr for detection of respiratory viruses in children comparison of the biofire filmarray rp, genmark esensor rvp, luminex xtag rvpv1, and luminex xtag rvp fast multiplex assays for detection of respiratory viruses key: cord-301340-lhh04pum authors: jamieson, frances b.; wang, elaine e. l.; bain, cindy; good, jennifer; duckmanton, lynn; petric, martin title: human torovirus: a new nosocomial gastrointestinal pathogen date: 1998-11-17 journal: j infect dis doi: 10.1086/314434 sha: doc_id: 301340 cord_uid: lhh04pum studies were undertaken to determine if human torovirus is associated with gastroenteritis and to examine the clinical features of torovirus illness in children. the fecal excretion of torovirus in patients with gastroenteritis was compared with that in matched asymptomatic controls in a case-control study. toroviruses were identified in 72 (35.0%) of 206 gastroenteritis cases compared with 30 (14.5%) of 206 controls (p < .001). clinical features of torovirus gastroenteritis in 172 patients positive for torovirus were compared with those of 115 patients infected with rotavirus or astrovirus. persons infected with torovirus were more frequently immunocompromised (43.0% vs. 15.7%) and nosocomially infected (57.6% vs. 31.3%). they also experienced less vomiting (46.4% vs. 66.7%) but had more bloody diarrhea (11.2% vs. 1.8%). an antibody response to torovirus developed mainly in older, nonimmunocompromised children (p < .01). these studies demonstrate an association between torovirus excretion and gastroenteritis in the pediatric population among immunocompromised hospitalized patients and in previously healthy patients. long. toroviruses were classified on the basis of the berne virus genome sequence as members of the family coronaviridae, which together with the family arteriviridae are now classified in the order nidovirales [12] [13] [14] . torovirus-like particles were first documented in 1984 by electron microscopy of fecal specimens of persons with gastroenteritis [15] . evidence that these were toroviruses came from their immunospecific cross-reactions with the breda virus and the ability of nucleic acid probes based on the berne virus sequence to hybridize with rna extracted from stool specimens positive for toroviruses by electron microscopy [16, 17] . studies of these viruses were limited because they cannot be grown in cell culture, they lack the distinct icosahedral structure of other gastroenteritis viruses, and their peplomers are less pronounced than those of coronaviruses [16] . in our diagnostic electron microscopy laboratory, pleomorphic fringed particles that resembled particles previously designated as toroviruses were observed in a substantial number of fecal specimens from children with gastroenteritis. these were shown to be toroviruses because of a high concordance between the identification of torovirus-like particles by electron microscopy and a positive enzyme immunoassay with breda virus reference antiserum [18] . torovirus-like particles purified from stool specimens were further shown to be toroviruses on the basis of their morphology, immunospecific interactions with breda virus antiserum, ability to elicit an immune response following infection, and the high degree of homology of the 3 end of their genome with that of the berne and breda viruses [19] . these data support the hypothesis that these toroviruses are infectious agents, but better evidence is needed to establish that toroviruses actually cause gastroenteritis. we therefore conducted two epidemiologic studies to determine the role of these virus particles in gastroenteritis. the first study was a case-control comparison of torovirus shedding in patients with gastroenteritis and in asymptomatic controls. the second compared the clinical features of symptomatic patients shedding torovirus and those shedding rotavirus or astrovirus. the studies were conducted between 1 october 1993 and 30 september 1995 in patients admitted to the hospital for sick children, a 410-bed primary, secondary, and tertiary care center. in this setting, fecal specimens are submitted for diagnostic virology for all patients admitted with symptoms of gastroenteritis or who develop these symptoms while hospitalized. specimens from ∼1500 patients are examined each year. in the first study, the frequency of torovirus identification in patients with gastrointestinal symptoms (cases) was compared with that of asymptomatic controls. once this was accomplished, a second study was undertaken to establish a more complete microbiologic diagnosis, examine the serologic response, and describe the progress of the infection. stool specimens from patients with gastroenteritis were examined for viruses by negative contrast electron microscopy [19, 20] . casepatients were selected from among this symptomatic group using a table of random numbers. for each case, a control patient was selected and matched by ward and date of admission within 1 week of the case. these matching criteria were used to ensure that the two populations were comparable with respect to underlying illness and season and duration of hospitalization. controls were not symptomatic for gastroenteritis and had experienced no changes in stool frequency or consistency on the day of selection. patients were excluded from enrollment if they had an underlying gastrointestinal disorder (e.g., crohn's disease). stool specimens from controls were collected within 72 h of enrollment of the case specimen and examined for viruses by electron microscopy; the electron microscopist was blinded to patient clinical status. all stool specimens were collected in a standard stool specimen container (25 ml) and processed within 48 h. control patients were reassessed on day 4 after enrollment to determine if they developed gastrointestinal symptoms subsequent to enrollment. case patients were excluded from the analysis when no matching control was available or when a stool specimen was not obtained from the matched asymptomatic control. at a different time from that of study 1, patients whose stool specimens were positive for torovirus, rotavirus, or astrovirus by electron microscopy were enrolled in study 2. information on clinical presentation and course throughout hospitalization was collected from patients or parents by questionnaire. after discharge, each patient was followed with a visit from the study nurse. in torovirus-positive patients, stool specimens were examined daily by electron microscopy during the hospitalization and at follow-up. acute sera from these patients were collected at the time of diagnosis, and convalescent sera were obtained 2-6 weeks after enrollment. diarrhea was defined as ≥3 loose stools in a 24-h period. nosocomial infection was defined as onset of gastrointestinal symptoms on or after day 3 of admission [3] . immunosuppressed patients included those with the following conditions: severe combined immunodeficiency, leukemia, other malignancies treated by chemotherapy, solid organ or bone marrow transplant recipients, human immunodeficiency virus infection or aids, and those receiving large doses of immunosuppressive therapy for any cause. seroconversion was defined as a rise in antibody titer to torovirus of ≥4-fold by hemagglutination-inhibition (hai) assay. stool specimens. these were prepared for diagnostic electron microscopy as described previously [19, 20] . in brief, a 10%-20% suspension of specimen was prepared in a 1% solution of ammonium acetate, applied to a polyvinyl carbon-coated 400-mesh electron microscope grid, and stained with a 2% solution of sodium phosphotungstate at ph 7.0. the specimen was examined under an electron microscope (phillips em300) at ϫ50,000 magnification. specimens were considered positive for torovirus if they contained spherical or kidney-shaped particles (100-140 nm in diameter) with a characteristic fringe of peplomers ∼10 nm long as shown in figure 1 [19] . other viral pathogens, including rotaviruses, adenoviruses, astroviruses, and norwalk virus-like agents, were identified by their morphologic features [20, 21] . all specimens were examined by an experienced electron microscopist blinded to specimen source. for patients participating in study 2, stool samples were also submitted for bacterial isolation [22] and tested for escherichia coli o157:h7 [23] , examined for ova and parasites [24] , and tested for clostridium difficile cytotoxin [25] . serology. antibody to torovirus was quantitated in acute and convalescent sera by hai assay as described previously [19] by use of a purified torovirus preparation from a single patient. the test was done without knowledge of the clinical and demographic features of the cases. the hai assay used a 0.5% suspension of rabbit erythrocytes and 4 hemagglutination units of virus per well. in the analysis of the results of these tests, age and immune status (immunocompromised vs. immunocompetent) were included in a logistic regression model to predict for seroconversion. statistical analysis. we used mcnemar's x 2 test to compare concordance of matched pairs of gastroenteritis patients and asymptomatic controls described in study 1. relative odds were calculated by dividing the number of cases with torovirus times the number of controls without torovirus by the number of controls with torovirus times the number of cases without torovirus. unpaired student's t tests for continuous measures and x 2 analysis or fisher's exact tests were used to compare clinical findings between patients positive for torovirus and patients positive for astrovirus or rotavirus. descriptive statistics were used to express duration of symptoms and shedding of virus. geometric means and 95% confidence intervals were used to describe serologic responses [26] . two hundred and six case-control pairs were included in this analysis. of the initial cases selected, 176 were excluded for one or more of the following reasons: underlying gastrointestinal disorder (5), lack of a control patient hospitalized at a comparable time (106), or lack of a stool specimen from the control patient (65). demographic characteristics were similar for cases and controls as summarized in ). since a substantial number of the patients were im-p ! .001 munocompromised, cases and controls of this subset were independently analyzed for excretion of torovirus (table 2) . in this subset, significantly more cases than controls were again positive for torovirus. this was also true among the immunocompetent patients. when controls were reassessed, 23 controls had developed gastroenteritis within 4 days of study enrollment. of these, 10 were positive for torovirus. when these 10 controls were excluded, only 20 (10.2%) of 196 controls were positive for torovirus. this study was designed to determine whether there was an association between torovirus excretion and gastroenteritis. accordingly, the patient and control specimens were examined only for viruses with the understanding that other microbial pathogens would be tested for in study 2. to obtain an insight as to whether the symptomatic patients may have had bacterial infections not diagnosed in study 1, a retrospective analysis was performed on a subset of symptomatic patients whose stool specimens were submitted for bacteriologic testing as part of the routine investigation outside the study protocol. this analysis revealed no bacterial pathogens in stool specimens from 28 cases for whom bacteriologic testing was done. study 2: clinical features of torovirus-positive gastroenteritis patients. between 1 october 1993 and 30 september 1995, clinical features were analyzed for 172 torovirus-, 102 rota-virus-, and 13 astrovirus-infected gastroenteritis patients. the monthly frequency distribution of viruses identified by electron microscopy in 1993 is shown in figure 2 . while rotavirus infections were most frequent during the winter and spring, the incidence of torovirus infections remained relatively constant throughout the year. as shown in table 3, torovirus-positive patients were older, developed nosocomial infections more often, and were more frequently immunocompromised than were patients infected with rotavirus or astrovirus. the clinical manifestations of torovirus were similar to those of rotavirus or astrovirus except that children infected with torovirus had less vomiting and more bloody diarrhea (table 4) . of the 19 torovirus-positive patients presenting with bloody diarrhea, 9 were immunocompetent, and 1 had a concomitant c. jejuni infection. nine additional torovirus-positive patients developed bloody diarrhea within 2-15 days after study enrollment and had no other pathogen in the stool sample. among those with rotavirus or astrovirus, only 2 presented with bloody diarrhea; another 6 developed bloody diarrhea after enrollment, 2 of whom were positive for c. jejuni. bacterial pathogens were detected in stool specimens from both groups of patients. among those infected with torovirus, yersinia enterocolitica was isolated from 1 and c. jejuni from 2. c. difficile cytotoxin was detected in 7 patients in a subset of 81 tested in the rotavirus-astrovirus group and in 8 of a subset of 128 torovirus-positive patients. one torovirus-positive patient (of 64 tested) had giardia lamblia cysts, and 1 rotaviruspositive patient (of 61 tested) had dientamoeba fragilis present in stool specimens. the average duration of symptoms was similar, although torovirus-positive patients required parenteral hydration for a longer period (6.4 vs. 4.8 days). stool specimens collected on a follow-up visit 2-6 months after study enrollment from 168 patients infected with torovirus showed that 14 were shedding the virus. when immunocompromised patients were compared with previously healthy torovirus-positive patients, they were significantly older and more likely to have acquired the infection in the hospital (78% vs. 42%) (table 5). three immunocompromised patients required admission to the intensive care unit after developing severe diarrhea and vomiting. seroconversion. in paired sera from 88 patients, 51 demonstrated seroconversion with a ≥4-fold increase in antibody titer (table 6) . of the 44 patients ≤2 years old, half (22) experienced seroconversion, whereas of the 44 patients ≥2 years old, 29 (66%) seroconverted. of the 30 immunocompromised patients, 11 seroconverted, and of the latter 11, 10 (91%) were older than 2 years. of the 58 immunocompetent patients, 40 seroconverted and 19 (47.5%) were older than 2 years. after controlling for age in the analysis, immunocompromised patients were significantly less likely to seroconvert than were immunocompetent patients ( ). the geometric mean ti-p ! .001 ter of convalescent sera was also significantly lower for immunocompromised patients (17.61) than for immunocompetent patients (33.97). these studies provide strong evidence for a causative role for torovirus in gastroenteritis. first, the relative odds of torovirus among gastroenteritis cases was 3.1-fold that for controls. however, if the 10 control patients who became symptomatic within 4 days of sampling are excluded, the relative odds become 4.7, which approaches those described with enteric adenovirus and astrovirus [4, 27] . secondly, over half of the torovirus-positive gastroenteritis patients developed an immune response to the virus. this suggests that the virus caused the infection rather than being a simple bystander or an electron microscopy artifact in patients with diarrhea. the higher frequency of seroconver-sion in older children in our study is similar to findings in animals reinfected with breda virus and suggests a booster response to repeated infections [28, 29] . finally, other known gastroenteritis pathogens were only sporadically identified in torovirus-positive patients. these were also found in children infected by rotavirus or astrovirus, established gastroenteritis viruses. the association of torovirus with acute and persistent diarrhea was recently reported in a smaller cohort of children in an urban brazilian slum [30] . in that study, 33 children had acute diarrhea and 41 had persistent diarrhea. there were 17 controls. torovirus appears to be a common agent of gastroenteritis at our institution. particles resembling toroviruses have been reported in stools of gastroenteritis patients, although in some cases they were designated "coronavirus-like particles" [15, [31] [32] [33] . during a 2-year period at our hospital, 20% of stool samples from gastroenteritis patients were positive for torovirus, more than double those positive for rotavirus (7%) and for astrovirus and norwalk-like viruses (2%). toroviruses were present all year and had seasonal distribution similar to that described for corona-like viruses [33] . these findings compare favorably with our recent report on the role of bovine torovirus in diarrhea of calves [34] . in that study, toroviruses were the predominant viral agent associated with diarrhea. torovirus is the principal identifiable cause of nosocomial diarrhea in immunocompromised and older children in our institution. although no deaths in the immunocompromised patients studied could be directly attributable to torovirus infection, enteric viral infections cause severe morbidity and mortality among such patients [35] . among asymptomatic controls, torovirus was detected at a lower rate in immunocompromised patients than in immunocompetent patients (9.0% vs. 15.6%; table 2), whereas among children with gastroenteritis, it was more common among immunocompromised than in immunocompetent patients. this further suggests that the recovery of torovirus by the immunocompromised is related to the illness rather than to a proclivity of immunocompromised patients to carry this organism. furthermore, this virus was a common cause of diarrhea in hospitalized patients who were previously healthy. an outbreak of gastrointestinal illness in a neonatal intensive care unit due to particles similar to toroviruses (coronavirus-like particles) has been described [36] . two clinical features that distinguish torovirus-infected patients are the lower frequency of vomiting and high frequency of bloody diarrhea. descriptions of illness in patients with enteric infection by coronavirus-like particles have indicated that diarrhea is the most prominent symptom, and patients often experienced frank or occult blood in stool [15, 32, 36] . a fatal case of gastroenteritis associated with coronavirus-like particles has also been reported [37] . bloody diarrhea is generally not a feature of viral gastroenteritis and is more commonly associated with bacterial infections such as those caused by campylobacter species or enterohemorrhagic e. coli [38] . although low platelet counts in immunocompromised patients could predispose such patients to bloody diarrhea, almost half of the patients with this symptom were immunocompetent and did not have thrombocytopenia, suggesting that this symptom cannot be explained wholly by host factors. studies of torovirus infections in calves (breda virus) indicate that these viruses infect differentiating epithelial cells in the crypts of the intestinal villi, especially in the large intestine [10, 11] . investigations of human torovirus infections are needed to compare the pathology of torovirus with that of other gastroenteritis viruses. because this study was of hospitalized patients, the importance of torovirus infection in the community is not known. while over half of the torovirus-positive gastroenteritis patients were considered to have a nosocomial infection, a substantial number were admitted for treatment of gastroenteritis. this suggests that torovirus causes gastroenteritis in the community. the brazilian study identified torovirus in the community but other pathogens were also found, including enteroaggregative e. coli [30] . in our study, bacterial coinfections were predominantly with cytotoxin-producing c. difficile in both torovirusand rotavirus-positive patients. other microbial infections, namely y. enterocolitica, g. lamblia, and d. fragilis, occurred singly and of the 2 c. jejuni isolates from torovirus-positive patients, 1 child had bloody diarrhea. these findings are consistent with our observation that nonviral microbial pathogens are likely to have been rare in the case and control specimens analyzed in study 1. our results support the hypothesis that human torovirus is an important cause of gastroenteritis. further epidemiologic studies to determine its frequency in the community and to identify mechanisms of transmission of this organism are indicated as are further studies to explain the pathophysiology of illness due to this agent. a study of illness in a group of cleveland families rotavirus as a cause of diarrheal morbidity and mortality in the united states the incidence of viral-associated diarrhea after admission to a pediatric hospital astroviruses as a cause of gastroenteritis in children sequence and genomic organization of norwalk virus viral gastroenteritis enteric adenovirus infection and childhood diarrhea: an epidemiologic study in three clinical settings calf diarrhea (scours) reproduced with a virus from a field outbreak the proposed family, toroviridae: agents of enteric infections studies with an unclassified virus isolated from diarrheic calves comparative studies on three isolates of breda virus of calves toroviruses: replication, evolution and comparison with other members of the coronavirus-like family genus torovirus assigned to the coronaviridae nidovirales: a new order comprising coronaviridae and arteriviridae an enveloped virus in stools of children and adults with gastroenteritis resembles the breda virus of calves preliminary characterization of torovirus-like particles of humans: comparison with the berne virus of horses and breda virus of calves diagnosis of torovirus infection enzyme-linked immunosorbent assay reactivity of torovirus-like particles in fecal specimens from humans with diarrhea characterization of torovirus from human fecal specimens viruses associated with acute gastroenteritis in young children methods for preparing specimens for electron microscopy processing and interpretation of bacterial fecal cultures sorbitol-macconkey medium for detection of escherichia coli o157:h7 associated with hemorrhagic colitis us government printing office; department of health and human services publication no. (cdc) clostridium difficile invasion and toxin circulation in fatal pseudomembranous colitis candidate adenoviruses 40 and 41: fastidious adenoviruses from human infant stool seroepidemiology of breda virus in cattle using elisa bredavirus (toroviridae) infection and systemic antibody response in sentinel calves association of torovirus with acute and persistent diarrhea in children pleomorphic virus-like particles in human feces coronavirus-like particles in human gastrointestinal disease an 8 year study of the viral agents of acute gastroenteritis in humans: ultrastructural observations and seasonal distribution with a major emphasis on coronavirus-like particles detection of bovine torovirus in fecal specimens of calves with diarrhea from ontario farms infectious gastroenteritis in bone-marrow-transplant recipients pleomorphic, enveloped, virus-like particles associated with gastrointestinal illness in neonates fatal gastroenteritis associated with coronaviruslike particles approach to patients with gastrointestinal tract infections and food poisoning the technical efforts of bo luan and maria szymanski are gratefully acknowledged. key: cord-350302-xmyqqgn5 authors: li, pengfei; liu, jiaye; ma, zhongren; bramer, wichor m; peppelenbosch, maikel p; pan, qiuwei title: estimating global epidemiology of low-pathogenic human coronaviruses in relation to the covid-19 context date: 2020-08-15 journal: j infect dis doi: 10.1093/infdis/jiaa321 sha: doc_id: 350302 cord_uid: xmyqqgn5 nan to the editor-coronaviruses (cov) comprise a large family of zoonotic rna viruses. among the 7 members known to infect humans, sars-cov-2, the causative agent of covid-19, together with sars-cov and mers-cov, cause severe respiratory syndrome. the other 4 members, including nl63, hku1, oc43, and 229e, are widely circulating in humans but predominantly cause mild respiratory tract illness [1] . thus we call these 4 viruses low-pathogenic human covs (lph-cov). two recent studies by nickbakhsh [2, 3] . interestingly, both studies detected the highest frequency of infection in children younger than 5 years. this is the opposite to the covid-19 pandemic where children are less commonly affected by sars-cov-2 [4] . these intriguing findings trigger important hypotheses on whether coinfection with lph-cov interferes with sars-cov-2 or exposure to lph-cov confers crossprotective immunity to some extent. as covid-19 is currently affecting the global population and research on lph-cov has been largely neglected in the past, we attempted to perform a systematic review and meta-analysis to map the global epidemiology of lph-cov. lph-cov-related studies from 1990 to march 2020 were systematically searched in medline, embase, web of science, cochrane central register of controlled trials (central), and google scholar. studies were included and data extracted only if they reported participants with symptoms of acute respiratory tract infections or influenza like illness. a 95% confidence interval (95% ci) was estimated using wilson score method. pooled prevalence (detection rate) was calculated using the dersimonian-laird random-effects model with freeman-tukey double arcsine transformation. in total, 128 studies with 205 421 individuals were included, and an overall infection rate was estimated as 5.21% (95% ci, 4.62%-5.83%; i 2 = 97%). the prevalence of lph-cov varied substantially among the reported 44 countries, from 0.73% (philippines; 95% ci, 0.09%-1.84%) to 21.51% (tunisia; 95% ci, 17.47%-25.83%) ( figure 1a and 1b). australia 74 125 30 30 149 1876 9 35 251 68 47 81 7 26 198 175 40 580 21 35 28 30 116 26 230 303 25 367 11 3 5 23 81 6 60 66 1652 48 71 20 131 80 29 18 1231 12 523 514 2277 526 561 6221 70 988 277 739 6064 1041 665 593 244 300 1910 4259 1059 7158 450 417 309 311 2060 1404 2693 3899 407 3910 172 411 114 369 2370 67 972 1528 40 150 686 1166 112 8462 372 2428 369 24 [2, 3] , our included studies only detected lph-cov in individuals with respiratory illness or symptoms. this suggests that the prevalence rate of lph-cov in the general population could be even lower, raising the question of how large the impact could be on covid-19. monto et al have nicely presented the seasonal distribution of the identified cases in michigan according to the 4 lph-cov types [3] . we performed similar analyses by pooling 5 studies with relevant data, and all these studies were from countries in the northern hemisphere. we confirmed their findings that more cases were detected in the winter season ( figure 1c ). however, we are cautious about the interpretation of these seasonal distribution data ( figure 1c ) [3] because they only specified the identified cases and not the rate of infection, as the total number of tested cases in each month was not given. more importantly, whether sars-cov-2 will develop into a seasonal and/or endemic virus only time will tell [3] . in summary, we have comprehensively estimated the global prevalence of lph-cov among 44 countries and mapped their seasonal distribution. our results further strengthen the epidemiological findings of nickbakhsh et al and monto et al, but also raise cautions about the interpretation of existing data. we agree that continued and enhanced monitoring of circulating hlp-cov is necessary to understand how they may have an impact on the epidemiology and outcome of covid-19 [5, 6] . z. m. and m. p. p. discussed the project and critically revised the manuscript. all authors reviewed the final version of the manuscript and approved for submission. disclaimer. funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. financial support. this work was supported by the netherlands organization for scientific research (grant number 91719300 to q. p.); and the china scholarship council (grant numbers 201808370170 and 201606240079 phd fellowships to p. l. and j. l.). potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. rhinovirus and coronavirus infection-associated hospitalizations among older adults epidemiology of seasonal coronaviruses: establishing the context for covid-19 emergence coronavirus occurrence and transmission over 8 years in the hive cohort of households in michigan coronavirus infections in children including covid-19: an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children who strategic and technical advisory group for infectious hazards. covid-19: towards controlling of a pandemic potential association between covid-19 mortality and health-care resource availability to the editor-we read with interest the letter by li et al [1] reporting on the global prevalence of endemic human coronaviruses. the last decade has witnessed an expansion of global surveillance efforts in influenza and respiratory syncytial virus infections, leading to an increased recognition of the importance of these viruses, particularly in low-and middle-income settings [2] . however, a paucity of epidemiological research exists for other respiratory viruses, as highlighted by the world health organization's battle against respiratory viruses initiative [3] .such knowledge is currently hindered by the lack of capacity of many diagnostic laboratories, including those key: cord-321132-xdpb3ukt authors: lhomme, sebastien; garrouste, cyril; kamar, nassim; saune, karine; abravanel, florence; mansuy, jean-michel; dubois, martine; rostaing, lionel; izopet, jacques title: influence of polyproline region and macro domain genetic heterogeneity on hev persistence in immunocompromised patients date: 2014-01-15 journal: j infect dis doi: 10.1093/infdis/jit438 sha: doc_id: 321132 cord_uid: xdpb3ukt hepatitis e virus (hev) can chronically infect immunocompromised patients. the polyproline region (ppr) and the macro domain of orf1 protein may modulate virus production and/or the host immune response. we investigated the association between the genetic heterogeneity of hev quasispecies in orf1 and the outcome of infection in solid-organ transplant patients. both sequence entropy and genetic distances during the hepatitis e acute phase were higher in patients whose infection became chronic than in those who cleared the virus. hence, great quasispecies heterogeneity in the regions encoding the ppr and the macro domain may facilitate hev persistence. hepatitis e virus (hev) can chronically infect immunocompromised patients. the polyproline region (ppr) and the macro domain of orf1 protein may modulate virus production and/or the host immune response. we investigated the association between the genetic heterogeneity of hev quasispecies in orf1 and the outcome of infection in solidorgan transplant patients. both sequence entropy and genetic distances during the hepatitis e acute phase were higher in patients whose infection became chronic than in those who cleared the virus. hence, great quasispecies heterogeneity in the regions encoding the ppr and the macro domain may facilitate hev persistence. keywords. hepatitis e; chronic infection; orf1; polyproline region; macro domain. hepatitis e virus (hev) is a major cause of enterically transmitted non-a, non-b hepatitis. it is also responsible for large outbreaks of waterborne acute hepatitis in tropical and subtropical countries as well as sporadic infections worldwide. hepatitis e is a zoonotic disease in industrialized countries, with pigs, wild boar, and deer being the major animal reservoirs of hev [1] . hev, genus hepevirus, family hepeviridae, is an unenveloped, single-stranded, positive-sense rna virus. like all rna viruses, hev exists as a mixture of closely related variants defining a quasispecies. the approximately 7.2 kb genome of hev has a coding region consisting of 3 open reading frames (orfs). orf2 encodes the capsid protein; orf3 encodes a small protein implicated in virus egress [2] , whereas orf1 encodes a nonstructural protein that contains several putative functional domains. these include at least 4 enzyme activities: methyltransferase, cystein protease, helicase, and rna-dependent rna polymerase [2] . homologies with other plant and animal positive strand rna viruses have been used to identify other domains: the y domain, the polyproline region (ppr), and the macro domain. the ppr is genetically very diverse and could correspond to an intrinsically disordered region involved in virus adaptation [3] . in addition, the ppr does not seem to be required for hev replication in vitro or in vivo [4] . nonstructural virus genes that are not essential for replication are usually involved in modulating host immune responses [5] . the genomes of several virus families, including all members of coronaviridae, rubella virus, and alphaviruses (togaviridae), and hev, have macro domains. the macro domain of the mouse hepatitis virus (mhv) influences the pathogenicity of the virus [6] . hev can lead to chronic hepatitis in solid-organ transplant (sot) patients [7] . but our knowledge of the factors associated with the development of chronic infection in sot patients exposed to hev is far from complete. our working hypothesis was that the genetic heterogeneity of the ppr or the macro domain play a role in the outcome of hev infection in immunocompromised patients, as the ppr could modulate the host immune response and the macro domain could influence virus pathogenicity. we therefore analysed the characteristics of hev quasispecies at the acute phase of hepatitis e in 2 groups of sot patients, one whose infection became chronic and the other who cleared the virus. we studied 14 sot patients who became acutely infected with hepatitis e between january 2004 and june 2009. the infection was diagnosed by the detecting hev rna using the real time polymerase chain reaction (pcr) and immunoglobulin m/immunoglobulin g anti-hev antibodies by a commercial enzyme-linked immunosorbent assay [8] . each patient was assigned to one of 2 groups according to the outcome of the infection. the first group (group i; 8 patients) had chronic infections, defined by persistently elevated liver enzyme activity and serum that was positive for hev rna for more than 6 months after diagnosis. the second group (group ii; 6 patients) had resolving infections. serum samples were collected from all patients at the acute phase of their infection and stored at −80°c. each patient underwent an exhaustive clinical and laboratory examination, including the immunosuppressive drugs used, the hepatic enzyme activities, and white blood cell count. the serum concentration of hev rna was measured by real-time pcr [9] . virus genotype was determined by sequencing a 189nucleotide fragment within the orf2 gene. the sequences were compared to reference hev strains (genbank) as described elsewhere [10] . nucleic acids were extracted from serum samples and analysed by 1-step rt-pcr with the sense primer hevorf1-s1 and anti-sense primer hevorf1-a1 using the super script iii enzyme (invitrogen, cergy-pontoise, france). the sequences of the primers are listed in supplementary table 1 . the sequence amplified included the ppr (nucleotide [nt] 2137-2340) and the macro domain (nt 2341-2829), using the genome 1b l08816 as a reference. the pcr products were purified using qiaprep (qiagen, courtaboeuf, france) miniprep kits and stored at −20°c. in total, 10 ng of the amplified sequence were directly ligated into 1 µl of pcr 4 vector (kit topo ta cloning; invitrogen) containing a gene conferring resistance to ampicilline. recombinant plasmids were used to transform escherichia coli-competent cells, and transformants were grown on ampicillin-coated plates at 37°c for 18 hours. the complementary dna (cdna) clones (20 from each patient sample) were analysed by pcr with the primers hevorf1-s1 and hevorf1-a1 (supplementary table 1 ). the pcr products of these amplifications were purified (quick-spin columns; qiagen) and sequenced using the fluorescent dye terminator method for big dyeterminator cycle sequencing (applied biosystems, paris, france) with the primers hevorf1-s1, hevorf1-s2, hevorf1-s3, hevorf1-a1 and hevorf1-a2 (supplementary table 1 ) on an applied biosystems abi 3130 xl analyzer. sequences were aligned using clustal x and compared with those of hev strains obtained from genbank. we quantified the complexity of the hev quasispecies by calculating the shannon entropy: s = −σ i ( p i ln p i ), where pi is the frequency of each sequence in the viral quasispecies. the normalized entropies for both nucleotides and amino-acids, sn, were calculated using sn = s/ln n, where n is the total number of sequences analysed. we quantified diversity as the mean genetic distance calculated for all pairs of nucleotide sequences using mega 4.0. the numbers of synonymous (ks) substitutions per synonymous site and nonsynonymous (ka) substitutions per nonsynonymous site were calculated with the jukes-cantor correction for multiple substitutions using mega 4.0. the ka/ks ratio is an indicator of the positive (>1) or negative (<1) selection pressure on a quasispecies [11] . proportions were compared by fisher exact test. quantitative variables were compared with the nonparametric mann-whitney test. correlations between complexities or diversities of quasispecies were estimated by calculating spearman rank correlation coefficient. a p-value below .05 was considered to be statistically significant. the sequences have been deposited in the genbank database under accession numbers kc911858 to kc912137. the clinical and biological characteristics of the patients are summarized in supplementary table 2 . there was no significant difference between patients with chronic infections and those with resolving infections in terms of gender, age, or immunosuppressant treatment. the alt activities of individuals with a chronic infection tended to be lower. patients whose infection became chronic had lower total, cd3, cd4, and cd8 lymphocyte counts, but the differences were not significant. the serum hev rna concentrations at the acute phase of the 2 groups of patients were similar. they were all infected with hev genotype 3f strains, except one whose infection was genotype 3c. we compared the sequence heterogeneity of 2 regions of orf1 in patients with chronic hev infection and those with resolving infections. the mean nucleotide sequence entropy (nt complexity) of the ppr was higher in group i [7] . the viral determinants associated with the persistence of such an infection are poorly documented. we therefore investigated the influence of the genetic heterogeneity of hev quasispecies on the outcome of infection, focusing on the ppr and the macro domain. both the complexity and diversity of the ppr and the macro domain were higher in viral population of the patients whose infection became chronic than in those who cleared the virus. it has been shown that the quasispecies heterogeneity in the orf2 region encoding the capsid protein during the acute phase of infection is associated with the development of a chronic hev infection [12] . our data support the finding that great genetic heterogeneity of the quasispecies in patients whose infection become chronic seems to favor the appearance of variants that can persist, as reported for hcv infections [13] . although the diversity of the region preceding the ppr was higher in patients with chronic infection, nt and aa complexities were not different in the 2 groups (data not shown), suggesting that the higher heterogeneity in chronic patients was not a general effect seen across the entire region studied. this great genetic heterogeneity could also reflect an inadequate control of the viral replication, but no correlation was found between quasispecies heterogeneity and viral concentrations. ppr appears to be dispensable for in vitro hev replication, but it is required for in vivo infection, suggesting that it is involved in infecting cells with innate immunity [4] . although the role of the macro domain in the replication of hev is unknown, it does not seem to be essential for the replication of coronavirus in cell culture [14] . it was recently suggested that genes that are dispensable for virus replication are involved in modulating the host immune response, like down-regulating interleukin 1β (il-1β) or tumor necrosis factor α (tnf-α) secretion [5] . the macro domain is also involved in the inflammation caused by the mouse hepatitis virus (mhv), and the substitution of a strictly conserved amino-acid residue is responsible for reducing the secretion of inflammatory cytokines [6] . the great quasispecies heterogeneity in patients whose infection became chronic may include some variants that reduce inflammatory cytokine production, which could facilitate hev persistence. this could explain the lower serum concentrations of interleukin 1 (il-1) receptor antagonist and tnf-α found in patients whose infection became chronic [12] . we find that the complexities and diversities of the ppr and the macro domain are correlated. these 2 regions may well have evolved together as the protein encoded by orf1 does not seem to be cleaved [15] . we also studied the correlations between the orf1 and orf2 as this study and our previous one on orf2 diversity [12] were carried out on the same patients (except for 3). we also find that the complexity and diversity of the regions in orf1 and orf2 that were studied are correlated (data not shown), suggesting that they, too, have evolved together. finally, we find no differences in the ka/ks ratios of the studied regions, even though the ka/ks ratios of the ppr seemed to be higher in patients who cleared the virus. alternatively, the ka/ks ratio may not allow us to infer differences in selection pressure. in both regions, ka/ks ratios indicate negative selection, probably due to structural or functional constraints. a limitation of our study is the small number of patients in each group and an implication of immunological factors in the evolution toward chronicity cannot be excluded. we conclude that the high genetic heterogeneity of the ppr and the macro domain at the acute phase of an hev infection is associated with persistence of the virus. this association may be due to the appearance of mutants able to modulate the host immune response. further investigation is now needed to confirm this association. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org/). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. molecular virology of hepatitis e virus the hepatitis e virus polyproline region is involved in viral adaptation deletions of the hypervariable region (hvr) in open reading frame 1 of hepatitis e virus do not abolish virus infectivity: evidence for attenuation of hvr deletion mutants in vivo immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response mouse hepatitis virus liver pathology is dependent on adp-ribose-1′′-phosphatase, a viral function conserved in the alpha-like supergroup hepatitis e virus and chronic hepatitis in organ-transplant recipients characteristics of autochthonous hepatitis e virus infection in solid-organ transplant recipients in france genotype 3 diversity and quantification of hepatitis e virus rna hepatitis e virus genotype 3 diversity statistical methods for detecting molecular adaptation hev quasispecies and the outcome of acute hepatitis e in solid-organ transplant patients the outcome of acute hepatitis c predicted by the evolution of the viral quasispecies adp-ribose-1′′-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture early secretory pathway localization and lack of processing for hepatitis e virus replication protein porf1 financial support. this work was supported by institut national de la santé et de la recherche médicale u1043.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-293299-gdew0ueo authors: jordan, william s.; dowdle, walter r.; easterday, bernard c.; ennis, francis a.; gregg, michael b.; kilbourne, edwin d.; seal, john a.; sloan, frank a. title: influenza research in the soviet union—1974 date: 1974-12-17 journal: j infect dis doi: 10.1093/infdis/130.6.686 sha: doc_id: 293299 cord_uid: gdew0ueo nan tamination procedures must be responsible. a means for interruption of such transmission must be sought. (g) risk figures for patients of hb agpositive physicians, surgeons, and dentists should be developed. if carriage of hg ag is hazardous to patients, the risk should be defined, and appropriate measures developed for reduction of the risk. (h) the effect of the season on dissemination of herpesviruses should be studied. in addition, the cytomegalovirus group should be evaluated for antigenic variants, since there may be considerable variation in potential for disease among different members of this group. (i) measures for the increase of specific resistance to representatives of the herpesvirus group should be evaluated. while the effectiveness of specific attenuated or inactivated vaccines may appear to be unlikely on theoretical grounds, transfer factor and other cellular immunological approaches should be studied. (3) personnel, physicians, and nurses must be trained in hospital epidemiology, surveillance, and control procedures, to implement and to increase the available knowledge. in addition, innovative approaches to the dissemination of knowledge concerning the usage of antimicrobial agents, hazards of various invasive procedures, and hospital control practices must be encouraged. william l. hewitt selected fields of health and medical science. the initial problems selected were malignant neoplasms, cardiovascular diseases, and environmental health. at its second session, the u.s.-u.s.s.r. joint committee for health cooperation agreed to further exploration in the field of influenza and other viral diseases; recommendations regarding areas of collaboration would be considered at the third session of the joint committee in moscow in june 1974. on behalf of the development of these recommendations, a delegation of scientists from the united states visited the soviet union during february 17-27, 1974, for discussions with soviet scientists in leningrad and moscow. the delegation was larger than the small group that had visited several institutes in these two cities in january 1973 [1] , and was able to collect additional information on soviet research. this summary presents a synthesis of information collected by members of the visiting delegation regarding soviet research in the five principal areas considered for possible joint collaboration. a long historical tradition for the use of livevirus vaccines in the soviet union dates from smorodintsev's experimental infection of volunteers with influenza virus in 1937 [2] . that the success of immunization with live virus has been limited is evidenced by continued research on new methods for attenuation and administration of virus and by the present emphasis on research in chemoprophylaxis. furthermore, mass immunization is not yet undertaken on more than a city or regional basis, and no more than 50 million doses of vaccine are used each year. the eventual goal is the achievement of mass immunization of about 70% of the population. such immunization would be nonselective, except that the vaccination of children would be emphasized. there is little interest in the use of inactivated vaccine by parenteral administration, although such vaccine is now being produced experimentally and is to be utilized, if approved, in hyperimmunization of volunteers for the production of immunoglobulin against influenza virus. administration and control. the development of new or modified vaccines is chiefly under the aegis of the all-union research institute of influenza in leningrad, although the institute for viral preparations in moscow also undertakes limited research and development in addition to its production activities. the initial testing of a new vaccine in volunteers is under the control of the institute involved in its development. protocols are first reviewed by an institute expert committee, then by a review council before testing of vaccines in volunteers. experimental vaccines are preliminarily tested under standard protocols for mycobacteria, mycoplasmas, and hemadsorbing viruses. initial testing in volunteers is principally for reactogenicity. testing is stepwise in relation to volunteer susceptibility and is restricted to young adults. thus, the live vaccine is successively tested in volunteers (five per group) with high, low, and undetectable levels of serum antibody, and then finally in groups of 20 antibody-negative subjects. application of the vaccine to nonvolunteer populations in field trials requires the permission of the ministry of health. selection of strains for the production of vaccines is made in the november through january period by a committee under the ministry of health; results of testing of the first three production batches in a total of 60 volunteers are submitted to the laboratory of biologic control, which is part of the tarasevich control institute for biological preparations of the ministry of health. allantoic fluid vaccine is distributed to sanitary-epidemiologic stations that arrange for and direct its use in organized population groups, such as industrial workers. neither allantoic nor tissue culture vaccines are available directly to polyclinics or other medical facilities. production. experimental vaccines are produced by the institute involved in their development. thus, smorodintsev's allantoic fluid vaccines are produced at the all-union research institute of influenza, while tissue culture vaccines originated by the institute for viral preparations are prepared there. on the other hand, the smorodintsev vaccine approved for general use is produced by the institute for viral preparations. about 12 million of the 45-50 million doses of influenza vaccine produced each year are produced in the latter institute. production of the remainder appears to be distributed among institutes in other cities. chick embryos used at both the all-union research institute and the institute 687 for viral preparations are furnished from flocks under supervision of the veterinary department. however, the all-union research institute minimizes the problem of avian leukosis, citing the negative evidence of 10,000 children who received mumps vaccine and were followed for 12 years, plus the impracticality of testing when 15 million eggs are used annually. the institute for viral preparations reported negative rip (resistance-inducing factor) tests on periodic testing of eggs. research. research in influenza vaccines appears to be entirely committed to live virus immunization. the primary site of this research is the all-union research institute of influenza, although some vaccine-related research is undertaken elsewhere, primarily at the institute for viral preparations. a significant amount of the genetic and molecular virologic research at the all-union research institute is oriented to problems of viral attenuation and virulence markers. until recently, the attenuation of viruses for use in the production of vaccine has been empirically achieved by serial passage in chick embryos and periodic testing in volunteers for reactogenicity. attention is now being directed to other means of attenuation and to the effects of employing a changed route of administration of low-passage, virulent virus for the reduction of toxic effects. preliminary experiments have attempted the recombination of freshly isolated virulent strains with attenuated "laboratory" viruses for the production of so-called "biological recombinants" that possess the antigens of the wild-type parent but are changed in other properties, such as virulence for mice. cold adaptation of viruses by passage at 25 c is also under study. a rapid-passage method in which allantoic fluids are harvested and passaged every 24 hr is also under investigation as a means of shortening the time required for attenuation. the greatest emphasis at present is on the use of the peroral (po) route of administration of live vaccine to children as a method of reducing symptoms associated with intranasal administration. comparative studies of the two routes have been done principally at the all-union research institute with allantoic fluid vaccines. these studies indicate that multiplication of virus is reduced after po adminstration, as evidenced by recovery of virus in low concentration only on the fifth day and a delay in antibody response when compared with that to intranasal vaccine. immunogenicity is also reduced by po administration, so that three separate applications of the virus at 14-day intervals are required to achieve a reasonable rate of seroconversion. at the institute for viral preparations, dr. a. k. alexeeva is investigating po live-virus vaccine prepared in tissue culture. the commitment to live influenza vaccines extends also to the immunoprophylaxis of other diseases, including infections with parainfluenza and respiratory syncytial viruses, adenoviruses, and mycoplasma. the ultimate research objective is the suppression of respiratory diseases, particularly in children, through the use of "combined immunization. " as previously reported [1] , passive immunotherapy is carried out through administration of y-globulin derived after specific immunization of man. earlier interest in the use of high-titered y-globulin intranasally as a prophylactic agent seems to have been abandoned. rather, y-globulin given parenterally was reported to be efficacious in alleviating high fever and other evidences of "toxicity," particularly in children. no new data on efficacy were provided. the y-globulin is prepared at the pasteur institute in leningrad from serum drawn from volunteers hyperimmunized with virus vaccines. the y-globulin is approved for use and is available at pharmacies without prescription at a cost of 3.75 rubles/2 ml. surveillance. most epidemiologic data presented covered the period from 1957 to the present, with by far the greatest emphasis on influenza a. the soviet union experienced major epidemics of influenza in 1957, 1959, 1962 (mixed a and b), 1965, 1967, 1968, and 1970 , reflecting roughly a two-to three-year cycle. the epidemics of 1957, 1962, and 1968 appear to have caused the greatest morbidity. sudden onset of countrywide outbreaks during the late fall or early winter months have characterized most epidemics, and the northern areas and urban populations have been most frequently and most severely affected. apparently most large cities (population of more than 200,000) experience relatively uniform increased morbidity during epidemics, with little asynchronous occurrence countrywide. influenza is a disease primarily of children most often affecting those between the ages of nine and 15 years. surveillance of influenza and other acute respiratory diseases (ard) is based on morbidity reports originating at the polyclinic or hospital level. large factories, mills, etc., also have clinics and provide data for the system. physicians are required to report cases seen at clinics or on house calls. report cards are filled out daily, collected by trained health personnel, tabulated, and sent to district sanitary-epidemiologic stations; from here reports are sent to successively higher administrative units: the rayon, the oblast, then to the republics, and finally to the ministry of health. although this system (which also applies to other reportable communicable diseases) is the formal, regular, national method of morbidity reporting, two other systems of influenza and ard reporting exist that serve somewhat different functions but utilize the same basic polyclinic source of data. these systems are operated by the all-union research institute, leningrad, and the ivanovsky institute, moscow. for the last two or three years the former institute has operated a surveillance system for influenza-ard consisting of regular daily morbidity reporting (by cable or telephone) from the capitals of the 15 republics. thirty-seven other cities submit regular reports weekly. another 100 cities are supposed to report weekly, but perhaps only 30-40 of these do so regularly. the morbidity data received by .the all-union research institute are identical to data reported in the national system. however, this institute receives these data either one day or one week before the regular system; it uses them primarily to determine the probability of influenza morbidity in the 52 cities under close surveillance. in 1957 the ivanovsky institute began receiving morbidity data every 10 days from 30 towns throughout the soviet union. the number of towns reporting grew to 55 but is now only 20. these 20 cities report the same data to the all-union research institute; the cities are distributed widely throughout the soviet union and have populations of 200,000 or more. fourteen of these cities report each week; six cities report by cable daily. all report the total number of patients seen at polyclinics or at home who are diagnosed as having influenza or ard plus the same morbidity data for children up to seven years of age. during influenza epidemics the ivanovsky institute also receives daily by cable the total number of hospitalized cases of influenza and the total number of deaths attributable to influenza. the daily and weekly morbidity data are considered provisional, and each month the 20 cities send an adjusted tabulation of cases which is final. a monthly summary of these data, along with laboratory results, is mailed to each city. a quarterly report is compiled and sent to the world health organization for publication. physicians are required to report all cases of influenza and ard they diagnose, and most do so; many ill persons are never seen by physicians. as a result of several apartment house surveys after two major epidemics, a rough estimate of overall morbidity of 35 % was obtained. approximately 50%-60% of these cases were seen by a physician. laboratory surveillance. the all-union and ivanovsky institutes receive regular laboratory reports from the 53 and 20 reporting cities, respectively. although it was not clearly confirmed, it appears that the gamaleya institute presently serves as a resource and administrative headquarters for most if not all of the laboratories that provide input to all three institutes. serologic tests are performed at the gamaleya institute on sera received weekly from 20 sanitary-epidemiologic stations. sera are obtained from patients of all ages presenting at clinics with noninfectious diseases, from blood donors, from military populations, and from children attending day-care centers and schools. 2, and 3) , and mycoplasma infections also are diagnosed by direct fluorescent antibody staining of nasal smears. the reagents are thought to be prepared at the all-union research institute and distributed to its stations. some of the stations also perform serodiagnostic tests, but many forward paired sera to the gamaleya institute for examination. paired sera are said to be difficult to obtain. some stations may attempt virus isolation in tissue culture; many apparently employ embryonated eggs for isolation of influenza viruses. computer modeling. soviet epidemiologists have devoted considerable effort to the 'development of a model for the prediction of registered numbers of cases of influenza or ard for 128 geographic units within the soviet union. since registered morbidity rather than a more direct measure of real influenza-ard morbidity is used, the model probably has more applicability for health planners, who need to anticipate increased requirements for delivery of health care associated with epidemics, and for industrial organizations that face future production losses, than for those concerned with anticipating changes in the health status of the population per se. critical to the quality of the data input is the proportion of the cases registered. many behavioral factors affect the proportion of cases registered. among these is probably the cost of iiiness. although both inpatient and outpatient services are provided at no direct cost to the patient, sick leave benefits do not always cover absences from work because of illness. for the first five years of employment, benefits cover 50% of the employee's salary; for the next three, the percentage increases to 80%; only after eight years on the job does sick leave cover the employee's entire salary during illness. thus, influenza imposes an indirect cost on many in terms of lost income. some may be reluctant to declare themselves ill because they do not want to incur this loss. once an influenza-ard epidemic has been identified in one location, the model enables one to predict future registrations for 128 observational units, encompassing 99 % of the soviet population. the model serves two roles: (1 ) prediction of daily registrations in a large number of locations; (2) evaluation of the effects of specific public programs (e.g., vaccination). the former application is particularly important in the soviet union, since physicians from all types of specialties and even medical students in advanced stages of training are mobilized to treat influenza during epidemics. although data from the morbidity registration system in the soviet union provide the basic information for this model, data from the 52 all-union reporting centers appear to be used as well, particularly for obtaining early warning of the location of the initial epidemic outbreak in the country. the model's theoretical structure was developed at the gamaleya institute by l. v. rvachev. rvachev's academic training is in mathematics and physics; the model is based on principles from physics. the basic equations have been presented by baroyan et al, [3] ; a much more rigorous theoretical development of the model has been published by rvachev [4] . the formal structure of the model will not be presented here, but brief mention will be made of the independent variables and parameters that serve as the basis for predictions. movement of persons among geographic areas provides the mechanism for the spread of influenza-ard. the model contains a large number of transportation coefficients (based on sales of airline, bus, and railroad tickets) relating interlocational flows of people. these data have been assembled through rather painstaking efforts on the part of staff. one of the soviet scientists estimated that it took three man-years to secure this information alone. two epidemic-specific parameters reflect the initial (i.e., at the time of epidemic outbreak) density of influenza-susceptible individuals and the speed of transmission within a location. these are estimated from data on registered morbidity in the location in which the initial outbreak occurs. it appears that data must be obtained for five to 14 days. rvachev maintains that the estimates are maximal likelihood estimates. once the epidemic-specific parameters have been estimated, predictions can be made for all locations in the soviet union. the interval between the date predictions are made and the peak of the influenza-ard epidemic in the median location is about 1.5 months. these two parameters are said to embody virological and immunological factors associated with the epidemic. the model, however, does not consider such factors explicitly. other variables and parameters in the model are the area's population and frequency distribution of length of influenza illness, obtained by averaging of data from a monograph by zhdanov et al. [5] . the predictive ability of the model has been evaluated with use of both historical data (for outbreaks in 1957, 1959, 1962, 1965, 1967, 1969, and 1970 ) and data generated for two years since the model was developed. the soviets show extensive plots of actual vs. predicted values of influenza-ard morbidity; these plots indicate that the model predicts quite well. timing of the epidemic and its intensity (measured by the number of registered cases in the location) have been used to assess predictive accuracy. a prediction is considered "accurate" if two conditions are satisfied. first, the day on which influenza-ard reaches its highest level must be within five days of the day on which the peak is predicted to occur. second, the following inequality must be satisfied: 0.7 < (highest predicted registered morbidity in location) / (highest actual registered morbidity in location) < 1.5. using this definition of accuracy, recent tests have shown the model to be "accurate" 80% of the time. compared with similar models in other fields, the model for prediction of influenza-ard epidemiology provides reasonably good predictions of the dependent variable. however, at present it provides very little time between the date on which predictions are made and the date on which epidemics occur. this interval might, of course, be lengthened if the epidemic-specific parameters were estimates based on influenza data from another country, at a time before the epidemic reached the soviet union. for this reason, there is some interest among the soviets in having the united states and other countries implement the model. discussions along this line are now underway with the german democratic republic and bulgaria. research on the ecology of influenza viruses is done at the ivanovsky institute under the direc-tion of d. k. lvov in collaboration with l. y. zakste1skaya. dr. lvov indicated that most of the work had been done since 1970. while there were earlier reports on animal influenza viruses, there appears to have been no organized program until 1970. dr. lvov has been investigating the ecology of arthropod-borne viruses for some time; he now is using some of the same procedures and materials in studying the ecology of influenza. most of his efforts have been directed toward avian species. thus far, most of these studies have been conducted in. the far east of the soviet union (specifically, the kamchatka region and the komandorskiye islands). other areas have included the eastern arctic, west central arctic, murmansk regions, byelorussia, and the caspian sea. all of the areas studied so far have been on the perimeter of the country. while the field aspects have been directed by drs. lvov and a. a. sazanov, the antigenic and serologic analyses and virus characterizations have been done under the direction of dr. zakstelskaya. viruses characterized as h3n2 have been isolated from the chicken, pig, dog, calf, and wild birds (terns and herons) in various parts of the soviet union. in the far east, antibody to the h3 antigen has been found in wild birds, fur seals, cattle, and mink. while there is serological evidence for the h3n2 virus in the far east, the only isolations of that virus from wild birds (terns and herons) have been in the caspian sea area. other information includes: (1) the demonstration of a/equine/miamij63 (heq2neq2) antibody in bird sera, (2) the demonstration of a/swine/ iowa/30 (hsw1nl) antibody in seal and some bird sera, (3) the presence of fowl plague virus (hav1) in chickens in 1970 and the presence of that antibody in wild birds in 1972, and (4) the isolation of swine influenza virus (a/swine/ tartu/1/70) from pigs in 1970. a virus (hav7n2) was isolated from terns in the caspian sea area in june 1973. it is also interesting to note that newcastle disease virus was isolated from wild birds (species not indicated) on kamchatka in 1972, the same year it was isolated from wild ducks in california by workers in the united states. several soviet scientists are interested in antiviral chemoprophylaxis and therapy, including use of 691 interferon and interferon inducers against influenza, and there is some overlap of interests by scientists at various institutions. however, there was little evidence of formal collaboration in this area of influenza research in the soviet union. laboratory research with anti-influenza drugs. dr. g. a. galegov of the ivanovsky institute, a biochemist, leads a group of scientists interested in testing antiviral agents. he hopes to achieve additive therapeutic effects without additive toxicity by using combinations of drugs or interferon plus a drug. dr. galegov has been a coauthor of several publications in this area [6] and is currently seeking an agent that will interfere with the rna polymerase of influenza virus. he is studying a disulfide agent that decreases the rna polymerase activity by about 40%. the name of the agent and the concentration required to produce this effect were not stated; no studies have been performed with it in animals. his laboratory apparently does not perform animal experiments with anti-influenza drugs but does measure their antiviral effects in tissue culture while attempting to delineate mechanisms of action. development of new anti-influenza drugs. very little information was acquired in this area. the two new chemical agents used in the soviet union to prevent influenza, oxolin and bonaphton, were supplied to clinicians for testing in volunteers by dr. pershin of the institute for chemo-therapeutic preparations in moscow; unfortunately, he was not a participant in the discussions. dr. d. m. zlydnikov of the all-union research institute had used these drugs to prevent or treat influenza but did not know what effect they had on influenza virus in vitro or on influenza infection in animal models. dr. zlydnikov suggested that dr. pershin might have done some studies in cell cultures or animals. the scientists who are concerned with influenza chemoprophylaxis and chemotherapy obtain the agents used from manufacturing facilities, such as dr. pershin's institute in moscow, or from outside the soviet union. the clinical investigators who used these agents in volunteers apparently had not done independent in vitro or animal studies. clinical studies. the research performed in the large and active volunteer unit at the all-union research institute of influenza can be briefly summarized. (l) amantadine. the observation made by scientists in the united states that amantadine was partially effective for prophylaxis against influenza type a2 infection in human volunteers was confirmed. no information was obtained about its mechanism of action in tissue culture or in animals. this drug has been used widely in epidemiologic field trials and is now approved for general use. the package insert states: "for the purpose of individual prophylaxis of influenza, the 0.25 % ointment is used in the form of daily two fold application to the nasal mucosa during the period of elevation and maximum development of the epidemic outbreak of influenza (for 25 days) or upon contact with an influenza patient." (3) bonaphton (called 6 bromonaphthoquinone-l, 2 ). dr. zlydnikov obtained this agent from dr. pershin and studied its effect in human volunteers challenged with partially attenuated influenza virus [a 2/hong kong/68 (h3n2)]. the drug was given the day before challenge and daily for six days after challenge. controls received a placebo. safety was studied in a total of 363 volunteers in many experiments. efficacy studies were limited to evaluation of clinical morbidity; no virus isolation or antibody studies were performed. a regimen of 50 mg given po two times daily, from the day before to six days after challenge, was said to decrease morbidity (e.g., 38.4% of 13 volunteers given bonaphton vs. 78.6% of 14 volunteers given placebo). large epidemiologic field trials were done, and approval for general use was obtained. as was the case with oxolin, no data were presented about the mechanism of bonaphton's effect, and it was not learned whether animal studies had been performed with this drug. (4) interferon and interferon inducers. soviet scientists are interested in the use of interferon, either by aerosolization of exogenous human interferon (widely used to treat influenza) or by induction with viral vaccines or chemical inducers. these studies were reviewed in detail in 1973 [1] ; no new information has been acquired. some interesting research is being done by two laboratories in the general area of nonspecific resistance to influenza. drs. t. g. orlova and l. m. mentkevich at the ivanovsky institute have studied the effects of interferon induction by viruses (including influenza) and nuc1eotides in irradiated cba mice, which received transplants of either syngeneic bone marrow or rat bone marrow [7] . induction of interferon by all inducers except influenza virus appeared to be related to production of interferon in bone marrow cells as measured by species specificity. influenza virus induced mouse interferon in irradiated mice that had received rat bone marrow transplants, a fact suggesting the possibility of extramedullary interferon induction by influenza. few laboratories in the soviet union appear to be concerned primarily with basic genetic studies of influenza viruses. recently, as noted, laboratories in the all-union research institute have begun to apply techniques of cold adaptation and recombination to attenuation of strains used for production of live-virus vaccine. in the laboratory of dr. d. b. golubev, antigenic and other variation of the neuraminidase antigens of h3n2 influenza viruses have been under study; his demonstration of differing temperature inactivation and temperature optima of these neuraminidases provides interesting genetic markers. this laboratory has continued studies on the possible relation between temperature optima of neuraminidase and virulence. a lower temperature optimum was described as characteristic of attenuated strains. dr. golubev also reported that, by use of the cf test and purified neuraminidase (n2) proteins, three major antigenic subgroups could be distinguished among influenza strains isolated since 1957. he described these groups as repre-senting "semi-shifts"; the three groups include strains isolated from 1957 to 1965, from 1967 to 1971, and from 1972 to the present, respectively. each semi-shift was accompanied by a change in neuraminidase inactivation temperature. each semi-shift also occurred at the time of a major epidemic in the soviet union. at the ivanovsky institute of virology, studies of influenza virus rna polymerase have been undertaken by dr. galegov, but these studies apparently are in their early stages. dr. u. z. ghendon's group at the institute for viral preparations has worked extensively with temperature-sensitive mutants of fowl plague virus since shifting attention from polioviruses to influenza. results of the assortment of the fowl plague ts mutants into five complementation groups have been published. more recently, ghendon has found evidence of variation in rna polymerase activity among wildtype strains of virus associated with epidemics in december 1971, january 1973, and early 1968. in contrast to observations of workers in the united states, ghendon has evidence against the association of rna virion polymerase activity with the ribonuclear protein. dr. f. i. yershov of the ivanovsky institute is attempting to establish persistent in vitro infections with influenza viruses. such studies may relate to possible mechanisms for survival of the virus during epidemic periods and for antigenic variation. work on such persistent infections with influenza viruses has just begun; major effort in the past involved tick-borne encephalitis virus and parainfluenza viruses. this report, without critique, has attempted to summarize objectively information helpfully provided by soviet colleagues. because of the press of time, important contributions may well have been missed by such a brief survey, and the possibility is acknowledged that some of the presented facts may have been misunderstood and are, therefore, misrepresented. apologies are offered for all such errors, with the expectation that they will be corrected as the collaboration recommended by the delegations from the united states and the soviet union becomes a reality. to these observers, it appears that the recognition given by the ministry of health of the soviet union to influenza as an important national 693 health problem exceeds that given by health authorities in the united states. the soviet investment in research on influenza probably exceeds investment in the united states. the development of a national system of morbidity surveillance and of computer forecasting as a means of allowing local health authorities to prepare for an increased demand for medical services recognizes the impact of the disease in the community. such morbidity reporting is superior to that in the united states and provides a hard-data base for the concern evidenced by the soviet ministry. clearly, neither country has solved the complex problem of influenza prevention; there is much to be gained through mutual cooperation. influenza and interferon research in the soviet union investigation of volunteers infected with the influenza virus possibilities for computer modeling of influenza epidemics in the u report on a visit to bulgaria, december 1-29, 1972. world health organization uchenie 0 grippe review of problems in viral chemotherapy interferon production and infection caused by influenza virus in radiation chimeras key: cord-002333-90f9vr0a authors: madan, anuradha; segall, nathan; ferguson, murdo; frenette, louise; kroll, robin; friel, damien; soni, jyoti; li, ping; innis, bruce l.; schuind, anne title: immunogenicity and safety of an as03-adjuvanted h7n9 pandemic influenza vaccine in a randomized trial in healthy adults date: 2016-12-01 journal: j infect dis doi: 10.1093/infdis/jiw414 sha: doc_id: 2333 cord_uid: 90f9vr0a background. almost 700 cases of human infection with avian influenza a/h7n9 have been reported since 2013. pandemic preparedness strategies include h7n9 vaccine development. methods. we evaluated an inactivated h7n9 vaccine in an observer-blind study in healthy adults aged 18–64 years. participants (420) were randomized to receive 1 of 4 as03-adjuvanted vaccines (low or medium dose of hemagglutinin with as03(a) or as03(b)), one nonadjuvanted vaccine, or placebo. the coprimary immunogenicity objective determined whether adjuvanted vaccines elicited an immune response against the vaccine-homologous virus, 21 days after the second vaccine dose per us and european licensure criteria in the per-protocol cohort (n = 389). results. all adjuvanted vaccines met regulatory acceptance criteria. in groups receiving adjuvanted formulations, seroconversion rates were ≥85.7%, seroprotection rates ≥91.1%, and geometric mean titers ≥92.9% versus 23.2%, 28.6%, and 17.2 for the nonadjuvanted vaccine. the as03 adjuvant enhanced immune response at antigen-sparing doses. injection site pain occurred more frequently with adjuvanted vaccines (in ≤98.3% of vaccinees) than with the nonadjuvanted vaccine (40.7%) or placebo (20.0%). none of the 20 serious adverse events reported were related to vaccination. conclusions. two doses of as03-adjuvanted h7n9 vaccine were well tolerated and induced a robust antibody response at antigen-sparing doses in healthy adults. clinical trials registration. nct01999842. periodic outbreaks of h7 avian influenza a virus infections occur in poultry worldwide, with sporadic transmission to humans. in 2003, an outbreak of h7n7 disease in the netherlands resulted in 89 human infections and 1 death, with evidence of limited human-to-human transmission [1] . human infections with h7n9 viruses were first reported in china in february 2013; to the present time, there have been 3 waves of infection [2] . as of december 2015, a total of 683 laboratory-confirmed cases, including 275 deaths, had been reported to the world health organization [2, 3] . the case fatality rate of h7n9 influenza is approximately 40% [2, 3] . the virus can cause rapidly progressive pneumonia, often complicated by extrapulmonary disease associated with hypercytokinemia [4] . genetic changes observed in the h7n9 virus suggest adaptation to mammals, carrying the risk of human-to-human transmission [5] . it has been shown that h7n9 and h7n1 influenza viruses are capable of airborne transmission in a mammalian host (ferret), without losing virulence [6, 7] . these observations suggest the potential for an h7 pandemic in humans, and support pandemic h7 vaccine development. several h7 inactivated influenza vaccines and live-attenuated influenza vaccines are in clinical development, but have not been highly immunogenic in humans [8] [9] [10] . adjuvanted vaccines have shown improved immunogenicity [11] [12] [13] [14] . a recent mix-and-match study demonstrated that a monovalent h7n9 vaccine adjuvanted with as03 induced a better immune response than the nonadjuvanted or mf59-adjuvanted formulations, when administered to adults according to a 2-dose schedule [14] . here, we present the findings of a study that evaluated h7n9 vaccine formulations with hemagglutinin (ha) antigen doses of 2.78 and 5.09 µg, given with as03 adjuvants of different potency and a nonadjuvanted formulation. the doses of as03-adjuvanted ha antigen were chosen for testing based on a clinical development program by gsk biologicals with an as03-adjuvanted split virus h5n1 marketed vaccine. this was a phase i/ii, randomized, placebo-controlled, multicenter trial evaluating an h7n9 influenza vaccine (nct01999842). the trial was approved by independent ethics committees or institutional review boards and was conducted in accordance with the declaration of helsinki, the international conference on harmonisation good clinical practice guidelines, and regulatory requirements of participating countries. participants provided written informed consent. the trial was observer blind and enrolled healthy participants 18-64 years of age in the united states and canada (inclusion criteria are detailed in supplementary text 1). the inactivated, split-virion vaccine, manufactured with a reverse geneticderived reassortant seed virus developed by world health organization collaborating centres and references laboratories from a/shanghai/2/2013 (h7n9) (gsk vaccines, quebec, canada), was adjuvanted with as03, an oil-in-water emulsion containing 5.93 mg (as03 b ) or 11.86 mg (as03 a ) of dl-αtocopherol. participants were randomized 1:1:1:1:1:2 to 1 of 6 groups receiving different ha antigen doses (mixed with adjuvant in groups 1-4) or placebo: (1) the antigen doses were less than the initially targeted concentrations of 3.75 and 7.5 µg, because the single radial immunodiffusion assay used to determine the antigen concentration during formulation overestimated the concentration in relation to subsequently available reagents provided by the center for biologics evaluation and research (cber) to evaluate vaccine potency. vaccines were administered twice, 21 days apart, by intramuscular injection in the deltoid muscle. the coprimary immunogenicity objective was to evaluate whether the adjuvanted a/shanghai/2/2013 (h7n9) vaccines elicited an immune response against the vaccine-homologous virus that met us cber and european committee for medicinal products for human use (chmp) immunogenicity targets at day 42 (21 days after the second vaccine dose). the primary immunogenicity objective of the study was met if the following criteria were fulfilled for any adjuvanted vaccine formulation: the lower limit of the 98.75% confidence interval was ≥40% for the seroconversion rate (scr) and ≥70% for seroprotection rate (spr), and chmp criteria were met if point estimates were >40% for scr, >70% for spr, and >2.5 for the mean geometric increase (mgi). the coprimary safety objective was to describe the safety and reactogenicity of the vaccines up to day 42. immunogenicity assessments were done with hi and mn assays at baseline (day 0), at 21 days after each dose (days 21 and 42), and at 6 months after the first vaccine dose (day 182), and with hi assay only at 12 months after the first vaccine dose (day 385). hi and mn antibody titers were assessed using horse red blood cells (rbcs). to remove nonspecific agglutinin to horse rbcs and nonspecific virus inhibitors introduced by the hemadsorption step, a receptor-destroying enzyme treatment step was added after horse rbc hemadsorption. humoral immune response assays were performed by a gsk biologicals laboratory (hi) and by viroclinic biosciences (mn). the following derived parameters related to the tested vaccine virus were estimated for hi titer: spr, gmt, scr, and mgi. spr was defined as the proportion of participants with reciprocal hi titers ≥40. gmts were defined as the antilog of the mean of the log 10 -transformed inverse titers. scr was defined as the proportion of participants with either a prevaccination reciprocal hi titer <10 and a postvaccination reciprocal titer ≥40 or a prevaccination reciprocal hi titer ≥10 and a ≥4-fold increase in postvaccination reciprocal titer. mgi was defined as the geometric mean of the within-participant ratios of the postvaccination to the prevaccination reciprocal hi titer. for mn titer, seropositivity rate and gmt were derived in a similar way as for hi. participants recorded solicited injection site and general symptoms between days 0 and 6 after each vaccination in diary cards, collected at the next visit. symptoms were graded by severity from 1 (mild) to 3 (severe). grade 3 was defined as "significant pain at rest, preventing everyday activities" for pain, "surface diameter >100 mm" for redness and swelling, temperatures of "≥39.0°c (≥ 102.2°f)" for fever, and "preventing normal activities" for all other solicited symptoms. blood samples for safety evaluations were collected on days 0, 7, 21, 28, and 42. participants recorded unsolicited symptoms (graded by severity) until 21 days after each vaccination. medically attended adverse events (aes), potentially immunemediated disorders ( pimds), and serious aes (saes) were followed up until the study end. participants were asked to report immediately any events perceived as serious. immunogenicity analyses were performed on the per-protocol cohorts ( participants who complied with the protocol, received vaccine, and had assay results available for antibodies against the vaccine-homologous ha antigen at the specified intervals). the safety analysis was descriptive and was performed on the total vaccinated cohort ( participants who received ≥1 dose of study vaccine or placebo). statistical methods are described in detail in supplementary text 2. a total of 420 participants were included in the total vaccinated cohort and 389 in the per-protocol cohort ( figure 1 ). demographics were similar in all study groups (supplementary table 1 ). the mean participant age was 40 years, 65% of participants were women, and most (85.5%) were of white european/caucasian ethnic origin. immunogenicity cber and chmp criteria against the vaccine-homologous virus were met for all adjuvanted vaccines at day 42, 21 days after the second vaccine dose. the scrs and sprs were similar in all adjuvanted vaccine groups; scrs were 85.7%-96.3% and figure s1 ). between 12.5% and 19.6% of participants were seropositive before vaccination, although all baseline gmt values were low (only 5 samples had a titer ≥40). after the peak observed on day 42, seropositivity rates and antibody titers declined at day 182 and further at day 385, but gmts remained above baseline levels in the adjuvanted groups, ranging from 10.8 to 14.3 (table 1 ; supplementary figure s1 ). the sprs at days 182 and 385 were 12.3%-31.5% and 7.8%-19.6%, respectively. an immune response against an h7n1 virus was also observed at day 42, albeit at lower levels than against the vaccine-homologous virus. in the adjuvanted groups, 21 days after the second vaccine dose, scrs were 57.7%-76.9%, mgis were 9.0-13.8 and gmts were 46.9-72.8, with the highest response observed in the as03 a adjuvanted groups (table 1; figure 2 ). immune responses assessed by the mn assay showed a similar kinetic; however, titers remained 1.5-4.3 times higher at day 182 compared with baseline ( figure 2 ). the vaccine response rate is presented in supplementary table s2 . the non-adjuvanted hd ha vaccine elicited a considerably lower immune response against both vaccine-homologous and vaccine-heterologous viruses (table 1; figure 2 ). adjuvant effect was demonstrated in all adjuvanted groups, as the lower limit of the 98.75% confidence interval for gmt ratio (adjuvanted over nonadjuvanted) exceeded 1.5, and the scr difference (adjuvanted minus nonadjuvanted) exceeded 10% in all groups at 21 days after the second vaccine dose (table 2 ). at days 182 and 385, no study participant had reciprocal hi titers ≥40. the immune response was low in all vaccine groups after administration of only 1 vaccine dose (table 1; figure 2 ). in a sub-analysis of the homologous immune response by age, the adjuvanted vaccine was immunogenic in both age groups, with sprs ≥80.0% in participants 41-64 years of age, despite lower gmts (table 3 ). the spr was 11.5% in participants 41-64 years of age who received the nonadjuvanted hd ha vaccine. the immune response was generally lower in participants who had previously received seasonal influenza vaccine than in nonrecipients. at day 42, gmt values in the adjuvanted vaccine groups were 134.5-220.2 in prior recipients and 70.3-129.0 in nonrecipients. spr values were 83.9%-96.6% in prior recipients and 95.8%-100% in nonrecipients. pain was the most common injection site solicited symptom, occurring more frequently in the adjuvanted vaccine groups than in the hd ha nonadjuvanted group (table 4 ). redness and swelling at the injection site occurred in 3.3%-6.7% of participants receiving vaccines adjuvanted with as03 b and in 13.3%-18.3% of those receiving vaccines adjuvanted with as03 a (table 4) . grade 3 injection site solicited symptoms were reported by up to 6.7% of the participants in the adjuvanted groups and by none of the participants in the nonadjuvanted and placebo groups. fatigue, headache, and muscle ache were the most frequently reported solicited general symptoms (table 4 ). fatigue and muscle ache occurred in 45.0%-55.0% of participants in all adjuvanted groups, compared with 25.4%-28.8% in the hd ha nonadjuvanted and 25.0%-20.8% in the placebo group. fever occurred infrequently and at a similar rate across all study groups. most solicited aes resolved spontaneously. grade 3 solicited and unsolicited events occurred at a low rate and few unsolicited aes were considered related to vaccination (table 4 ). twenty saes were reported in 13 participants up to the study end, and none of them were assessed as vaccination related. nine pregnancies occurred during the study. one participant (exposed to the vaccine during the first trimester) underwent elective abortion, not related to the vaccination. for the other 8 pregnancies, the exposure to study vaccine occurred before the pregnancy; 6 gave birth to live neonates, and for 2 the outcome was unknown. one potentially immunemediated disorder assessed by the investigator as not related to vaccination-autoimmune thyroiditis-was reported 303 days after administration of the second dose of placebo. no deaths were reported during the study. for hematological and biochemical parameters, results outside of the normal laboratory range were evenly distributed across all time points (including baseline) and vaccine groups, and no clear clinical trends were observed (supplementary tables s3 and s4 ). the results of this phase i/ii randomized, placebo-controlled trial showed that 2 doses of the h7n9 as03-adjuvanted vaccine elicited a robust immune response in healthy adults up to 64 years of age, with an acceptable safety profile. adjuvantation with as03 enabled an immune response that satisfied regulatory acceptance criteria at antigen-sparing concentrations of ha, a prerequisite for a pandemic influenza vaccine. a dose as low as 2.8 µg ha elicited a robust hi antibody response. hi responses were low after the first vaccine dose in all vaccine groups, indicating that 2 doses are required to induce an adequate immune response. three weeks after the second vaccine dose, sprs and scrs against the vaccine-homologous virus were ≥85.7% in the adjuvanted vaccine groups. the non-adjuvanted vaccine elicited a poor immune response, and an adjuvant effect was demonstrated in all adjuvanted groups in terms of gmt ratio and scr difference. the gmt and mgi for the as03-adjuvanted vaccines against the vaccine-homologous virus were highest in the md ha/as03 a group, followed by the ld ha/as03 a group, the hd ha/as03 b group, and finally the ld ha/as03 b group. thus, the potency of the as03 adjuvant (11.86 or 5.93 mg tocopherol in as03 a or as03 b , respectively) seems to have more influence than antigen content on immunogenicity. this was also observed in a study of a h7n9 ha antigen produced by a different manufacturer mixed with gsk's as03 a at the point of use [14] and in a study of with the as03-adjuvanted h5n1 pandemic vaccine [15] . immune responses in older participants (aged 41-64 years) were generally lower than in younger participants (aged 18-40 years) and lower in participants who had previously received seasonal influenza vaccine than in nonrecipients, consistent with findings in other studies of pandemic influenza vaccines [13, 14, 16, 17] . up to 19.6% of study participants were seropositive before vaccination. detectable levels of hi antibody in the general population before vaccination with pandemic influenza vaccines have been reported in several studies [18] [19] [20] , suggesting either natural immune response to previous exposure or crossreactivity between virus strains. previous exposure is unlikely, because the h7n9 virus circulated in china and no infections in humans were reported in the north american population [21] . in addition, human antibody response to this strain has been shown to be very poor [22] . the same study suggests cross-reactivity of h1 and h3 seasonal influenza subtypes to the h7n9 virus, and this is the most likely explanation of our finding. the cd8 + t cells to seasonal influenza are reported to recognize h7n9 epitopes and to exhibit cross-reactivity with the h7n9 virus [23] . in our study, cross-reactivity could originate either from previous infection or seasonal vaccination. of note, a total of 218 study participants had received vaccination against influenza within the previous 3 seasons, although the virus strain to which the participants were previously exposed was not documented. the study shows that the as03 adjuvant enhances the immune response against h7 antigens at antigen-sparing doses. previous studies of h7 vaccines that were nonadjuvanted or adjuvanted with aluminum hydroxide have shown poor immunogenicity in humans [8, 9] . clinical studies of adjuvanted h7n9 vaccines from different manufacturers have found higher immunogenicity compared to nonadjuvanted formulations [11] [12] [13] [14] . a phase ii study of an h7n9 vaccine mixed at the point of use with mf59 adjuvant resulted in seroconversion in 59% of participants with the ld ha dose; higher antigen doses did not elicit an increase in immunogenicity [13] . a recent phase ii study compared different doses of an h7n9 vaccine mixed at the point of use with as03 a or mf59 or administered without an adjuvant [14] . as in the present study, the immune response with all formulations was low after 1 vaccine dose. after 2 doses, the immune response was superior with the as03-adjuvanted formulations compared with the mf59-adjuvanted or standard or high-dose nonadjuvanted formulations [14] . for as03-adjuvanted vaccines, similar immunogenicity was attained with antigen content varying between 3.75, 7, and 15 µg of ha [14] . the present study also demonstrated cross-reactivity of the vaccine against a vaccine-heterologous strain. phylogenetic analysis has shown a high degree of homology in the ha gene sequence of various h7 viruses [24] . an as03-adjuvanted h7n1 vaccine, engineered by reverse genetics from an h7n3 virus, has been developed by gsk vaccines, and therefore the present study evaluated cross-reactivity against an h7n1 vaccine-heterologous virus. robust cross-reactivity was seen, with sprs ranging from 57.7% to 76.9%. in preclinical studies in mice, an h7n1/as03 vaccine elicited antibodies cross-reacting with h7n9, h7n7, and h7n3 viruses [25] , and an h7n9 viruslike particle vaccine produced in insect cells using a baculovirus vector elicited antibodies cross-reacting with an h7n3 virus [26] . to our knowledge, this is the first report on the immune response against h7n9 in humans beyond day 42 after vaccination. based on the blood sampling schedule, hi antibody titers against the vaccine-homologous virus peaked at 21 days after the second dose. at day 182, a notable decrease in gmts was observed, although of different magnitude across groups. however, seropositive rates remained above 90% at day 182 and above 70% at day 385 in all adjuvanted groups, compared with 50.0% and 22.6% in the nonadjuvanted group and 4.5% and 1.9% in the placebo group. in addition, recent studies seem to suggest that a robust immune response to the ha head and stalk domains, as measured with enzyme-linked immunosorbent assay, may be induced even in the absence of hi and mn response [27] , so the clinical relevance of the decline in antibody titers is not clear. the observed kinetics of the immune response in adjuvanted groups in our study is similar to that elicited by other influenza vaccines, in particular the adjuvanted h5n1 vaccines [28] , for which a strong anamnestic response was elicited by a heterologous booster dose, up to 3 years after priming [29, 30] . the h7n9 as03-adjuvanted vaccine was generally well tolerated. pain was the most common solicited injection site symptom, as observed in other studies of adjuvanted influenza vaccines [12-17, 19, 31, 32] . tolerability seemed to be acceptable, as most participants returned for the second vaccine dose. most aes were low grade and resolved spontaneously. none of the saes was assessed as related to vaccination. in this study, a trend was observed for higher reactogenicity with as03 a -adjuvanted vaccines than with as03 b formulations. however, all formulations were well tolerated and no safety signal was identified during the study. the use of the as03 a adjuvant led to an improved immune response compared with that induced by as03 b -adjuvanted formulations, so the risk-benefit ratio in using a high potency adjuvant seems clinically acceptable. pandemic preparedness strategies include development of vaccines that are antigen sparing, because the time available to produce sufficient antigen to provide effective vaccine coverage is likely to be limited. in addition, vaccines that elicit a broad cross-reactive immune response are needed to allow pre-pandemic priming and use of prime-boost vaccination schedules. formulation of pandemic vaccines with an effective adjuvant is an accepted strategy to accomplish both goals [33] and to ensure adequate immunogenicity for the elderly and those with reduced immune responsiveness due to concurrent illness. the assessment of immunogenicity for this study is based on hi and mn assays; no additional evaluation such as cellmediated immunity was performed which could bring additional information but for which no regulatory acceptance criteria have been established. no formal comparisons between adjuvanted vaccine groups were performed, and analyses were descriptive. these could constitute potential limitations to the study. nevertheless, the type i error was adjusted for the evaluation of the primary immunogenicity objective, as well as the secondary objective related to adjuvant effect. in conclusion, 2 doses of an as03-adjuvanted h7n9 vaccine induce a robust anti-h7 immune response with an acceptable safety profile. when balancing antigen sparing with effective immunization, a 2.78-µg antigen dose (or 3.75 µg to align with the already licensed h5n1 vaccines made by the same process) with as03 a adjuvantation seems the most desirable formulation. this vaccine candidate could be beneficially deployed should the h7n9 virus acquire the ability for sustained human-to-human transmission or to protect persons at risk. supplementary materials are available at http://jid.oxfordjournals.org. consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be 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influenza vaccines: a new cornerstone of pandemic preparedness plans acknowledgments. we are indebted to the participating study volunteers, clinicians, nurses, and laboratory technicians at the study sites. we are grateful to the principal investigators, alan kravitz and jack yakish. we also thank janine linden for writing the study protocol; stephanie sharp (veristat on behalf of gsk vaccines) for writing the study report; thierry ollinger for contributing to immunological data generation; andré key: cord-338776-2wa30218 authors: zhao, xiaoyu; chu, hin; wong, bosco ho-yin; chiu, man chun; wang, dong; li, cun; liu, xiaojuan; yang, dong; poon, vincent kwok-man; cai, jianpiao; chan, jasper fuk-woo; to, kelvin kai-wang; zhou, jie; yuen, kwok-yung title: activation of c-type lectin receptor and (rig)-i-like receptors contributes to proinflammatory response in middle east respiratory syndrome coronavirus-infected macrophages date: 2020-02-15 journal: j infect dis doi: 10.1093/infdis/jiz483 sha: doc_id: 338776 cord_uid: 2wa30218 background: human infection with middle east respiratory syndrome coronavirus (mers-cov) poses an ongoing threat to public health worldwide. the studies of mers patients with severe disease and experimentally infected animals showed that robust viral replication and intensive proinflammatory response in lung tissues contribute to high pathogenicity of mers-cov. we sought to identify pattern recognition receptor (prr) signaling pathway(s) that mediates the inflammatory cascade in human macrophages upon mers-cov infection. methods: the potential signaling pathways were manipulated individually by pharmacological inhibition, small interfering ribonucleic acid (sirna) depletion, and antibody blocking. the mers-cov-induced proinflammatory response was evaluated by measuring the expression levels of key cytokines and/or chemokines. reverse transcription-quantitative polymerase chain reaction assay, flow cytometry analysis, and western blotting were applied to evaluate the activation of related prrs and engagement of adaptors. results: mers-cov replication significantly upregulated c-type lectin receptor (clr) macrophage-inducible ca(2+)-dependent lectin receptor (mincle). the role of mincle for mers-cov-triggered cytokine/chemokine induction was established based on the results of antibody blockage, sirna depletion of mincle and its adaptor spleen tyrosine kinase (syk), and syk pharmacological inhibition. the cytokine and/or chemokine induction was significantly attenuated by sirna depletion of retinoic acid-inducible-i-like receptors (rlr) or adaptor, indicating that rlr signaling also contributed to mers-cov-induced proinflammatory response. conclusions: the clr and rlr pathways are activated and contribute to the proinflammatory response in mers-cov-infected macrophages. middle east respiratory syndrome coronavirus (mers-cov) has been identified as a novel pathogen causing human respiratory infections since 2012 [1] . most mers patients presented with viral pneumonia, and some of them developed acute respiratory distress syndrome and multiorgan failure with a case-fatality rate over 30% [2] . cytological examination of bronchoalveolar lavage fluids from mers patients exhibited large numbers of neutrophils and macrophages, indicating a massive pulmonary inflammation [3] . the postmortem study of a mers patient revealed compatible pathological changes, including edematous alveolar septa with infiltration of lymphocytes, neutrophils, and macrophages, as well as diffuse alveolar damage [4] . a detailed examination of lung tissues of mers-cov-infected nonhuman primates including common marmosets and rhesus macaques revealed similar pathological changes, reminiscent of prominent lung disease in severe mers patients. thus, the authors reached a central conclusion that robust viral replication, together with an intense local immune response to mers-cov infection, may result in severe respiratory disease in these experimental animals [5] . macrophages are important sentinel cells of the innate immune system. macrophages sense and recognize invading pathogens or endogenous ligands through a broad range of sensors, and they generally elicit effective clearance via phagocytosis. on the other hand, macrophages may fail to eliminate invading microbes. instead, they become inappropriately activated and initiate dysregulated inflammatory responses in some cases [6, 7] . we have previously reported that mers-cov productively infected human monocyte-derived macrophages (mdms) and triggered aberrant proinflammatory response [8] , providing direct evidence to explain the massive inflammation observed in severe mers patients and experimentally infected nonhuman primates. upon viral infection, host germline-encoded pattern recognition receptors (prrs) including retinoic acid-inducible gene (rig)-i-like receptors (rlrs), toll-like receptors (tlrs), nucleotide-binding oligomerization domains-like receptors (nlrs), and c-type lectin receptors (clrs) detect the presence of foreign motifs or ligands known as pathogen-associated molecular patterns [9] [10] [11] [12] . the intracellular signaling cascades triggered by these prrs lead to transcriptional activation of type i interferons (ifns) and inflammatory mediators that coordinate the elimination of pathogens and infected cells and, meanwhile, contribute to the inflammation and clinical symptoms of viral infections. the rlrs, such as rig-i and melanoma differentiationassociated gene 5 (mda5), have been extensively characterized as essential cellular sensors to recognize rna viruses [11, 13] . ding et al [14] demonstrated the rlr-mediated signaling for nuclear factor (nf)-κb activation in transmissible gastroenteritis virus infection. zalinger et al [15] elucidated the importance of mda5 to host defense during murine cov infection. meanwhile, clr signaling has shown the increasing importance for triggering inflammatory response upon viral infections [16, 17] . the transcription factor nf-κb is an essential mediator of inducible gene expression of cytokines and chemokines. clr induced signal transduction seems to mainly activate and modulate nf-κb functions [17, 18] . the mechanisms by which covs evade host innate antiviral response, including mers-cov and severe acute respiratory syndrome (sars)-cov, have been extensively investigated [19] [20] [21] . however, it is poorly understood how the highly pathogenic mers-cov triggers the aberrant proinflammatory response, one of the pathological bases of severe respiratory diseases in mers patients and experimentally infected animals. in this study, we sought to elucidate the contribution of rlr and clr signaling pathway for mediating the exuberant inflammatory responses in macrophages upon mers-cov infection. the mers-cov of strain emc/2012 [1] was propagated in vero e6 cells. three days after virus inoculation, the cell-free media were collected and stored at −80°c in aliquots. ultraviolet (uv) inactivation of mers-cov was performed by exposing the virus to uv cross-linker for 10 minutes as described previously [22] . peripheral blood was obtained from healthy blood donors at the hong kong red cross blood transfusion center according to a protocol approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster. monocyte preparation and differentiation were performed according to a well established protocol described previously [23] . for viral infection, treated or mock-treated mdms were inoculated with mers-cov at a multiplicity of infection (moi) of 2 or were mock inoculated for 1 hour at 37°c. cell lysates were harvested at 24 hours postinfection (hpi) for quantification of messenger rna (mrna) expression levels of cellular genes and detection of viral load. tank-binding kinase 1 (tbk1) and inhibitor of nf-κb kinase epsilon (ikkε) inhibitor amlexanox, spleen tyrosine kinase (syk) inhibitor r406, and caspase-1 inhibitor vx-765 were purchased from invivogen. the individual inhibitors or a neutralization antibody against human mincle (invivogen, mabg-hmcl) or mouse monoclonal immunoglobulin (ig)g2b (mab004; r&d) were administered to mdms 1 hour before virus inoculation at the indicated concentrations and supplemented in the culture media throughout after infection. silencer select small interfering rna (sirna) targeting human mitochondrial antiviral-signaling proteins (mavs) (s33179; thermo fisher scientific), syk (s13681), rig-i (s223615), mda5 (s34499), mincle (s25297), and scrambled sirna were transfected to mdms using lipofectamine 3000 (thermo fisher scientific) according to the manufacturer's instruction. in brief, mdms were transfected with 200 nm sirna for 2 consecutive days. at day 3 after sirna transfection, the cells were inoculated with mers-cov. detection of cellular mrna expression and viral load was performed as described previously [24] . in brief, cell lysates were applied to rna extraction, followed by reverse transcription (rt) using oligo(dt) or the virus-specific primer. the resultant complementary deoxyribonucleic acids (cdnas) were used for quantitative polymerase chain reaction (qpcr) assay to measure mrna expression level of cellular gene and viral load. the primer sequences used in qpcr assay are shown in the supplementary table. flow cytometry analysis, immunofluorescence staining, and western blot immunostaining and flow cytometry analysis were performed according to the standard protocol as described elsewhere [25, 26] . in brief, after detachment, fixation, and permeabilization, mdms were labeled with a mouse antibody against human mincle (ab100846; abcam) and in-house made antibody against mers-cov np or antibody of isotype control, followed by the corresponding secondary antibodies. flow cytometry analysis was performed using a bd facscanto ii flow cytometer (bd biosciences), and the data were analyzed using flowjo vx (tree star). for immunofluorescence staining, mdms seeded on glass coverslips were inoculated with mers-cov at 5 moi. at 24 hpi, the cells were fixed with 4% paraformaldehyde and labeled with anti-np and anti-mincle, followed by corresponding secondary antibodies. slides were mounted with prolong gold antifade reagent with 4' ,6-diamidino-2-phenylindole (themo fisher scientific) and imaged with a carl zeiss lsm 800 confocal microscope. the whole-cell extracts of infected and mock-infected macrophages were separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. after blocking, the membranes were probed with antibodies against mavs (ab31334; abcam), syk (ab40781), rig-i (ab45428), mda5 (ab126630), mincle (ab100846), and card9 (ab124922), np antibody or mouse β-actin antibody (a5441; sigma), followed by secondary staining. the blots were visualized by luminata classico western hrp substrate (wbluc0500; millipore). quantification was performed using imagej software. a human ip-10 elisa kit (ab173194; abcam) was used for quantification of ip-10 secretion in the culture media. an unpaired t test was performed for data analysis using graphpad prism 6. p < .05 was considered to be statistically significant. data are presented as mean and standard deviation of representative experiments. we first used the inhibitors of rlr and clr signaling pathway to evaluate their potential contribution for mediating the proinflammatory response in mers-cov-infected macrophages. amlexanox, an inhibitor of rlr signaling pathway, specifically supresses the noncanonical ikb kinases ikkε and tbk1, and both are essential players for the coordination of ifn regulatory factor 3 (irf3)-and nf-κb-mediated innate immune response [27, 28] . r406 is a specific, atp-competitive inhibitor of syk, the essential adaptor of clr pathway [29] . caspase-1 inhibitor vx-765, a commonly used inhibitor for nlr signaling, is used for comparison [30] . mtt assay was used to detect the 50% cytotoxic concentration (cc 50 ) of each inhibitor in mdms (supplementary figure 1) , to ensure that the concentrations used for inhibition of prrs have no adverse effect on macrophage viability. based on the effective concentration of each inhibitor and its cc 50 , the indicated working concentrations ( figure 1a ) were used throughout the study. to assess the contribution of rlr or clr pathway to induce proinflammatory response, we measured the expression levels of a series of key proinflammatory cytokines and/or chemokines in mers-cov-infected mdms in the presence of these inhibitors ( figure 1b ). consistent with our previous observation, mers-cov infection globally stimulated an array of proinflammatory cytokines and chemokines including interleukin (il)-6, tumor necrosis factor (tnf)-α, macrophage-inflammatory protein (mip)-1α, rantes, ifn-γ, and ip-10 [8] . the induction of cytokine and/or chemokine was dependent on viral replication because the inoculation of the uv-inactivated viruses was unable to trigger these inflammatory mediators, except rantes (supplementary figure 2) . it is notable that mers-cov-mediated induction of tnf-α, mip-1α, ifn-γ, and ip-10 was generally diminished in the presence of amlexanox and r406, although amlexanox treatment only marginally reduced tnf-α production. in addition, the treatment of amlexanox, but not r406, significantly attenuated the induction of il-6 and rantes. however, nlr signaling inhibitor vx-765 seemed to have minimal effect on these inflammatory mediators. because the replication of mers-cov is the driving force for the induction of most cytokines and/or chemokines, we evaluated the viral replication in the presence of the inhibitors. it was shown that the addition of these inhibitors did not affect viral replication (supplementary figure 3) . accumulating evidence suggested that rlrs and clrs are inducible upon stimulation or microbial infection [14, [31] [32] [33] . among clrs, dectin-1, dectin-2, and mincle are highly expressed in myeloid cells such as monocytes, macrophages, and dendritic cells [17] . thus, we measured the mrna profiles of rlrs and myeloid clrs in mers-cov-infected macrophages. as shown in figure 2a , the mrna expression levels of rlrs including rig-i, mda5, and lpg2, as well as clrs mincle and dectin-2 were significantly upregulated at 24 hpi. the positive modulation pattern of rlr and clr, especially the increased expression of rig-i and mincle, was manifested as early as 6 hpi ( figure 2b ), suggesting an accelerating activation of these prrs during mers-cov infection. overall, the above results suggested that rlr and clr signaling might be involved in viral recognition and trigger the proinflammatory response upon mers-cov infection in macrophages. to dissect the role of rlr and clr signaling in inducing proinflammatory response, we depleted the rlr adaptor, mavs, or the clr adaptor, syk, by sirna knockdown and examined mers-cov-elicited cytokine and/or chemokine response. the effective depletion of both adaptors was shown by rt-qpcr assay at 48 hours post-sirna transfection, to approximately 20% for syk and 40% for mavs relative to the control cells ( figure 3a ). the reduced expression of the adaptors was also verified by western blot. subsequently, we infected the transfected cells with mers-cov and measured the expression profiles of the cytokines and/or chemokines. as shown in figure 3b , sirna depletion of either mavs or syk significantly diminished the induction of all tested inflammatory molecules including il-6, tnf-α, mip-1α, rantes, ifn-γ, and ip-10. rig-i, mda5, and mincle were upregulated the most among rlr and clr receptors ( figure 2 ). we then tested whether sirna depletion of rig-i, mda5, and mincle have any effect on mers-cov-triggered immune activation. as shown in figure 4a , the receptors were depleted to approximately 30% at transcriptional level, which were verified by western blot analysis. in rig-i-, mda5-, and mincledepleted cells, the induction of ifn-γ and ip-10 was generally abolished; and mip-1α and rantes expression were significantly reduced ( figure 4b) . a significantly attenuated il-6 induction was observed in rig-i-depleted cells, but not in mda5-or mincle-depleted cells; whereas tnf-α production was basically unaffected after depletion of these receptors individually. the induction of ifn-γ and ip-10 was constantly and significantly reduced in various assays, including the inhibitor treatment and sirna depletion. in addition, these 2 proinflammatory mediators are highly stimulated in severe sars and mers patients [34] [35] [36] . thus, we examined mers-cov-induced secretion of ifn-γ and ip-10 after genetic depletion of the above-mentioned rlr and clr receptors and adaptors. ip-10 secretion was readily detectable from both mers-cov-infected and mock-infected mdms; whereas ifn-γ secretion was below the detection limit, even in the virus-infected cells. thus, we examined mers-cov-induced ip-10 secretion in mdms upon sirna transfection. as shown in figure 4c , genetic depletion of rig-i, or syk and mincle resulted in a signification reduction of ip-10 secretion. knockdown of rig-i adaptor mavs also marginally attenuated ip-10 production. taken together, we found that disrupting rlr or clr signaling, especially at the adaptor level, largely dampened the production of mers-cov-induced proinflammatory cytokines and chemokines. the interplay of clr signaling and human covs has not been appreciated yet. to further characterize the role of mincle for immune activation in mers-cov-infected macrophages, we measured the mers-cov-related ifn-γ induction in the presence of increasing concentrations of a neutralization antibody against mincle. the results showed that the addition of α-mincle significantly suppressed mers-cov-triggered ifn-γ induction in a dose-dependent manner ( figure 5a ). in addition, treatment of α-mincle significantly diminished the induction of il-6, rantes, ifn-γ, and ip-10 ( figure 5b ). however, the inhibitory effect was negligible for tnf-α and mip-1α. taken together, in line with the observations from mincle sirna depletion, clr receptor mincle contributed to the immune activation in mers-cov-infected macrophages. to characterize the engagement of mincle in mers-cov-infected cells, we assessed mincle protein expression by flow cytometry analysis and immunofluorescence staining. as shown in figure 6a , in the mock-infected cells, approximately 50% of macrophages were mincle + , indicating that mincle is indeed highly expressed in macrophages as reported previously [17] . the percentage of np + (infected) cells increased from 2.9% at 3 hpi to 37.8% at 24 hpi, indicating a productive viral replication. we then compared mincle expression in np + cells versus that in np − cells at 24 hpi. in np − cells, mincle protein expression, ie, the percentage of mincle-expressing cells and the abundance of mincle protein (mean fluorescence intensity), remained similar in the np − cells and the mock-infected cells. it is notable that mincle protein expression was significantly elevated in the np + cells ( figure 6b ). mincle upregulation in the virus-infected cells was verified by costaining of mincle and np. the np + macrophages displayed the substantially elevated mincle expression ( figure 6c ). therefore, the results suggested that mers-cov replication significantly enhanced mincle expression in macrophages. engagement of adaptor syk with clrs, via card9, activates mitogen-activated protein kinases and transcription factor nf-kb and results in the induction of proinflammatory cytokines [9] . card9 activation is not only mediated by phosphorylation and ubiquitination processes, but it is also enabled by upregulation [37] . r406 had a negligible effect on card9 expression in the naive macrophages. however, card9 was significantly upregulated in mers-cov-infected macrophages. in contrast to the mockinfected cells, inhibition of clr signaling by r406 significantly suppressed the degree of card9 activation. thus, the results revealed the importance of card9 as an essential adaptor to relay clr signaling in mers-cov-infected macrophages. mers-cov, the most virulent human cov identified so far, has posed an ongoing threat to public health worldwide. it has been recognized that the rapid viral replication in lung tissues, massive inflammation, and elevated proinflammatory cytokine and chemokine response collectively contribute to acute lung injury and underlie the high pathogenicity of this life-threatening human cov [2, 5, 34, 38] . the postmortem examination of mers-cov patient's lung tissues, which were obtained by needle sampling shortly after death, revealed severe pneumonia with diffuse alveolar damage and mixed inflammatory cell infiltration. immunostaining revealed numerous intra-alveolar macrophages and infiltrating t lymphocytes in the parenchyma [4] , which are consistent with the histopathological changes in lung tissues of mers-covinfected marmosets. it is notable that profound macrophage proliferation was the most prominent pathological change in the postmortem examination of lung tissues of fatal sars patients [39] . it was postulated that the proinflammatory cytokines and chemokines released from the proliferative alveolar macrophages play a prominent role in the pathogenesis of sars [40] [41] [42] . we have reported that mers-cov can productively infect human macrophages and trigger exuberant proinflammatory response [8] . it is conceivable that the proliferating macrophages in lung tissue of a mers patient and the dysregulated immune response in these cells may contribute significantly to the high pathogenicity of mers-cov. in this study, we sought to identify the cellular signaling pathway(s) that mediate the proinflammatory response in mers-cov-infected human macrophages. the initial observations from the effect of the pathway inhibitors and profiling of receptor activation hinted at the possible involvement of rlr and clr signaling in mers-cov-induced immune activation in macrophages. we then performed a series of experiments to characterize the role of rlr and clr, especially clr, in mers-cov-infected macrophages, with the intention to better understand the pathogenesis of human mers. the role of rlr and clr for mers-cov for eliciting immune activation was noted upon genetic depletion of the rlr adaptor mavs or the clr adaptor syk. the genetic depletion generally compromised or abolished the induction of cytokines and/or chemokines. the attenuated production of most proinflammatory mediators including mip-1α, rantes, ifn-γ, and ip-10 was replicated when rlr receptors (rig-i or mda5) or clr receptor (mincle) were depleted individually. however, the dampened inflammatory response was more prominent in adaptor depletion than receptor depletion, which could be ascribed to the fact that the activation signals from multiple receptors (eg, rig-i and mda5) converge at 1 adaptor (eg, mavs). upon mers-cov infection, multiple prrs were activated in the macrophages ( figure 2 ). therefore, depleting individual receptors was unable to entirely block the activation signals. the rlrs are ubiquitously expressed in many cell types and are among the first sensors to detect many viral infections. they are localized in the cytoplasm and recognize the genomic rna of double-stranded rna (dsrna) viruses and dsrna generated as the replication intermediate of single-stranded rna viruses. the rig-i and mda5 are inducible upon ifn treatment or virus infection. the activated rig-i or mda5 interacts with an essential rlr adaptor, mavs, initiating a signaling cascade that includes the activation of tbk1 and ikkε kinases, which subsequently phosphorylate and activate the transcription factor irf3 and nf-κb. in this study, we demonstrate that rig-i or mda5 were both induced in macrophages upon mers-cov infection. the engagement of the receptors and subsequent signaling transduction were manifested in sirna depletion of both receptors and their adaptor mavs, as well as tbk1/ikkε inhibition. thus, rlrs indeed recognize replicating mers-cov and mediate the proinflammation response in macrophages. recent studies have identified clrs as an important family of prrs that are involved in the induction of inflammation response to specific pathogens. dectin-1, dectin-2, and mincle are members of a family of c-type lectins that are typically expressed by dendritic cells and macrophages [17] . mincle is known to associate noncovalently with the adaptor molecule fcrγ. the phosphorylation of the adaptor serves to recruit syk and induces downstream nf-κb activation through the syk-card9-bcl10-malt1 complex. the in vivo genetic studies have revealed an essential role of clr-triggered card9 signaling in host protection. card9-deficient mice exhibited deregulated innate responses and failed to control infection of mycobacterium tuberculosis [43] . in addition, mincle was identified as a lipopolysaccharide-inducible protein in mouse macrophages and was transcriptionally upregulated after exposed to various stimuli and cellular stress [44] . in human monocytes, fungus infection elevated mincle expression [31] . in this study, we demonstrated that mers-cov replication upregulated mincle expression. the antibody blockage of mincle, sirna depletion of mincle, syk pharmacological inhibition, and card9 activation invariably highlighted the role of mincle for mediating the proinflammatory cascade in mers-cov-infected macrophages. in terms of cellular inflammatory response, 4 major family of prrs, including tlrs, rlrs, clrs, and nlrs, share overlapping ligand specificities and converge on common downstream pathways. the cooperative detection by different prrs has been reported in other dna and rna viruses [33, 45] and bacteria [46] . in rhinovirus-infected bronchial epithelial cells, the innate immune response requires a coordinated recognition, initially via tlr3 or trif and followed by rna helicases rig-i and mda5 [33] . our results indicate that clr and rlr signaling may be involved in mediating the immune activation in mers-cov-infected human macrophages. however, the possible crosstalk or coordination of different pathways has yet to be investigated. taken together, our study has advanced current knowledge of the recognition of human covs by cellular prrs. in agreement with previous literature on other human covs, our data revealed the important role of rlrs in mediating the proinflammatory response upon mers-cov infection. more important, our study further investigated the involvement of clrs in sensing mers-cov and provided the first evidence that clrs contributed to the recognition of mers-cov. in addition, we demonstrated that a significant proportion of mers-cov-triggered proinflammatory response was governed by mincle. in this regard, clrs may play an important role in modulating the pathogenesis of mers-cov. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. supplementary figure 1 . determination of 50% cytotoxic concentration (cc 50 ) of 3 pattern recognition receptor (prr) pathway inhibitors. monocyte-derived macrophages (mdms) were treated with the indicated concentrations of inhibitors and incubated at 37°c for 24 hours. the cells were then applied to mtt assay. the results are used for calculation of the cc 50 of each inhibitor. data are presented as mean ± standard deviation (sd) of triplicate wells of mdms from 3 different donors. isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study an acute immune response to middle east respiratory syndrome coronavirus replication contributes to viral pathogenicity protective and pathogenic functions of macrophage subsets disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in crohn's disease active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis signaling in innate immunity and inflammation pattern recognition receptors and inflammation differential roles of mda5 and rig-i helicases in the 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death by pyroptosis drives cd4 t-cell depletion in hiv-1 infection mincle polarizes human monocyte and neutrophil responses to candida albicans murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda5 co-ordinated role of tlr3, rig-i and mda5 in the innate response to rhinovirus in bronchial epithelium pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology an interferon-gammarelated cytokine storm in sars patients comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity regulation of dectin-1-mediated dendritic cell activation by peroxisome proliferator-activated receptor-gamma ligand troglitazone middle east respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis lung pathology of fatal severe acute respiratory syndrome immunopathogenesis of coronavirus infections: implications for sars multiple organ infection and the pathogenesis of sars dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice the adaptor molecule card9 is essential for tuberculosis control a novel lpsinducible c-type lectin is a transcriptional target of nf-il6 in macrophages innate immune sensing of modified vaccinia virus ankara (mva) is mediated by tlr2-tlr6, mda-5 and the nalp3 inflammasome mycobacterium paratuberculosis is recognized by toll-like receptors and nod2 supplementary figure 2 . inactivated middle east respiratory syndrome coronavirus (mers-cov) was unable to induce proinflammatory cytokines and chemokines. ultraviolet (uv) inactivation was performed by exposing the viruses to uv crosslinker for 10 minutes at 37°c. the inactivation was confirmed after inoculation in vero e6 cells. monocyte-derived macrophages (mdms) were infected with mers-cov or the uv-inactivated mers-cov at a multiplicity of infection (moi) of 2 or were mock infected. at 24 hours postinoculation, cells were harvested for detecting the expression levels of cytokines and chemokines by reverse transcription-quantitative polymerase chain reaction (rt-qpcr) assay. data are presented as mean ± standard deviation (sd) of mdms from 3 different donors.supplementary key: cord-342996-honeavwj authors: mair-jenkins, john; saavedra-campos, maria; baillie, j. kenneth; cleary, paul; khaw, fu-meng; lim, wei shen; makki, sophia; rooney, kevin d.; beck, charles r.; nguyen-van-tam, jonathan s. title: the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis date: 2015-01-01 journal: j infect dis doi: 10.1093/infdis/jiu396 sha: doc_id: 342996 cord_uid: honeavwj background. administration of convalescent plasma, serum, or hyperimmune immunoglobulin may be of clinical benefit for treatment of severe acute respiratory infections (saris) of viral etiology. we conducted a systematic review and exploratory meta-analysis to assess the overall evidence. methods. healthcare databases and sources of grey literature were searched in july 2013. all records were screened against the protocol eligibility criteria, using a 3-stage process. data extraction and risk of bias assessments were undertaken. results. we identified 32 studies of sars coronavirus infection and severe influenza. narrative analyses revealed consistent evidence for a reduction in mortality, especially when convalescent plasma is administered early after symptom onset. exploratory post hoc meta-analysis showed a statistically significant reduction in the pooled odds of mortality following treatment, compared with placebo or no therapy (odds ratio, 0.25; 95% confidence interval, .14–.45; i(2) = 0%). studies were commonly of low or very low quality, lacked control groups, and at moderate or high risk of bias. sources of clinical and methodological heterogeneity were identified. conclusions. convalescent plasma may reduce mortality and appears safe. this therapy should be studied within the context of a well-designed clinical trial or other formal evaluation, including for treatment of middle east respiratory syndrome coronavirus cov infection. as of 23 may 2014, the world health organization (who) had been informed of 635 persons with laboratory-confirmed middle east respiratory syndrome coronavirus (mers-cov) infection, of whom 193 (30%) have died [1] . the current approach to clinical management of mers-cov infection centers on general supportive care, with provision of critical care and organ support when necessary [2] . it has recently been suggested that administration of convalescent plasma or hyperimmune immunoglobulin will yield a clinical effect for treatment of mers-cov infection [3] . however, numerous uncertainties remain because the clinical course, viral replication kinetics, and host interactions are yet to be fully established [4] . furthermore, the underlying evidence is based on studies of varying size and quality that describe clinical experience in treating other viral infections, including those due to sars coronavirus (sars-cov), spanish influenza a(h1n1), avian influenza a(h5n1), and 2009 pandemic influenza a (h1n1) (hereafter, "influenza a[h1n1]pdm09") [5] [6] [7] [8] [9] . we conducted a systematic review and exploratory meta-analysis to evaluate the clinical effectiveness of convalescent plasma, serum, or hyperimmune immunoglobulin for the treatment of severe acute respiratory infections (saris) of viral etiology, to help inform clinical management of mers-cov infection. this systematic review was conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines [10] . the study protocol was registered with the national institute for health research international prospective register of systematic reviews [11] . the study eligibility criteria are available elsewhere [11] . briefly, the study population of interest was human subjects of any age or sex who were hospitalized with saris with a laboratory-confirmed or suspected viral etiology. the intervention of interest was convalescent plasma, serum, or hyperimmune immunoglobulin derived from convalescent plasma. comparator treatments included placebo, sham therapy, or no intervention; studies with no comparator group were also included. outcome measures were derived from the protocol research questions to ascertain the clinical effectiveness of therapy [11] . two reviewers (j. m.-j. and m. s.-c.) executed the search strategy in july 2013. the sources of information searched and search construct are available elsewhere [11] . adaptations were made for search interfaces that did not allow use of complex constructs. all search records were imported to endnote x5 software (thomson reuters, san francisco, ca) or screened manually, using paper records. following the removal of duplicate entries, a 3-stage screening process was followed to identify eligible records through the sequential examination of each title, abstract, and full text. two reviewers (j. m.-j. and m. s.-c.) screened each record, with provision for arbitration from a third reviewer (c. r. b.). data were collected independently by paired reviewers, using a piloted form. consensus agreement for each extracted data item was reached by discussion, with provision for arbitration from a third reviewer (j. m.-j., m. s.-c., and c. r. b.). the data extraction form is available as an appendix to the study protocol [11] . risk of bias assessments were performed at the outcome measure level during data collection. the cochrane collaboration tool was used for experimental and prospective cohort studies [12] , the newcastle-ottawa scale was used for observational studies (excluding prospective cohort studies) [13] , and a tool published by the us agency for healthcare research and quality was used for systematic reviews [14] . records limited to abstracts were not assessed, because of the paucity of information contained therein. odds ratios (ors), case-fatality rates (cfrs), absolute differences in cfrs, and difference in means were calculated as summary statistics with 95% confidence intervals (cis). study characteristics and outcome measures were tabulated. a recognized framework for narrative synthesis was adopted [15] . because of potential concerns with clinical heterogeneity, analyses were stratified by viral etiology for each research question in accordance with the protocol [11] . an exploratory, post hoc, random-effects-model metaanalysis was conducted to describe the pooled or of mortality, irrespective of sari etiology, following treatment with convalescent plasma or serum, using the odds after receipt of placebo or no therapy as a reference. results were adjusted by adding 0.5 to each cell of the contingency table when no deaths occurred in the exposed group in individual studies [12] . meta-analysis of crude cfrs, using a random-effects model, was undertaken. statistical heterogeneity was ascertained using the i 2 statistic, and meta-analyses were abandoned when this reached 85% [16] . sensitivity analyses were undertaken to investigate the impact of excluding studies with ≤5 patients in the exposed group. publication bias was assessed through construction of funnel plots and by use of the egger test. all statistical analyses were conducted using stata software, version 12.1 (statacorp, college station, tx), except for meta-analysis of pooled proportions, for which we used statsdirect software, version 2.8.0 (statsdirect, altrincham, united kingdom). statistical significance was assumed at the 5% level. the search process yielded 3406 records ( figure 1 ). after sifting 1449 unique records against the protocol eligibility criteria, we identified 32 studies from 50 reports (supplementary table 1 ). three studies could not be obtained [17] [18] [19] , although results from a study by bass et al [17] were reported elsewhere [20] , which enabled their inclusion. french (n = 1), german (n = 2), and korean (n = 2) records were screened by single reviewers because of a lack of multilingual collaborators. the study characteristics are summarized in supplementary table 2 . three systematic reviews met our protocol eligibility criteria [7, 21, 22] . data on 1327 patients from 6 case studies [23] [24] [25] [26] [27] [28] , 20 case series [8, 17, 20, [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] , 2 case-comparison studies [46, 47] , and 1 prospective cohort [48] were included. we identified 13 observational studies published between 1918 and 1920, which studied 980 patients who received a clinical diagnosis of influenza-associated pneumonia or spanish influenza a (h1n1) infection [17, 20, 33-35, 38-44, 47] . it is unclear whether some of these studies recruited patients with secondary bacterial pneumonia. sixteen observational studies that met our protocol eligibility criteria were published between 2003 and 2011. four studies reported outcomes for 29 patients infected with avian influenza a(h5n1) [23, 26, 27, 36] , 4 studies reported outcomes for 104 patients infected with influenza a (h1n1)pdm09 [24, 30, 37, 48] , and 8 studies reported outcomes for 214 patients with sars [8, 25, 28, 29, 31, 32, 45, 46] . the clinical status of patients at the time of treatment administration varied, as did concomitant treatments and comorbidities. convalescent plasma was used in all observational studies of sars-cov, influenza a(h1n1)pdm09, and avian influenza a(h5n1) infections (supplementary table 2 ). for spanish influenza a(h1n1) infection, 2 observational studies used convalescent plasma, and 11 used convalescent serum (supplementary table 2 ). no studies that used hyperimmune immunoglobulin met our protocol eligibility criteria. the use of sham treatments or placebos was not reported. two systematic reviews were at low risk of bias [7, 21] , whereas one was at moderate to low risk of bias across most domains ( table 1 ) [22] . data extraction was judged to be a moderate source of bias in all systematic reviews. search strategies were also a moderate source of bias in 2 systematic reviews, as grey literature and non-peer-reviewed sources were not considered [7, 22] . the risks of bias of 2 outcomes in a single prospective cohort study were considered to be moderate ( table 2 ) [48] . the lack of randomized treatment allocation may have introduced systematic error, and the viral load outcome was at high risk of bias because of incomplete follow-up of patients. figure 2 summarizes the risk of bias assessments for 44 outcomes from 25 observational studies. studies reported outcomes that were either at moderate risk (11 outcomes) or moderate to high risk of selection bias (33 outcomes). the majority of studies lacked a comparator group, and 28 studies were at high or very high risk of reporting bias. this suggests that the observational study data included are at moderate to high risk of bias. three studies were not assessed for risk of bias, because they presented insufficient data [17, 29, 45] . table 3 summarizes our narrative synthesis, and supplementary table 3 shows results of the individual studies that included an all-cause mortality outcome. meta-analyses, sensitivity analyses, and assessments of publication bias, by viral etiology, proved unfeasible due to a paucity of suitable data. there were no data available to address study questions relating to organ failure and sepsis or to hospital readmission and recurrence of severe disease. table 3 and supplementary 3 summarize 8 observational studies at moderate to high risk of bias that reported improved mortality after patients received various doses of convalescent plasma [8, 25, 28, 29, 31, 32, 45, 46] . a retrospective case-comparison study showed a cfr reduction after plasma treatment that reached statistical significance (absolute reduction in cfr, 23%; 95% ci, 6%-42%; p = .049) [46] . a second study with a comparator group described a cluster of 29 cases of sars-cov infection in which 1 patient received convalescent plasma and survived (absolute reduction in cfr, 7%; 95% ci, −2% to 17%; p = .93) [32, 49] . three small studies reported treatment of 5 patients with no deaths, and a case series by cheng et al reported a cfr of 12.5% (10 of 80 patients) following treatment (supplementary table 3 ) [8, 9, 25, 28, 31, 45, 50] . within this series, a subgroup analysis of 30 patients found that those treated when pcr-positive but seronegative for sars-cov were more likely to be discharged within 22 days of admission than those who were seropositive at the time of plasma infusion (67% vs 20%; p = .001). a further subgroup analysis of 48 patients found that receipt of convalescent plasma treatment <14 days after onset of symptoms improved the likelihood of discharge within 22 days of admission (58% vs 16%; p < .001); this remained significant after adjustment for age, viral status, time of administration, and lactate dehydrogenase level, suggesting that early treatment with convalescent plasma may be beneficial. however, allocation of treatment was mostly based on the physician's decision and the availability of plasma, and this study was at high risk of bias. four observational studies [24, 30, 37, 48] and 1 systematic review [22] reported data on severe cases of influenza a(h1n1)pdm09 infection treated with convalescent plasma (table 3 and supplementary table 3 ). hung et al [48] performed a prospective cohort study in which patients received a single 500-ml dose of convalescent plasma with a neutralizing antibody titer of >1:160. univariate analysis showed a significant absolute reduction in cfr of 35% (95% ci, 14%-56%; p = .01) after treatment. multivariable analysis also showed a significant reduction in the relative risk of mortality (or, 0.20; 95% ci, .06-.69; p = .011), although the factors adjusted for were not clearly stated. both groups received other treatments, such as neuraminidase inhibitors and steroids (supplementary table 2 ). this nonrandomized study was at moderate risk of bias. a small study by chan et al [30] at moderate risk of bias reported exclusively on patients who received extracorporeal membrane oxygenation (ecmo) and showed a nonsignificant absolute reduction of 33% (95% ci, −20% to 87%) in the cfr after convalescent plasma treatment. in a case series at high risk of bias, in which 2 of 26 patients receiving convalescent plasma, a nonsignificant absolute reduction of 70% (95% ci, 52%-89%; p = .11) in the cfr was observed (supplementary table 3 ) [36] . three case reports reported recovery among patients who were treated with convalescent plasma [23, 26, 27] . the dose of convalescent plasma varied across each study, and the neutralizing antibody titer was reported for only 1 case (1:80) [26] . all studies were at high to moderate risk of bias and had patients who were given other therapies concomitantly (including steroids and antivirals), which could have influenced the reported clinical effect. a systematic review and meta-analysis by luke et al [21] showed that treatment with convalescent plasma, serum, or blood was associated with a significant absolute reduction of 21% (95% ci, 15%-27%) in the pooled cfr. statistical heterogeneity was low (i 2 = 29.3%), although interventions were clinically heterogeneous. of the 6 studies included in the meta-analysis, 2 reported use of convalescent whole blood; however, these studies only contributed 84 patients (25%) in the treatment group. when timing of treatment was recorded, patients who received early treatment (<4 days from pneumonia onset) had a cfr of 19% (28 of 148), compared with 59% (49 of 83) for those treated later [21] . only 2 studies of convalescent serum reported a comparator group [38, 47] . both reported absolute reductions in cfr after treatment, with a reduction of 19% (95% ci, 11%-48%) in one and 22% (95% ci, 11%-32%) in the other; the reduction in the latter reached statistical significance (p = .008). the remaining studies observed a cfr ranging from 0% (0 of 2) to 48% (12 of 25) after treatment (supplementary table 3) . a significant absolute reduction in the cfr was observed in a case series of 157 cases, 46 of whom received convalescent plasma (absolute reduction in the cfr, 18%; 95% ci, 8% to 30%; p = .0075) [33] . a further study of patients treated with convalescent plasma reported a cfr of 50% (7 of 14) [41] . the majority of studies on spanish influenza a(h1n1) infection were found to have high risk of bias due to the use of now archaic research methods and a risk of wartime censorship and publication bias [21] . the post hoc meta-analysis evaluated pooled data from 8 comparative studies: 2 studies of sars-cov infection [32, 46] , 2 of influenza a(h1n1)pdm09 infection [30, 48] , 1 of avian influenza a(h5n1) infection [36] , and 3 of spanish influenza a(h1n1) infection [33, 38, 47] . there was a statistically significance lower risk of mortality in the group treated with convalescent plasma or serum ( pooled or, 0.25; 95% ci, .14 to .45; p < .001; i 2 = 0%; figure 3 ). examination of the funnel plot and findings of the egger test showed no evidence of publication bias. sensitivity analyses that excluded studies with ≤5 cases demonstrated little variation in the pooled or or change in statistical heterogeneity (figure 4) . meta-analysis of the crude cfr in treated patients was rejected due to excessive statistical heterogeneity (i 2 = 85%). sensitivity analysis that excluded studies with ≤5 cases did not account for this and was similarly abandoned (i 2 = 91%). convalescent plasma treatment was associated with a significant increase in the proportion of sars-cov-infected patients discharged within 22 days of admission in 1 center (absolute difference, 54%; 95% ci, 25%-85%; p = .004) after excluding patients with comorbidities from the analysis (table 3 ) [46] . a further sars-cov infection case series [31] reported that 47% of patients (15 of 33) were discharged by day 22, and initiation of therapy was significantly earlier among patients discharged by that time (mean number of days from symptom onset, 11.67 vs 16.04; p < .001). both studies were at moderate to high risk of selection bias and confounding by indication. a case-comparison study at moderate risk of bias [30] reported no significant difference in length of hospital stay between treatment and control patients with severe pandemic influenza a (h1n1) infection who required ecmo ( table 3) . a retrospective observational study [30] reported that convalescent plasma treatment made nonsignificant reductions to the length of time spent in the intensive care unit, days of no data were reported in identified studies. significantly lower viral load after treatment was observed at days 3, 5, and 7 after icu admission in subgroup analysis of 1 prospective study, which was at moderate to high risk of bias. one noncomparative study found a reduction in viral load after treatment. no adverse events or complications were reported after treatment. nonsignificant benefits following intervention were reported in 1 study with comparator data. three case reports reported no deaths. no comparative data were reported. the length of hospital stay was 94 d in a case report at high risk of bias. no comparative data were reported. one case report, which had a high risk of bias, cited that treatment allowed discontinuation of mechanical ventilation. specific antibodies were detected between day 7 and day 16 after treatment in a case report at high risk of bias. no comparative data were reported. three studies reported reductions in viral load after treatment. no adverse events or complications were reported after treatment. a pooled absolute reduction of 21% (95% ci, 15%-27%)in the cfr was reported by a meta-analysis at low risk of bias. this pooled 6 studies, including 2 studies using convalescent blood. subgroup analyses suggested that early treatment was beneficial. the absolute reduction in the risk of mortality ranged from 18.66% (95% ci, 10.62%-47.95%) to 21.60% (95% ci, 11.2%-31.93%) in 3 studies at high risk of bias. ten noncomparative studies found that the cfr varied from 0% (0/2) to 50% (7/14) . no data were reported in identified studies. no data were reported in identified studies. no data were reported in identified studies. no data were reported in identified studies. three studies reported chills, increased temperature, and sweats after infusion. abbreviations: cfr, case-fatality rate; ci, confidence interval; ecmo, extracorporeal membrane oxygenation; icu, intensive care unit; influenza a(h1n1)pdm09, 2009 pandemic influenza a(h1n1); sars-cov, severe acute respiratory syndrome coronavirus. a all studies reported use of convalescent plasma, except 11 studies, in which convalescent serum was used to treat spanish influenza a(h1n1) infection, and 1 meta-analysis of 6 studies, 2 of which reported use of convalescent blood to treat spanish influenza a(h1n1) infection. additional data pertaining to individual studies (including comparator data, where presented) are available in the supplementary materials. mechanical ventilation, or number of days of ecmo for 6 patients with severe pandemic influenza a (h1n1)pdm09 infection (table 3) . two other case reports of pandemic influenza a (h1n1)pdm09 infection [24] and avian influenza a(h5n1) infection [27] also suggested that convalescent plasma may have aided clinical improvement and reduced the duration of mechanical ventilation. we identified limited evidence relating to levels of viral antibodies after convalescent plasma treatment; studies did not use a comparator and were at high risk of bias. peaks in sars-cov antibody levels occurred 3-5 days following receipt of a single dose of convalescent plasma in 3 healthcare workers (table 3 ) [8] . however, it is likely that other treatments, such as intravenous immunoglobulin, ribavirin and steroids, may have influenced the relationship between plasma and antibody levels. a case report of a patient with avian influenza a(h5n1) infection also found that virus-specific antibodies appeared 7-16 days following administration of convalescent plasma [23] . the sars-cov load in the respiratory tract decreased at a higher rate in patients who received convalescent plasma in a subgroup analysis of 44 patients with influenza a(h1n1)pdm09 infection in a prospective cohort study (table 3) ; [48] viral loads were significantly lower 3, 5, and 7 days after intensive care unit admission. however, there was a high risk of selection bias for this outcome, and concomitant treatments, including oseltamivir, zanamivir, and corticosteroids, may have confounded the results. further studies reported that viral load became rapidly undetectable in the blood of 3 patients with sars-cov infection [8] and in respiratory tract specimens from a patient infected with influenza a(h1n1)pdm09 [24] after treatment. similar decreases in viral loads in serum and respiratory tract specimens were observed in 3 cases of avian influenza a(h5n1) infection, with virus becoming undetectable 2-3 days after initiation of convalescent plasma treatment for 2 cases and 7-16 days after treatment initiation for the third case (supplementary table 3 ) [23, 26, 36] . no studies reported a serious adverse event, and few studies reported information about treatment complications, although minor complications may be underreported in the literature. two observational studies [8, 46] concerned with sars-cov infection reported that treatments did not cause harm when administered to patients. one study involving influenza a(h1n1) pdm09 infection reported that no adverse events were observed in the treatment group [48] . three studies from 1918-1920 (involving 101-157 patients with influenza) reported minor infusion complications, including chills, increased temperature [34, 44] , and sweats [33] . a study of 14 patients did not report chills or any serious complications. the methods and reporting of these studies reflect the period during which they were conducted, and the studies are therefore at high risk of bias. our analyses suggest that convalescent plasma may have a clinically relevant impact in reducing the rate of mortality and viral load in patients with sari of viral etiology. post hoc pooled meta-analysis across all viral etiologies showed a statistically significant 75% reduction in the odds of mortality among those who were treated with convalescent plasma or serum. we found no evidence of serious adverse events or complications due to therapy and limited evidence of a reduction in the use of critical care resources and the length of hospital stay. of interest is the evidence for a survival benefit after early administration. a recent multicenter, prospective, double-blind, randomized control trial compared the use of hyperimmune immunoglobulin (derived from influenza a(h1n1)pdm09 convalescent plasma) to intravenous immunoglobulin manufactured before the 2009 pandemic [5] . for 22 patients from this study who received treatment within 5 days of symptom onset and were excluded per protocol, a multivariate subgroup analysis demonstrated that hyperimmune immunoglobulin had a protective effect (or, 0.14; 95% ci, .02-.92) [5] . evidence from studies of sars-cov infection [31] and spanish influenza a(h1n1) infection [21] showed a survival benefit following convalescent plasma treatment within 14 days and 4 days of symptom onset, respectively. these findings suggest that early initiation of treatment may be of critical importance to reducing mortality in patients with sari of viral etiology. a lack of high-quality studies and a paucity in the volume of relevant literature limited our analyses. observational studies were predominately case reports or series, had no control groups, and had a moderate to high risk of bias. findings were commonly at high risk of confounding by indication. although selection or reporting bias may favor the intervention, recruiting patients who are clinically deteriorating or moribund would bias the result in the opposite direction. adequate methodological or statistical measures were infrequently used to control bias and confounding, and we identified numerous sources of clinical and methodological heterogeneity. we cannot be assured that all spanish influenza a(h1n1) infection studies were included since our protocol did not include hand searching of literature from 1918-1920. although our post hoc metaanalyses were undertaken to help inform clinical decision making, the theoretical rationale for pooling mortality data from different viral etiologies remains to be fully established. the results obtained must be considered experimental and interpreted with an appropriate level of caution. we did not identify any reports of convalescent plasma use for patients with mers-cov infection. the evidence for a reduction in mortality associated with convalescent plasma is strongest for sars and influenza a(h1n1)pdm09 infection. although it is clinically rational to consider novel therapies for critically ill patients, there is evidence that maximum benefit from convalescent plasma might be realized through early initiation of therapy. however, many treatment protocols currently mention convalescent plasma as a treatment of last resort. if this treatment is considered for mers-cov-infected patients with sari, it should ideally only be administered in acute centers able to manage potential treatment-related complications, such as transfusionrelated acute lung injury. we consider this a precautionary approach because of the limited clinical experience of administering convalescent plasma to this patient group. improved knowledge regarding the mode of action of convalescent plasma and the virologic and immunologic kinetics of novel respiratory infections that cause sari (such as mers-cov) are needed. this would help clarify the potential benefits and harms of treatment, identify optimal dosage, and ascertain whether repeated treatments are relevant factors for clinical practice. randomized controlled trials or observational studies that adopt a standardized minimum data set are needed to better evaluate convalescent plasma as a therapeutic option for mers-cov infection before it can be fully recommended or before refinements can be made over its current use, other than our current recommendation for early use. the who and the international severe acute respiratory and emerging infection consortium are currently developing a clinical trial protocol to investigate the effectiveness of passive immunotherapy for patients with sari. available evidence suggests that convalescent plasma is likely to reduce mortality during saris of viral etiology, with larger treatment effects if it commenced early after symptom onset. however, this is based on predominately low-quality, uncontrolled studies. our review supports the use of convalescent plasma in critically ill mers-cov-infected patients as part of a well-designed clinical trial or other formal evaluation. we thank the following reviewers from the convalescent plasma study group, who evaluated non-english-language articles on the basis of protocol eligibility criteria and undertook risk of bias assessments and data extraction: dr ana l. p. mateus supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole 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from the national institute for health research to set up a pandemic influenza clinical trial and has received unrestricted funding from pfizer for a study in adult pneumonia. the university of nottingham health protection research group (with which j. s. n.-v.-t. and c. r. b. are affiliated) is an official who collaborating center for pandemic influenza and research and receives limited funding from the who in support for specific activities. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-324701-7vjdlt1v authors: dahlmann, franziska; biedenkopf, nadine; babler, anne; jahnen-dechent, willi; karsten, christina b.; gnirß, kerstin; schneider, heike; wrensch, florian; o'callaghan, christopher a.; bertram, stephanie; herrler, georg; becker, stephan; pöhlmann, stefan; hofmann-winkler, heike title: analysis of ebola virus entry into macrophages date: 2015-10-01 journal: j infect dis doi: 10.1093/infdis/jiv140 sha: doc_id: 324701 cord_uid: 7vjdlt1v ebolaviruses constitute a public health threat, particularly in central and western africa. host cell factors required for spread of ebolaviruses may serve as targets for antiviral intervention. lectins, tam receptor tyrosine kinases (tyro3, axl, mer), t cell immunoglobulin and mucin domain (tim) proteins, integrins, and niemann-pick c1 (npc1) have been reported to promote entry of ebolaviruses into certain cellular systems. however, the factors used by ebolaviruses to invade macrophages, major viral targets, are poorly defined. here, we show that mannose-specific lectins, tim-1 and axl augment entry into certain cell lines but do not contribute to ebola virus (ebov)-glycoprotein (gp)–driven transduction of macrophages. in contrast, expression of mer, integrin αv, and npc1 was required for efficient gp-mediated transduction and ebov infection of macrophages. these results define cellular factors hijacked by ebov for entry into macrophages and, considering that mer and integrin αv promote phagocytosis of apoptotic cells, support the concept that ebov relies on apoptotic mimicry to invade target cells. viruses of the genus ebolavirus within the family filoviridae can cause severe disease in humans and nonhuman primates [1] . ebola virus disease (evd) is endemic in sub-saharan and western africa, with the largest ever recorded evd outbreak currently unfolding in guinea, sierra leone, and liberia [2] . the import of ebolaviruses into europe and the united states via infected travelers or infected nonhuman primates highlight the fact that evd constitutes a global health threat [1, 3, 4] . however, neither approved vaccines nor therapeutics are currently available to combat evd. host cell factors exploited by ebolaviruses for viral amplification constitute potential targets for antiviral intervention, and a blockade of the factors required for the first step in viral replication, entry into target cells, might be particularly attractive. the glycoprotein (gp) of ebolaviruses drives viral entry into host cells. the gp1 subunit contains an nterminal receptor-binding domain [5] and interacts with host cell factors, and the gp2 subunit drives fusion of the viral membrane with the membrane of host cell endosomes. cellular entry of ebolaviruses is thought to commence upon viral binding to incompletely defined factors expressed at the host cell surface, followed by uptake of virions and processing of gp by endosomal cysteine proteases [6] . processed gp1 then interacts via its receptor-binding domain with the cholesterol transporter niemann-pick c1 (npc1) [7, 8] . finally, an incompletely characterized additional stimulus activates gp2 for membrane fusion [9] , resulting in delivery of the viral nucleocapsid into the host cell cytoplasm. several host cell factors have been implicated in the cellular entry of ebolaviruses. first, binding of lectins such as dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (dc-sign) [10, 11] presented in part: sixth international symposium on filoviruses, galveston, texas, 30 march to 2 april 2014. to glycans on gp can enhance gp-driven entry [12] . in addition, certain integrins were found to promote gp-mediated entry, and evidence of gp binding to these factors has been obtained [13] . furthermore, tam receptor tyrosine kinases (tyro3, axl, mer) were shown to promote gp-mediated entry [14] , possibly by stimulating viral uptake via macropinocytosis [15] . direct interactions between tam receptors and gp have not been detected, to our knowledge. two members of the t cell immunoglobulin and mucin domain (tim) protein family, tim-1 and tim-4, were also shown to promote viral entry, and binding of gp to tim-1 has been demonstrated [16] . however, binding to gp might not account for tim-1-mediated augmentation of gp-driven entry. thus, tim proteins directly [17] and tam receptors indirectly [18] bind to phosphatidylserine (ptdser) on apoptotic cells, and binding to ptdser on the virion surface was shown to be important for augmentation of gpdriven entry [15, [19] [20] [21] [22] [23] . finally, npc1 has been identified as an endosomal binding partner for gp [7] [8] [9] , as outlined above. however, many of the above-mentioned studies focused on the analysis of cell lines, whereas the cellular factors required for entry of ebolaviruses into macrophages, the major viral target cells, are poorly defined and were thus in the focus of the present study. cell lines were maintained in dulbecco's modified eagle medium or roswell park memorial institute 1640 medium, supplemented with 5%-10% fetal bovine serum and antibiotics. thp-1 monocytes were differentiated into macrophages by exposure to phorbol-12-myristate-13-acetate (sigma). primary human monocyte-derived macrophages (mdms) were cultured in monocyte differentiation medium: x-vivo 10 (lonza) supplemented with 1% human fibrin-depleted plasma and antibiotics. all cells were grown in a humidified atmosphere at 37°c and 5% carbon dioxide. first, mdms were prepared from thrombocyte-depleted blood from healthy human donors by means of ficoll density gradient centrifugation. subsequently, cells were cultured in monocyte adhesion medium (roswell park memorial institute 1640 medium with 7.5% human fibrin-depleted plasma and antibiotics), nonadherent cells were removed by washing. the next day, cells were detached, reseeded in monocyte differentiation medium, and cultivated for 7 days. differentiation into mdms was controlled by analyzing cd14 expression. expression plasmids for viral gps [24, 25] , lectins [24, 26] , ctype lectin domain family 5 member a (clec5a) [27] , npc1 [7] , tim-3 [28] , angiotensin-converting enzyme 2 [24] , and folate receptor α [29] were described elsewhere. the plasmid encoding axl was purchased (imagegenes). expression plasmids for human tim-1, tim-4, and scavenger receptor a (sr-a) were generated by reverse-transcriptase polymerase chain reaction (rt-pcr) and cloning of products into plasmid pcaggs. the integrity of all pcr-amplified sequences was confirmed by automated sequence analysis. antibodies against entry factors were purchased from r&d systems. 293t cells, transfected to express entry factors, were incubated with concentrated cellular supernatants containing comparable amounts (as determined by western blot analysis) of ebov-gp1 and severe acute respiratory syndrome coronavirus spike protein, subunit s1 (sars-s1) fused to the fc portion of human immunoglobulin, as described elsewhere [24] . after washing, cells were stained with fluorescein isothiocyanatelabeled secondary antibody (dianova) and analyzed by means of fluorescence-activated cell sorting (facs). for mer expression, thp-1 cells were differentiated into macrophages and small interfering rna (sirna) transfected, as described below, and then incubated for 24 hours followed by facs analysis of mer expression. for sr-a expression, mdms were sirna transfected as described below and lysed in radioimmunoprecipitation assay buffer. proteins were then separated by sodium dodecyl sulfate gel electrophoresis and blotted onto nitrocellulose membranes (hartenstein), and sr-a expression was detected with a sr-a-specific antibody and horseradish peroxidase-coupled secondary antibody (jackson research). signal intensities were quantified using the imagej software program version 1.47 (national institutes of health). for detection of tim-1, axl, and npc1 transcripts with quantitative rt-pcr, rna was extracted from cell lines and mdms and analyzed using an abi 7500 fast real-time pcr system (applied biosystems), taqman and hs99999908_m1 [β-glucuronidase]). the mean cycle threshold (ct) value for each individual assay was calculated from triplicate measurements and mean ct values calculated for tim-1, axl, and npc1 were normalized by subtraction from the ct values obtained for the housekeeping reference β-glucuronidase. template-free complementary dna reactions were analyzed in parallel, and no specific signal was detected in any of these experiments. lentiviral vectors pseudotyped with heterologous viral gps ( pseudotypes) were generated as described elsewhere [11] and normalized for comparable infectivity for 293t cells. for transduction experiments, target cell lines were seeded in 96-well plates and incubated for 8 hours with vector preparations. the mdms were spinoculated at 2000 rpm and 21°c for 2 hours and then incubated at 37°c for 6 hours. subsequently, the medium was replaced by fresh culture medium. transduction efficiency was determined by quantifying luciferase activities in cell lysates 72 hours after transduction using a commercial kit (promega). to assess the impact of sirna knock-down on transduction, the target cells were lipofectamine 2000 (invitrogen) transfected with 5 pmol sirnas (santa cruz) at 24 hours before transduction. to determine whether u18666a (biomol), tannic acid, and mannan (both sigma-aldrich) modulated transduction efficiencies, the inhibitors were incubated with target cells for 30-60 minutes before addition of pseudotypes. all experiments with replication competent ebola virus (ebov, formerly zaire ebolavirus), strain mayinga, were performed in the bsl-4 facility of the institute of virology at the philipps-university marburg. first, mdms seeded in 8-well chamber slides (ibidi) were transfected with sirna in duplicates and infected with ebov at a multiplicity of infection of 10. inoculum was replaced by fresh culture medium at 1 hour after infection and infection was stopped after 16 hours by treatment of cells with medium containing 4% paraformaldehyde (pfa). after virus inactivation by pfa, cells were permeabilized and stained for nucleoprotein, and nuclei were stained with 4′,6-diamidino-2-phenylindole. to determine infection efficiency, 100 pictures of each well were taken with an automated microscope, and the number of nucleoprotein-positive cells relative to the total number of cells was determined in every tenth photomicrographs. tim proteins and tam receptor tyrosine kinases are expressed on certain ebov target cells and were previously implicated in host cell entry of these viruses [10, 14, 16] . to confirm and extend these observations, we compared the ability of lectins, tim proteins, and the tam family member axl to augment ebov-gp-driven entry into 293t cells, which can be efficiently transfected and are highly susceptible to transduction mediated by ebolavirus gps [11] . for transduction, we opted for pseudotypes bearing the gp of the highly pathogenic ebov and an ebov-gp variant lacking the mucin domain (ebov-gpδmuc), which was reported to yield higher vector titers than the wild-type (wt) protein [30] . expression of the c-type lectins dc-sign, dc-sign-related, asialoglycoprotein receptor 1 (asgpr-1), and liver and lymph node sinusoidal endothelial cell c-type lectin (lsectin) increased ebov-gp-and ebov-gpδmuc-mediated transduction up to 37-fold but had no effect on entry mediated by vesicular stomatitis virus glycoprotein (vsv-g) ( figure 1a ), as expected [10, 11, 24, 26] . an increase in transduction efficiency was also observed on expression of tim-1, whereas expression of tim-4 and axl had no effect on transduction mediated by ebov-gp but slightly enhanced transduction driven by ebov-gpδmuc ( figure 1b) . finally, expression of folate receptor α (which does not augment gp-driven entry [29, 31] ), tim-3, and clec5a (a lectin that interacts with dengue virus [32] ), did not modulate transduction under all conditions tested. these results demonstrate that expression of certain lectins and tim-1 on a susceptible cell line can increase ebov-gp-driven entry, supporting a role for these proteins in host cell entry of ebovs. we next tested whether the entry factors examined in our study interact directly with the gp1 subunit of the ebov-gp. the expression of all entry factors tested was readily detectable on transfected cells (figure 2a ), and binding of severe acute respiratory syndrome coronavirus spike protein, subunit s1, fused to the fc portion of human immunoglobulin to dc-sign-and angiotensin-converting enzyme 2-expressing cells was markedly above the background signal ( figure 2b ), as expected [33] . the ebov-gp1 subunit was also able to bind to dc-sign, in keeping with published results [10, 11, 33] , but binding to the other proteins tested was within the background range, at least under the conditions used ( figure 2b ). thus, augmentation of gp-driven host cell entry by lectins but not by tim-1 and axl might rely on efficient binding to the gp1 subunit, in keeping with the concept that the latter factors augment ebov-gp-driven entry by binding to ptdser in the viral envelope [20, 23] . dc-sign is only expressed on a narrow spectrum of target cells of ebov infection, including dendritic cells and certain tissue macrophages. therefore, we focused our subsequent analyses on tim-1 and axl, which are broadly expressed on cell lines, as confirmed by facs analysis (figure 3a) , and primary cells. however, a comparison between endogenous tim-1 and axl expression on cell lines and susceptibility to ebov-gp and ebov-gpδmuc-driven transduction revealed no correlation ( figure 3b) . similarly, the effect of sirna-mediated knockdown of axl and tim-1 expression ( figure 3c ) on transduction efficiency was dependent on cell type ( figure 3d ). thus, augmentation of ebov-gp-mediated transduction by axl and tim-1 is restricted to certain cell lines, a finding that matches previous results [15, 16] . the npc1 protein, an intracellular ebov entry factor, is ubiquitously expressed. we asked whether npc1, in contrast to axl and tim-1, is universally required for ebov-gp-mediated transduction. indeed, knock-down of npc1 expression ( figure 4a ) and treatment of target cells with u18666a, an inhibitor that induces . an isotype-matched control antibody was included as a negative control. the geometric mean channel fluorescence (gmcf) measured in a single representative experiment performed with unicates is shown; similar results were obtained in 3 separate experiments. b, cells analyzed in a were incubated with the indicated soluble, fc-tagged gps, and binding efficiency was determined with facs. results of a single representative experiment performed with single samples are shown; similar results were obtained in a separate experiment, performed with a different gp preparation. abbreviations: ace2, angiotensin-converting enzyme 2; fr, folate receptor; sars-s1, severe acute respiratory syndrome coronavirus spike protein, subunit s1. a npc1 knockout phenotype in treated cells [34] , efficiently and specifically reduced ebov-gp-and ebov-gpδmuc-driven transduction ( figure 4b ). moreover, we found that npc1 messenger rna (mrna) was readily detectable in c8166 t lymphocytes and raji b cells ( figure 4c ), which were resistant to ebov-gp-driven transduction ( figure 4d ), indicating that lack of npc1 does not account for the well-documented resistance of lymphocytes to ebov-gp-mediated entry [35] . macrophages are targeted early and throughout ebov infection [36, 37] . therefore, we next asked whether axl, tim-1, and npc1 contribute to gp-driven transduction of mdms. quantitative rt-pcr analysis revealed that mdms express only low amounts of axl and tim-1 compared with the cell lines hela and huh7 ( figure 5a ). in keeping with this finding, sirna-mediated knock-down of axl and tim-1 expression had no appreciable effect on ebov-gp-driven transduction of mdms ( figure 5b ). in contrast, knock-down of npc1 significantly reduced mdm transduction mediated by ebov-gp but not vsv-g. similarly, transduction of mdms by ebov-gpbut not vsv-g-bearing pseudotypes was reduced efficiently and in a concentration-dependent manner by u18666a ( figure 5c ), indicating that npc1 might be required for ebov infection of macrophages. finally, the mannose-polymer mannan, which inhibits ligand binding to dc-sign and related lectins, did not inhibit gp-driven transduction of mdms, although it markedly reduced transduction of 293t cells expressing dc-sign ( figure 5d ), indicating that mannose-specific lectins do not promote gp-dependent entry into macrophages. a, hela and huh7 cells were transfected with the indicated small interfering rnas (sirna) and then transduced with the indicated pseudotypes. at 72 hours after transduction cells were lysed and analyzed for luciferase activity. results of a single representative experiment performed with triplicate samples are shown; similar results were obtained in 3 separate experiments. error bars represent standard deviations. statistical significance was calculated using the 2tailed student t test. †p < .01. b, the depicted cell lines were incubated with solvent (dimethyl sulfoxide) or u18666a at a final concentration of 10 µmol/l followed by transduction with the indicated pseudotypes, as described above. transduction of each cell line in the absence of inhibitor was set at 100%. results of a single representative experiment are shown; similar results were obtained in 2 separate experiments. c, total rna isolated from huh7, 293t, and the lymphocytic cell lines c8166 and raji was analyzed for npc1 expression by reverse-transcriptase (rt) polymerase chain reaction, in the presence (+) or absence (−) of rt. expression of glyceraldehyde 3-phosphate dehydrogenase (gapdh) was examined as a control. d, the cell lines analyzed in c were transduced with the indicated pseudotypes and analyzed for luciferase activity, as described above. results of single representative experiment performed with triplicate samples are shown; results were confirmed in a separate experiment. abbreviation: vsv-g, vesicular stomatitis virus glycoprotein. the results obtained so far indicate that, of the previously identified ebov entry factors, only npc1 is essential for efficient gpmediated transduction of mdms. notably, 2 factors required for optimal transduction of cell lines, axl and tim-1, are intimately involved in the recognition of apoptotic cells [17, 18] . therefore, we asked whether ebov-gp might exploit other cellular factors with a comparable activity for macrophage entry. we focused our analysis on integrin αv, cd14, cd36, lox-1, sr-a, and mer, all known to be expressed in macrophages and to play a role in the detection of apoptotic cells [38] . moreover, integrin αv and mer were previously implicated in ebov-gp-mediated entry [13, 14] . transfection of sirnas directed against integrin αv, sr-a, and mer expression significantly reduced ebov-gp-but not vsv-g-mediated transduction of mdms ( figure 6a, left) . moreover, a combination of sirnas against mer, sr-a and npc1 reduced transduction more efficiently than the single sirnas ( figure 6a, right) , suggesting that mer, sr-a, npc1, and integrin αv might all be required for robust ebov-gpmediated transduction of macrophages. we next assessed the efficiency and specificity of the sirnamediated knock-down of mer and sr-a expression. we analyzed the effects of sirna transfection on mer expression using thp-1 cell-derived macrophages, because mer protein was readily detected on these cells but not on mdms, owing to high background signals measured for the latter. mer expression was modestly reduced upon transfection of mer-specific but not npc1-or sr-a-specific sirna ( figure 6b) . similarly, expression of sr-a in mdms was reduced by sirna specific for sr-a but not npc1. transfection of sirna directed against mer had a slight but reproducible effect on sr-a expression ( figure 6b ), suggesting that full expression of sr-a might ; results on the right, the mean of 3 independent experiments; error bars indicate standard errors of the mean. b, thp-1 cells induced with phorbol-12-myristate-13-acetate were transfected with the indicated sirnas, and expression of mer (white bars) was analyzed with flow cytometry. mdms were transfected with the indicated sirnas, and sr-a expression (black bars) was analyzed by means of western blotting. the signals measured were quantified with imagej software. expression of mer and sr-a in cells transfected with control sirna was set at 100%. results represent the mean of 4 (mer expression) to 6 (sr-a expression) independent experiments. c, hela cells, which do not express endogenous mer or sr-a, were transfected with the respective sirnas and transduced with the indicated pseudotypes. luciferase activities in cell lysates were analyzed 72 hours after transduction. results represent the mean of 4 independent experiment performed with triplicate samples. the transduction of cells transfected with control sirna was set at 100%. d, mdms were incubated with the indicated concentrations of tannic acid transduced with the indicated pseudotypes, and transduction efficiency was determined as described above. transduction of cells incubated with solvent (water) was set at 100%. results represent the mean of 3 separate experiments performed with triplicate samples. statistical significance was calculated using the 2-tailed student t test. *p < .05; †p < .01; ‡p < .001. abbreviation: vsv-g, vesicular stomatitis virus glycoprotein. depend on parallel expression of mer, a scenario in keeping with published findings [39] . to assess the specificity of entry modulation by sirnas, we investigated hela cells, which are negative for sr-a [40] and mer [41] .npc1-specific sirna markedly reduced ebov-gp-but not vsv-g-driven entry ( figure 6c ), as expected. in contrast, sirna against mer had no significant effect on ebov-gp-mediated transduction and slightly augmented transduction driven by vsv-g. finally, sirna directed against sr-a reduced ebov-gp-but not vsv-g-driven transduction by 45% ( figure 6c ), suggesting off-target effects. to further investigate the role of sr-a, we asked whether tannic acid, which blocks ligand binding to sr-a [42] , inhibits transduction of mdms. indeed, tannic acid reduced ebov-gp-but not vsv-g-mediated transduction in a robust and concentration dependent manner ( figure 6d ). collectively, these results are in keeping with a role of sr-a and mer in ebov-gp-dependent macrophage transduction, although offtarget effects of the sr-a sirna cannot be excluded. it is possible that gp-pseudotyped retroviral particles might not mirror all facets of ebov entry into host cells. therefore, we assessed whether the sirnas directed against mer and sr-a reduced mdm infection by replication-competent ebov. we also tested sirna against integrin αv and npc1. transfection of single sirnas slightly but reproducibly decreased infection efficiency, and a marked drop in the infection rate was observed upon parallel knock-down of mer, sr-a and npc1 (figure 7) , indicating that the expression of npc1, together with several factors involved in the phagocytosis of apoptotic cells, is required for efficient ebov infection of macrophages. the present study confirms that tim-1 and axl can promote ebov-gp-driven entry into certain cell lines and shows that these proteins do not augment entry into macrophages. in contrast, npc1 was required for entry into macrophages and all cell lines tested. finally, evidence was obtained that also mer, integrin αv, and possibly sr-a promote ebov infection of macrophages. the comparative analysis of ebov entry factors revealed that lectins are most efficient at increasing gp-driven entry into already-susceptible 293t cells. however, mannose-specific lectins did not promote gp-dependent transduction of mdms and have little impact on ebov infection of monocyte-derived dendritic cells [12] . these in vitro results argue against a major contribution of mannose-specific lectins to ebov entry into macrophages and dendritic cells in vivo. nevertheless, one should keep in mind that lectins such as human macrophage c-type lectin specific for galactose and n-acetylgalactosamine, lsectin, and asgpr-1, which recognize carbohydrates other than mannose and augment ebov-gp-driven infection in cell culture [24, 26, 43] , might affect viral cell tropism in vivo. tam receptor tyrosine kinases and tim proteins promote entry of ebolaviruses [14, 16] and other enveloped viruses [19, 20, 23, 44] by binding to ptdser in the viral envelope. upon expression in 293t cells, tim-1, but no other tim proteins or axl, increased ebov-gp wt-mediated entry. augmentation of gpdriven entry upon tim-1, axl, and tim-4 expression was either more pronounced or exclusively observed for ebov-gpδmuc compared to ebov-gp wt, suggesting that the presence of the mucinlike domain might modulate augmentation of gpdriven transduction by ptdser-binding proteins. the inability of tim-3 to augment entry is due to inadequate presentation of its ptdser-binding domain [45] . in contrast, it is currently unclear why tim-1 augments entry driven by ebov-gp but not vsv-g (and a few other viral gps), a finding also reported by others [19] , and several hypotheses have been proposed [19, 23] . our finding that endogenous expression of tim-1 and axl augments gp-driven entry into a subset of susceptible cell lines matches previous reports [16, 22] and raised the questions whether these factors contribute to gp-mediated entry into macrophages. quantitative rt-pcr revealed that less axl and tim-1 mrna is produced in mdms compared with susceptible cell lines, and sirna-knock-down showed that expression of these factors is dispensable for gp-driven entry into mdms. in contrast, mer but not axl expression was required for efficient gpdriven transduction and ebov infection of mdms. similarly, mer but not axl or tyro3 was found to be essential for efficient phagocytosis of apoptotic cells by macrophages [46] , indicating that mer is the tam receptor kinase active in macrophages. several previous reports indicate a key role of npc1 in host cell entry of ebolaviruses [7] [8] [9] . our finding that npc1 was required for gp-mediated transduction of all target cells tested and was essential for efficient ebov infection of macrophages suggests that npc1 may be universally required for host cell infection by ebolaviruses. in this regard, it should be noted that the lymphocytic cell lines examined here expressed robust levels of npc1 mrna, suggesting that lack of npc1 expression does not account for the resistance of lymphocytes to gp-driven entry. the relatively modest effect of mer knock-down on gpdriven entry into mdms and ebov infection of mdms suggested that other factors might contribute to these processes, and we speculated that they may also recognize apoptotic cells. indeed, we found that sirnas against integrin αv and sr-a reduced gp-driven entry and ebov infection of mdms. integrins αvβ3 and αvβ5 interact with milk fat globule-egf factor 8, which in turn recognizes ptdser [47] , suggesting that these factors might promote gp-driven entry in a fashion similar to tam receptor kinases and their ptdserbinding ligands growth arrest-specific protein 6 or protein s [44] . such a scenario could be reconciled with a previous study reporting that recombinant α5β1 integrin or antibodies against β1 integrin interfere with gp-driven entry [13] . however, subsequent work indicated that expression of α5β1 integrin is required for full activity of cathepsin l, which processes gp for binding to npc1 [13, 48] . integrin αv expression might thus promote macrophage entry of ebolaviruses via several mechanisms. sr-a is expressed in macrophages and dendritic cells and contributes to the macrophage uptake of a broad spectrum of ligands, including apoptotic cells [49] . ebov-gp1 did not bind to cells expressing sr-a and directed expression of sr-a did not augment gp-driven transduction (not shown) but treatment of mdms with sr-a-specific sirna consistently reduced ebov-gp-but not vsv-g-driven entry as well as ebov infection. furthermore, gp-driven transduction of mdms was also inhibited by tannic acid and fucoidan (not shown), sr-a ligands [42, 50] , arguing for a role of sr-a in entry of ebovs into macrophages. on the other hand, sirna against sr-a also reduced gp-driven entry into sr-a-negative hela cells, and alveolar macrophages isolated from sr-a knockout and wt mice were equally susceptible to gp-driven transduction (not shown). further studies are therefore required to fully define the potential role of sr-a in macrophage entry of ebolaviruses. collectively, our results support the concept that ebolaviruses use apoptotic mimicry for infection of cell lines and macrophages and suggest that the set of factors exploited by the viruses for this purpose may be cell type dependent. ebola haemorrhagic fever world health organization. ebola virus disease, west africa-update association of ebolarelated reston virus particles and antigen with tissue lesions of monkeys imported to the united states isolation and partial characterisation of a new strain of ebola virus conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor endosomal proteolysis of the ebola virus glycoprotein is necessary for infection ebola virus entry requires the cholesterol transporter niemann-pick c1 small molecule inhibitors reveal niemann-pick c1 is essential for ebola virus infection ebola virus entry requires the host-programmed recognition of an intracellular receptor c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells analysis of the interaction of ebola virus glycoprotein with dc-sign (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) and its homologue dc-signr downregulation of beta1 integrins by ebola virus glycoprotein: implication for virus entry tyro3 family-mediated cell entry of ebola and marburg viruses the tyro3 receptor kinase axl enhances macropinocytosis of zaire ebolavirus t-cell immunoglobulin and mucin domain 1 (tim-1) is a receptor for zaire ebolavirus and lake victoria marburgvirus tim genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity tam receptors in apoptotic cell clearance, autoimmunity, and cancer tim-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine role of the phosphatidylserine receptor tim-1 in enveloped-virus entry the mechanism of axl-mediated ebola virus infection tyrosine kinase receptor axl enhances entry of zaire ebolavirus without direct interactions with the viral glycoprotein the soluble serum protein gas6 bridges virion envelope phosphatidylserine to the tam receptor tyrosine kinase axl to mediate viral entry lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus the signal peptide of the ebolavirus glycoprotein influences interaction with the cellular lectins dc-sign and dc-signr differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr structural flexibility of the macrophage dengue virus receptor clec5a: implications for ligand binding and signaling a highly conserved tyrosine of tim-3 is phosphorylated upon stimulation by its ligand galectin-9 folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha clec5a is critical for dengue-virusinduced lethal disease dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus cholesterol synthesis inhibitor u18666a and the role of sterol metabolism and trafficking in numerous pathophysiological processes characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection the role of antigen-presenting cells in filoviral hemorrhagic fever: gaps in current knowledge the innate immune system and the clearance of apoptotic cells human protein s inhibits the uptake of acldl and expression of sr-a through mer receptor tyrosine kinase in human macrophages host glycoprotein gp96 and scavenger receptor srec interact with porb of disseminating neisseria gonorrhoeae in an epithelial invasion pathway the c-mer gene is induced by epstein-barr virus immediate-early protein brlf1 inhibition of antigen trafficking through scavenger receptor a human macrophage c-type lectin specific for galactose and n-acetylgalactosamine promotes filovirus entry the role of phosphatidylserine receptors in enveloped virus infection characterizing functional domains for tim-mediated enveloped virus entry phagocytosis and clearance of apoptotic cells is mediated by mer identification of a factor that links apoptotic cells to phagocytes β 1 -integrin controls ebolavirus entry by regulating endosomal cathepsins role for the class a macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro growth within macrophages increases the efficiency of mycobacterium avium in invading other macrophages by a complement receptor-independent pathway acknowledgments. we thank h. s. earp, w. kuehn, k. chandran, and g. simmons for the mer, t cell immunoglobulin and mucin domain-3, niemann-pick c1, and folate receptor α expression plasmids, respectively. we also thank a. büscher for excellent technical assistance, t. legler for supplying thrombocyte-depleted blood, and s. kimmina, e. munk, h. verkennis, and f. j. kaup for collaboration and support.financial support. this work was supported by the german research foundation (grant po 716/8-1 to s. p.), the german ministry for research and education (subproject within ebocon; grant to s. p.), sfb 900 (grant to rita gerardy-schahn), the leibniz graduate school for emerging infectious diseases (to s. p.), and the leibniz foundation.potential conflicts of interest. all authors: no potential conflicts of interest.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-351776-otx5qwyu authors: ibáñez-samaniego, luis; bighelli, federico; usón, clara; caravaca, celia; carrillo, carlos fernández; romero, miriam; barreales, mónica; perelló, christie; madejón, antonio; marcos, aránzazu caballero; albillos, agustín; fernández, inmaculada; garcía-samaniego, javier; calleja, josé luis; bañares, rafael title: elevation of liver fibrosis index fib-4 is associated with poor clinical outcomes in patients with covid-19 date: 2020-06-21 journal: j infect dis doi: 10.1093/infdis/jiaa355 sha: doc_id: 351776 cord_uid: otx5qwyu background: covid-19 is a potentially severe disease caused by the recently described sars-cov-2. whether liver fibrosis might be a relevant player in the natural history of covid-19 is currently unknown. we aimed to evaluate the association between fib-4 and the risk of progression to critical illness in middle-aged patients with covid-19. methods: in this multicenter, retrospective study with prospective follow-up of 160 patients aged 35–65 years with covid-19, fib-4, clinical, and biochemical variables were collected at baseline. fib-4 ≥2.67 defined patients with risk for advanced liver fibrosis. results: risk for advanced fibrosis was estimated in 28.1% of patients. patients with fib-4 ≥2.67 more frequently required mechanical ventilation (37.8% vs 18.3%; p = .009). in multivariate analysis, fib-4 ≥2.67 (odds ratio [or], 3.41; 95% confidence interval [ci], 1.30–8.92), cardiovascular risk factors (or, 5.05; 95% ci, 1.90–13.39), previous respiratory diseases (or, 4.54; 95% ci, 1.36–15.10), and c-reactive protein (or, 1.01; 95% ci, 1.01–1.02) increased significantly the risk of icu admission. bootstrap confirmed fib-4 as an independent risk factor. conclusions: in middle-aged patients with covid-19, fib-4 may have a prognostic role. the link between liver fibrosis and the natural history of covid-19 should be evaluated in future studies. coronavirus disease 2019 (covid-19) is a potentially severe disease, caused by the recently described sars-cov-2, with a broad spectrum of clinical manifestations, including acute respiratory distress syndrome (ards) and death. most health systems have been overwhelmed due to the rapid spread of the virus and the high demand of patients for medical attention, especially intensive care requirements [1] . approximately 5% of the patients with symptomatic covid-19 will progress to critical illness, mostly due to the development of ards [2] . patients at higher risk of ards or intensive care unit (icu) admission are those with advanced age or comorbidities, including previous history of metabolic risk factors such as type 2 diabetes mellitus (t2dm) or hypertension. alteration of liver biochemistry (ie, elevation of aspartate aminotransferase [ast]/alanine aminotransferase [alt]) or liver function tests (ie, hyperbilirubinemia or hypoalbuminemia) are frequent findings in patients with covid-19 and have been linked to major adverse clinical outcomes [2, 3] . prevalence of liver fibrosis (≥ stage 2), mostly attributed to metabolic-associated fatty liver disease (mafld), is frequent in the general population (2.8%-5.6%) and increases significantly in high-risk populations (up to 18% in patients with t2dm) [4] . mafld is the most frequent cause of chronic liver disease in western countries and is closely related with the features of the metabolic syndrome and with numerous extrahepatic complications (ie, cardiovascular disease, neoplasms, chronic kidney disease, etc.). advanced liver fibrosis is the main determinant of progression to cirrhosis, liver failure, and hepatocellular carcinoma [5, 6] . furthermore, patients with advanced fibrosis (stages 3 and 4) present a higher risk of mortality compared with the reference population [7] . however, there is no information related to the prevalence and influence of liver fibrosis in covid19. noninvasive tests, based on routine biochemical and clinical parameters, are useful tools for the assessment of liver fibrosis and risk stratification. fib-4 is a simple fibrosis score that has been validated in several etiologies of liver disease and was shown to be superior to other noninvasive markers of fibrosis [8, 9] . fib-4 has been associated with extrahepatic clinical outcomes in patients with liver disease and, importantly, in various nonliver-related conditions such as intracerebral hemorrhage or atrial fibrillation [10, 11] . finally, mafld has been reported in up to 38% of patients with covid-19 and its presence has been associated with worse evolution of the respiratory disease [12] . we hypothesize that liver fibrosis might be a relevant player in the covid-19 natural history. therefore, we aimed to assess noninvasively the presence of advanced liver fibrosis in patients with covid-19 and to evaluate the contribution of advanced fibrosis to clinical outcomes. this observational study included patients with a confirmed sars-cov-2 infection at 5 tertiary-level hospitals in the region of madrid from 26 february to 20 march 2020. study inclusion was retrospective and follow-up was prospective until discharge from hospital, death, or end of follow-up. diagnosis of the infection was based on rna detection of sars-cov-2 in a nasopharyngeal swab sample by real time reverse-transcriptase polymerase chain reaction (rrt-pcr) followed by a second positive rrt-pcr confirmation in all patients. patients were excluded for any of the following criteria: previous diagnosis of myopathy or platelet disorders, recipients of solid organ transplant, and patients treated with drugs known to produce myelotoxicity. patients younger than 35 years or older than 65 years (the range of ages in which fib-4 is less accurate) were also excluded [13] . in addition, patients with sars-cov-2 infection who were hospitalized for reasons other than covid-19 were excluded. the study was performed in agreement with the declaration of helsinki and was approved as consent-waived by the ethics and clinical research committee of hospital universitario puerta de hierro. demographic characteristics (age, sex, and race) and laboratory tests (alt, ast, bilirubin, γ-glutamyl transpeptidase [ggt], lactate dehydrogenase [ldh], c-reactive protein (crp), international normalized ratio [inr] , platelets, hemoglobin, and white cell count) were recorded at the same time as sars-cov-2 detection at the emergency room. additional information was recorded regarding concomitant medication, previous history of cardiovascular risk factors (t2dm, dyslipidemia, hypertension, smoking habit, and obesity), as well as other relevant past medical history, including chronic obstructive pulmonary disease, obstructive sleep apnea/hypopnea syndrome, ischemic or valvular heart disease, chronic kidney disease, chronic advanced liver disease, cancer (type of tumor and remission status), autoimmune disorders, and psychiatric or neurologic diseases. fib-4 was calculated according to the following equation [age × ast (iu/l)]/[platelets (×10 9 ) × √alt (iu/l)] from blood tests taken at the time of hospital admission, before starting any specific covid-19 therapies. previously published cutoffs were used to exclude and diagnose advanced fibrosis [14] . specifically, a value of fib-4 below 1.30 is considered as low risk for advanced fibrosis; a value of fib-4 over 2.67 is considered as high risk for advanced fibrosis; and fib-4 values between 1.30 and 2.67 are considered as intermediate risk of advanced fibrosis. in order to minimize overestimation of predicted advanced fibrosis, patients belonging to the intermediate fib-4 category were considered negative for advanced fibrosis in the multivariate analysis (see results section). the primary endpoint was to evaluate noninvasively the proportion of patients with covid-19 at risk for advanced liver fibrosis (fib-4 ≥2.67). the secondary aims were (1) to evaluate baseline characteristics of patients according to fib-4 categories (high risk vs low/intermediate risk for advanced fibrosis), and (2) to evaluate whether fib-4 is associated with the need for mechanical ventilation (mv). data were reported as the median and interquartile range (iqr) or mean and standard deviation (sd) for continuous variables, while frequency and percentage were used for discrete variables. categorical variables were compared with χ 2 test and continuous variables with the student t test. nonparametric alternatives (mann-whitney u or fisher exact test) were used for non-normal distributions. for secondary endpoint univariate and multivariate logistic regression analyses were performed. independent variables with significance p ≤ .10 in the univariate analysis, together with selected covariates based on their biologically plausible potential to act as confounders, were introduced in covariate-adjusted multivariate analyses (backward likelihood ratio regression analysis) to provide an optimal control for risk factors and confounders. to increase the robustness and assess the accuracy of the results provided by the logistic regression model we performed a bootstrap analysis. from the original cohort, 1000 different samples were generated by random selection and replacement using the conditional resampling method. this procedure provides a more reliable estimate of the coefficients of each covariate and therefore increases the internal validity of the results. additionally, we specifically checked for the modifier effect of fib-4 categories on the covariates by including interaction terms in the models. a significant interaction would indicate that the effect of covariates was different between fib-4 categories. odds ratios (or) and their 95% confidence intervals (ci) were estimated. values were considered statistically significant when p < .05. spss statistics (version 19.0; ibm corporation) was used in all analyses. this observational study was conducted in accordance with the strobe (strengthening the reporting in observational studies) statement. between 26 february and 20 march 2020, 449 patients with a confirmed sars-cov-2 infection attended the participating centers. patients meeting exclusion criteria (n = 236) and those in whom fib-4 could not be calculated at baseline (n = 53) were excluded ( figure 1 ). the cohort considered for the analysis comprised 160 patients. the median length of follow-up was 29 days (iqr, 26-33 days) and no patient was lost during follow-up. the baseline characteristics of the population are given in table 1 . briefly, the median age was 55 years (iqr, 48-60 years) with a lower proportion of women (41.3%). overall, 39.4% of patients presented with at least 1 cardiovascular risk factor, the most frequent being obesity (37%), hypertension (20%), and smoking (19.3%). patients with a previous diagnosis of respiratory or heart disease were 12.6% and 21.3% of the population, respectively. previous history of liver disease was reported in 13 patients (8 mafld, 3 hepatitis c virus, 1 hepatitis b virus, and 1 alcohol-related liver disease). no active cases of malignancy were reported. median fib-4 was 1.87 (iqr, 1.34-2.90). a total of 23.8% (38/160) required mv with a median time of 7 days (iqr, 4-11 days) from hospitalization to icu admission. forty-five patients (28.1%) showed a fib-4 ≥2.67. as expected, individual components of fib-4 were significantly higher in the fib-4 ≥2.67 group. an almost significant higher prevalence of cardiovascular risk factors was noted in the group of patients with fib-4 ≥2.67 (23 [51.1%] vs 40 [34.8%]; p = .057). previous heart, lung, and renal diseases showed a balanced distribution between groups with fib-4 <2.67 and fib-4 ≥2.67 ( table 2) . levels of acute-phase response proteins, such as cpr, were higher in the group at risk for fibrosis (87 mg/l [sd 66] vs 76 mg/l [sd 100]; p = .020). other features of systemic inflammatory response were also more pronounced in the group at risk for fibrosis, that is a lower lymphocyte count (0. 9 in the group of patients at risk for liver fibrosis, need for mv occurred more frequently (37.8% vs 18.3%; p = .009) and time from diagnosis of covid-19 to icu admission was shorter (5 [sd 4] days vs 10 [sd 5] days; p = .05). to evaluate the influence of covid-19 on fib-4 values and its individual components we retrieved previous values of ast, alt, and platelets in 24 (15%) patients of the cohort. these laboratory tests had been done within the previous 6 months before the diagnosis of covid-19 as part of scheduled tests at primary care. baseline characteristics between patients with or without available blood test were similar. ast and alt increased significantly at the time of covid-19 diagnosis, while platelet counts remained stable from previous during hospitalization, 23.8% (38/160) of patients were transferred to icu facilities for invasive (n = 36) or advanced noninvasive mv (n = 2). differences in baseline characteristics between patients requiring or not mv are depicted in supplementary table 1 . we observed that patients needing mv presented more frequently with chronic respiratory diseases (p = .018) as well as cardiovascular risk factors (hypertension (p = .041) and obesity (p = .003)). these patients also exhibited more frequently features of systemic inflammatory response, such as higher crp (p = .001) and lower lymphocyte count (p = .003). univariate association between potential predictors and need for mv are shown in table 3 we constructed 2 additional multivariate models to evaluate specifically the influence of ast on the endpoint. the first model included ast (instead of fib-4 to avoid collinearity) with the aim of evaluating the effect of ast individually. the second model included both ast and fib-4 to evaluate the influence of fib-4 in the presence of ast as a covariate. in both multivariate models, ast was excluded confirming that ast was not independently associated with the endpoint. no significant interactions between the endpoint, fib-4 categories, and other covariates were found. in this study, we evaluated the association between fib-4, a liver fibrosis index, and the risk of progression to critical illness in middle-aged patients with covid-19. the first important result of our series is that the estimated risk of liver fibrosis by fib-4 was greater than expected, reaching 28.1%. furthermore, patients with higher fib-4 were more likely to require mv. finally, our results indicate that after adjusting for other wellknown risk factors that negatively influence the natural history of covid-19 (age, comorbidities, and markers of acute inflammation), fib-4 ≥2.67 independently increases (or, 3.41) the need for mv in middle-aged patients. our results are in line with those recently published where an association between fib-4 categories and the risk of severe covid-19 was found in patients with mafld [15] . liver fibrosis is a strong predictor of all-cause mortality and increased liver-related morbidity (liver failure, portal hypertension, and hepatocellular carcinoma) in patients with chronic liver disease [6, 16] . in the last decades, a significant number of noninvasive tests have been developed to noninvasively assess liver fibrosis. we selected fib-4 because it is a simple score composed of age and 3 readily available laboratory parameters (ast, alt, and platelets), which can be obtained at the emergency room. in addition, fib-4 cutoffs have been validated in different etiologies of liver disease. this is a convenient feature for our study because we could not assess properly the etiology of a potential underlying liver disease in all patients. previous history of liver disease was reported in 13 patients; however, prevalence of mafld was presumably underestimated in our cohort in light of the high prevalence of metabolic and cardiovascular risk factors, and therefore mafld probably accounted for a greater proportion of patients, as previously reported [12] . unfortunately, due to the circumstances of data acquisition, noninvasive diagnosis of liver steatosis could not be obtained in this cohort. fib-4 is also useful to identify patients with liver diseases who are likely to have a liver-related adverse clinical outcome [17, 18] . importantly, fib-4 has been shown to predict nonliver-related clinical outcomes like cardiovascular mortality or risk of atrial fibrillation in patients with mafld [10, 19] . likewise, fib-4 has been shown to predict mortality in the general population [20] and clinical outcomes in nonliver-related clinical settings [11] . it should be emphasized that the aim of our study was not to elaborate a prognostic model for the need for mv in covid-19 but to point out the possible influence of underdiagnosed liver disease in the natural history of covid-19. transient elevations of transaminases have been reported during covid-19 infection [2] . therefore, evaluation of transaminases at the time of covid-19 might not be representative of the pre-covid-19 status and thus fib-4 may not be an accurate estimator of liver fibrosis. to overcome this problem, we analyzed our data in different ways: (1) we retrieved available information on blood test done within 6 months before covid-19 diagnosis in a relatively small number of patients (15% of the total series): at the time of covid-19 diagnosis, ast and alt increased significantly while platelets remained stable as compared with previous values; however, there were no significant changes in fib-4 categories; (2) we evaluated specifically the prognostic value of isolated baseline ast: in contrast to previous reports, ast was not an independent predictor either at univariate level or when adjusted by other clinical and laboratory covariates; and (3) finally, we evaluated specifically the association between the elevation of ast (ie, ast above the upper limit of normality) and the need for mv, which identified that ast elevation was an independent risk factor. however, this assessment deserves a cautious interpretation as it incorporates collinearity in the model and complicates the interpretation of the results. importantly, it should be emphasized that estimating the presence of fibrosis with an approach different from biochemical markers is very complex during covid-19. liver biopsy, the current gold standard for assessing liver fibrosis, is clearly unfeasible and probably unethical and elastography is difficult to perform. the most intriguing finding of our study is the association between elevated fib-4 and poor covid-19 outcomes. strikingly, patients classified as at risk for fibrosis required mv more frequently. in this context, it is possible to speculate that advanced fibrosis may enhance the risk for development of exacerbated inflammatory response, a characteristic finding of severe covid-19. in fact, advanced liver disease is characterized by a persistent stimulation of immune cells by pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (damps) that activate immune cells and upregulates the production of cytokines, chemokines, and growth factors, which are released to recruit and activate additional inflammatory cells, perpetuating a state of chronic lowgrade systemic inflammation [21, 22] . a similar state of low-grade inflammation has been reported in patients with obesity and insulin resistance. in fact, serum levels of interleukin-6 (il-6) have been correlated with the degree of obesity and with the risk of t2dm development [23] . in the setting of an acute infection, activated macrophages secrete il-6, which is the major inducer for the synthesis of acutephase response proteins in the hepatocyte (crp, ferritin, complement, clotting factors, etc.). the acute-phase proteins produced by the hepatocyte have direct effector function on innate immunity therefore promoting pathogen clearance [24] . unfortunately, we do not have information regarding il-6 levels in this cohort to evaluate specifically the interaction between il-6 and fib-4. we have shown, however, that elevation of fib-4 was associated with higher levels of crp, suggesting that inflammatory response is aggravated in patients with higher fibrosis markers. although we report data from a large cohort of patients with covid-19 from tertiary-level hospitals, there are several limitations that should be discussed. first, fib-4 components are not liver specific and may be affected by disorders other than liver disease. to overcome this problem, we excluded patients previously diagnosed with myopathies and platelet disorders. second, our study only included patients aged between 35 and 65 years. however, for patients younger than 35 years noninvasive assessment of liver fibrosis should be done with tests other than fib-4 (ie, elastography) because fib-4 may underdiagnose fibrosis. on the other hand, specificity of fib-4 for advanced fibrosis in patients older than 65 years decreases significantly and may overestimate fibrosis [13] . furthermore, the need for mv was an endpoint of the study. management and decision making in the overwhelming and exceptional setting of covid-19 was not homogeneous over time in patients with advanced age, a finding that has been reported previously [1] . however, the range of age selected in the study is not generally affected by this factor. finally, a significant proportion of patients (48%) were categorized in the intermediate fib-4 category, which is similar to that previously reported [25] . in this group of patients additional tests should be carried out to determine the risk of advanced fibrosis [24] . this pragmatic approach, however, could not be adopted in the setting of coronavirus. although some patients in the grey zone would be positive for advanced fibrosis, we decided to categorize this subset of patients as negative for advanced fibrosis in multivariate analysis in order to minimize the risk of overestimation of fibrosis severity. in conclusion, our results suggest that in middle-aged patients with covid-19, fib-4 may have a relevant prognostic role. whether fib-4 really accounts for liver fibrosis or it is just a change induced by covid-19 will need to be clarified in future studies. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. fair allocation of scarce medical resources in the time of covid-19 risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china [published online ahead of print 13 clinical characteristics of coronavirus disease 2019 in china screening for liver fibrosis in the general population: a call for action fibrosis severity as a determinant of cause-specific mortality in patients with advanced nonalcoholic fatty liver disease: a multi-national cohort study association between fibrosis stage and outcomes of patients with nonalcoholic fatty liver disease: a systematic review and meta-analysis liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease apricot clinical investigators. development of a simple noninvasive index to predict significant fibrosis in patients with hiv/hcv coinfection validation of fib-4 and comparison with other simple noninvasive indices for predicting liver fibrosis and cirrhosis in hepatitis b virus-infected patients impact of the fibrosis-4 index on risk stratification of cardiovascular events and mortality in patients with atrial fibrillation: findings from a japanese multicenter registry liver fibrosis indices and outcomes after primary intracerebral hemorrhage non-alcoholic fatty liver diseases in patients with covid-19: a retrospective study age as a confounding factor for the accurate non-invasive diagnosis of advanced nafld fibrosis sanyal aj; nash clinical research network. comparison of noninvasive markers of fibrosis in patients with nonalcoholic fatty liver disease risk of severe illness from covid-19 in patients with metabolic dysfunction-associated fatty liver disease and increased fibrosis scores the long-term pathological evolution of chronic hepatitis c prognostic value of non-invasive fibrosis and steatosis tools, hepatic venous pressure gradient (hvpg) and histology in nonalcoholic steatohepatitis simple noninvasive systems predict long-term outcomes of patients with nonalcoholic fatty liver disease the risk of atrial fibrillation in patients with non-alcoholic fatty liver disease and a high hepatic fibrosis index liver fibrosis scores predict liver disease mortality in the united states population macrophages in obesity and non-alcoholic fatty liver disease: crosstalk with metabolism cirrhosis-associated immune dysfunction: distinctive features and clinical relevance c-reactive protein, interleukin 6, and risk of developing type 2 diabetes mellitus hepatocytes: a key cell type for innate immunity fibrosis-4 (fib-4) score at the primary care level: an analysis of over 160 000 blood samples we thank all healthcare workers of the participating centers that, despite great difficulties, have done their very best to attend patients with covid-19.author contributions. l. i. s., j. l. c., and r. b. designed the study and are guarantors of the article. l. i. s. and r. b. drafted the manuscript, and carried out statistical analyses and interpretation. all authors curated the data, critically reviewed the manuscript, and approved the final version of the article, including the authorship list.potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-330645-46qaljq1 authors: waghmare, alpana; krantz, elizabeth m; baral, subhasish; vasquez, emma; loeffelholz, tillie; chung, e lisa; pandey, urvashi; kuypers, jane; duke, elizabeth r; jerome, keith r; greninger, alexander l; reeves, daniel b; hladik, florian; cardozo-ojeda, e fabian; boeckh, michael; schiffer, joshua t title: reliability of self-sampling for accurate assessment of respiratory virus viral and immunologic kinetics date: 2020-07-25 journal: j infect dis doi: 10.1093/infdis/jiaa451 sha: doc_id: 330645 cord_uid: 46qaljq1 the sars-cov-2 pandemic demonstrates the need for accurate and convenient approaches to diagnose and therapeutically monitor respiratory viral infections. we demonstrated that self-sampling with foam swabs is well-tolerated and provides quantitative viral output concordant with flocked swabs. using longitudinal home-based self-sampling, we demonstrate nasal cytokine levels correlate and cluster according to immune cell of origin. periods of stable viral loads are followed by rapid elimination, which could be coupled with cytokine expansion and contraction. nasal foam swab self-sampling at home provides a precise, mechanistic readout of respiratory virus shedding and local immune responses. the covid-19 pandemic is an unprecedented event in modern history. as of may 27, there are 5.6 million documented covid-19 cases and 350,000 deaths worldwide with rapidly expanding outbreaks ongoing in dozens of countries [1] . in all likelihood, this highly contagious and lethal respiratory virus will likely circulate widely for years to come [2] . a critical research priority is to develop rapid molecular tests that provide accurate diagnosis, determine infectiousness and transmissibility, and allow for monitoring of viral load during therapy [3] . for numerous viral infections, including influenza, viral load correlates with disease severity and secondary household attack rate [4] [5] [6] . early studies suggest that peak viral load differentiates mild from severe covid-19 [7] .furthermore, viral load monitoring during antiviral therapy is a mainstay for various human infections including hiv, hepatitis b, cytomegalovirus and hepatitis c infections [8] [9] [10] [11] [12] [13] [14] . for viruses such as sars-cov-2 for which severe clinical outcomes are rare, viral load may serve as a useful surrogate marker to design smaller, but still sufficiently powered treatment studies [7, 15] . another major unmet medical need is the ability to frequently measure the local mucosal immune response during infection. it is increasingly recognized that tissue resident t-cells and antigen presenting cells are phenotypically and functionally distinct from circulating immune cells, especially in respiratory viral infections [16] [17] [18] . therefore, measuring immune responses in blood can fundamentally misclassify the agents responsible for local viral elimination. important shifts in the immune response against respiratory viruses likely occur rapidly and in stages during the early and late phases of viral shedding [19] and serial measurement of local cytokines may provide a window into the local cellular response [20] . a c c e p t e d m a n u s c r i p t self-testing for respiratory viruses has been successfully performed both in research and primary care, but regulatory agencies have been slow to accept patient collected samples as valid, especially in the home setting. currently licensed flocked swabs may not be optimal for patients with vulnerable mucosal membranes and low platelet counts (e.g. following cytotoxic chemotherapy) because they are associated with discomfort and possible bleeding. moreover, discomfort may deter participants from collecting longitudinal samples. importantly, a reliable and comfortable home-based self-testing methodology is needed to prevent potentially infected individuals from entering healthcare facilities to be tested and transmitting virus to healthcare workers and other patients. initial data on foam swabs are promising, suggesting a broader role for home-based self-swabbing for respiratory viral pandemics [21, 22] . here we report data on a novel respiratory virus detection method using self-collected nasal foam swabs. this methodology expands our testing armamentarium with easily collected and comfortable swabs that can be applied to viral load and cytokine kinetic studies. most importantly, they can be easily scaled and used at home in this time of severe testing shortages and dangerous transmission risk. the study was approved by the institutional review board at fred hutchinson cancer research center. flocked vs foam swab study: participants with respiratory symptoms (table s1) for less than 3 days were enrolled in the study. each participant completed 2 sample collections, each separated by one hour. at each time point, the participant collected either a) two self-collected a c c e p t e d m a n u s c r i p t copan flocked swabs (#23-600-966), one from each nostril or b) two self-collected puritan foam swabs (puritan medical red #25-1805-sc 2), one from each nostril. swab collection was randomized by order of swab type. details of the swab collection and processing are provided in the supplemental methods. following sample collection, participants were asked to complete a survey to assess tolerability and acceptability. mcnemar's test with exact p-values was used to compare survey responses. longitudinal sampling study: separately, new participants with respiratory symptoms (table s1) for less than 3 days were enrolled in the study. each participant collected two puritan foam nasal swabs, one from each nostril, per day for 14 days after enrollment or until symptoms resolved, whichever was longer. details of the swab collection and processing are provided in the supplemental methods. participants completed a daily electronic symptom survey (table s1 ) and an end of study survey. sample processing: each conical vial containing a swab was vortexed and 500ul of buffer was removed and stored at -80°c for pcr analysis. swabs were processed and stored at -80°c for cytokine testing as described in the supplemental methods. viral testing: nasal swab specimens were tested using a multiplex pcr testing for 11 respiratory viruses [adenovirus a-f, human rhinovirus (hrv), influenza a and b, parainfluenza viruses (piv) 1-4, human coronavirus (cov), bocavirus (bov), respiratory syncytial virus (rsv) and human metapneumovirus (mpv)] as previously described [23] . statistical analysis. pcr results that were positive but below the limit of detection (lod) were imputed as 500 copies per ml, using the lod divided by two. negative results were assigned a value of 0. the concordance correlation coefficient (ccc) was used to measure agreement of quantitative results between paired samples [24] . cytokine results below the fitted curve range were assigned the value of the lower lod divided by two and results above the fitted curve range were assigned the value of the upper lod. symptoms are represented as the total number of symptoms present for each day, out of a total of 26 (table s1) we performed a cluster analysis where each sample is an array of 20 measured cytokine concentrations. first, we checked for cluster tendency of the samples using hopkin statistic (h) [25, 26] , where values close to 1 indicate that the samples are highly clustered and values close to 0.5 indicate random samples. when calculated h (get_clust_tendency function in r3) was greater than 0.5, we did a linkage hierarchical clustering with euclidean distances of the samples [27] . we used an acute viral infection model that distinguishes between early and late responses to fifteen participants were enrolled in the foam versus flocked nasal swab study. four participants were negative for any respiratory virus from all swabs (table 1) (table s2) . discrepant results occurred exclusively in samples with low viral load (<4 log10 viral copies/ml) ( table 1) . agreement between samples collected by foam and flocked swabs from the same nostril was generally high, particularly with high viral load samples, with no evidence of higher yield with one method versus the other (fig 1a) . in the same dataset, we compared swab samples obtained with the same swab type from separate nostrils with a total of 15 paired samples. viral loads were notably higher in one nostril than the other and were less in agreement (fig 1b) . moreover, the value from the highest nostril strongly agreed with the sum of the two nostrils suggesting that a majority of sampled virus comes from one side (fig 1c) and that sampling the other side underestimates viral load. therefore, bilateral sampling is likely required for optimal yield and accurate quantitation. survey responses suggested participants found foam swabs more comfortable ( a c c e p t e d m a n u s c r i p t we next enrolled 9 otherwise healthy, adult study participants who self-sampled their nasal passage serially for 14 days. one participant contributed serial samples twice. overall compliance was high: median number of sample days was 14 (range 11-19 days). after study completion, the majority of participants agreed or strongly agreed that foam swabs were comfortable (70%), easy (90%) and that they would participate in future research with foam swabs (80%). serial home-based testing appears to be a well-accepted methodology. in longitudinal sampling, we were able to detect 14 viruses including seven human rhinovirus (hrv), two coronavirus (cov), one bocavirus (bov), two adenovirus (adv), one human metapneumovirus (mpv) and one respiratory syncytial virus (rsv). there were four instances of viral co-infection, though in each case a dominant virus was evident based on greater duration of shedding and higher viral load (fig 2a) . during most extended periods of hrv, rsv and hmpv shedding, viral loads were remarkably stable (fig 2a) . for hrv, a pattern of viral load steady state or slight gradual decline followed by rapid elimination was noted. the case of rsv had a similar profile but with an initial high viral load peak and shorter duration of shedding. the case of mpv had a more protracted decline with a single re-expansion phase. these transiently observed periods of steady state viral loads are highly unlikely to occur by chance if true viral loads fluctuated or exhibited stochastic noise. thus, the sampling method appears highly reliable. these data also suggest a brief period of equilibrium between the virus and local immune system before viral elimination. a c c e p t e d m a n u s c r i p t in general, the level of symptoms appeared to track with detectable virus, particularly for cov, hrv and mpv. for the single case of rsv, a high number of symptoms persisted beyond viral elimination (fig 2a) . for several cytokines, particularly those in the th2, th17 and non-defined pathways (il-2, il-4, il-5, il-10, il-13, il-17a and eotaxin), there was notable stability within and between study participants, independent of viral shedding (fig 2b, fig s2b) . this result demonstrates consistency in swabbing technique and again validates the precision of our approach. other molecules, particularly those associated with cytotoxic t-cell responses (granzyme b, perforin, tnfα and ifn) and macrophage responses (mip-1α, il-1α, il-6, il-18) showed monotonic expansion or clearance in response to most infections, with particularly dynamic shifts during rsv, mpv and one instance of hrv (p21) with the highest initial viral load (fig 2b, fig s2b) . in 6 participants with hrv, we correlated cytokine patterns to infer cellular origin. high positive correlations were noted among analytes associated with a cytotoxic t-cell response (granzyme b, perforin, tnfα, and il-6), among macrophage or epithelial cell-derived cytokines (mip-1α, il1α, il-6, il-12p70, il-21), and among th2-associated responses (il-5, il-10, il-17). the th2 associated cytokines also correlated with many of the cytolytic t-cell and macrophageassociated cytokines (fig 3a) . hrv viral load was only moderately correlated with granzyme b, perforin, and ip10. this suggests that hrv may not induce an intense local immune response in a dose-dependent fashion. similar results occurred with inclusion of all samples from all participants in the cohort (fig s3a) . in two participants infected with more inflammatory viruses, rsv and mpv, we noted similar correlative trends as with hrv. correlations among related pairs were higher for rsv/mpv than for hrv (fig 3b) and temporal kinetics were often strikingly similar suggesting an equivalent cellular source (fig 2b, fig s2b) . overall there was a lack of correlation between cytokines associated with t-cell responses and with epithelial cells and macrophages. viral load correlated with many cytokines of t-cell origin (fig 3b) , suggesting that rsv and mpv may induce inflammation in a dose-dependent fashion. we sorted all hrv samples using linkage clustering analysis and demonstrated three classes of samples that were distinguished by levels of t-cell and macrophage-associated cytokines (fig 3b) . the minority of samples (blue class) with the highest levels of granzyme b, perforin, il-6, il-1α, mip-1α and ifn all had high viral loads, all from two participants. all six participants had some samples in the least inflammatory class (grey) and 5 participants had samples in the moderate inflammatory class (green). these data indicate that the inflammatory milieu in the hrv-infected nasal passage is dynamic over time, but tilts toward higher inflammation with higher viral loads. similar results were observed when all samples were analyzed though only two classes were distinguished (fig s3b) . we next sorted the rsv and mpv samples and could not identify the optimal number of clusters. we selected two clusters which were differentiated according to concentrations of most cytokines, again including granzyme b, perforin, il-6, il-1α, mip-1α and ifnhere the more inflammatory cytokine cluster clearly associated with high viral loads for both rsv and mpv (fig 3d) . a c c e p t e d m a n u s c r i p t we developed the ordinary differential equation model in equation (1) to link rsv and mpv viral load and early and late immune responses and evaluated which cytokines may track those responses (fig s4-s6) . the models suggest that ifnγ and il-21 may play a major role in rsv and mpv control in vivo but do not rule out the effects of other cytokines and molecules in limiting infection. here we demonstrate that home self-sampling with nasal foam swabs is well-tolerated and provides reliable results for monitoring viral load and molecular immune responses to respiratory virus infection. these results have enormous practical implications. self-collection at home is safe, non-invasive and easily learned, allowing a reliable method for diagnosis and therapeutic monitoring. because our kits could easily be used at home or in a drive through testing environment, they provide an avenue to eliminate contact between an infected and contagious person, and health care providers. they could also be used in the hospital or clinic setting, thereby saving personnel time and personal protective equipment. the use of comfortable, safe and affordable foam swabs also highlights the possibility of scaling this approach to pediatric, adult, elderly and immunocompromised populations. for the current sars-cov-2 pandemic, and future deadly respiratory virus epidemics, home self-swabbing will be a vital tool. the simplicity of the sampling approach also facilitates large scale research studies of viral pathogenesis and transmission dynamics in which participants self-sample for months. a c c e p t e d m a n u s c r i p t we have previously demonstrated increased sensitivity of self-collected foam nasal swabs compared to nasal washes in immunocompetent adults with respiratory viral infections [21] , and in longitudinal studies in solid organ transplant recipients [22] , with good compliance and participants reporting no issues with swab discomfort. the specific swab used in these prior studies and our present study were custom designed to limit discomfort while maintaining adequate sensitivity; we have demonstrated stability with these swabs with and without transport media after storage at room temperature for 7 days [21] , making them ideal for home self-testing followed by shipment directly to a testing lab. our data also demonstrate that bilateral swabbing increases yield and allows for more accurate quantification than swabbing a single nostril. we also demonstrate an ability to accurately sample local cytokines with our swab, present at picogram levels. the combination of precise virologic and immunologic readouts of local infection is highly relevant for developing clinical severity scores and biomarkers. while studies are beginning to show that viral load may be predictive of covid-19 severity [7] , it is equally plausible that the intensity and phenotype of the early local cellular immune response plays a causal role in limiting the extent of infection [28] . by following the molecular immune response closely with daily sampling intervals, we also provide adequate data for mathematical models that can link specific arms of the cellular immune response to pathogen control in real time [20] , a goal that has been difficult to attain for a majority of viral infections in humans. our study demonstrates several novel features of respiratory virus kinetics. rsv infection achieves a brief, extremely high, viral load, followed by a steady state and a final rapid phase of elimination. hrv also has a remarkably stable viral load in most participants before being rapidly eliminated. during a majority of our observed episodes, viral shedding is strongly correlated with symptoms. as viral load decreases, symptoms tend to dissipate. m a n u s c r i p t in summary, we establish a foam swab-based sampling method that is optimal for patient selftesting, both at home and in the clinical setting, permits serial therapeutic monitoring, and is suitable for tracking the natural virologic and immunologic course of respiratory virus infections. we recommend that this method be adapted to future clinical and research applications, including for the study of sars-cov-2. m a n u s c r i p t m a n u s c r i p t an interactive web-based dashboard to track covid-19 in real time coronavirus mapped: which countries have the most cases and deaths? available at defining the epidemiology of covid-19 -studies needed viral load is strongly associated with length of stay in adults hospitalised with viral acute respiratory illness clinical correlation of influenza and respiratory syncytial virus load measured by digital pcr effects of oseltamivir treatment of index patients with influenza on secondary household illness in an urban setting in bangladesh: secondary analysis of a randomised, placebo-controlled trial evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections herpes simplex virus shedding rate: surrogate outcome for genital herpes recurrence frequency and lesion rates, and phase 2 clinical trials end point for evaluating efficacy of antivirals viral kinetic correlates of cytomegalovirus disease and death after hematopoietic cell transplant cytomegalovirus viral load and mortality after haemopoietic stem cell transplantation in the era of pre-emptive therapy: a retrospective cohort study use of viral load as a surrogate marker in clinical studies of cytomegalovirus in solid organ transplantation: a systematic review and metaanalysis the use of plasma hiv rna as a study endpoint in efficacy trials of antiretroviral drugs sofosbuvir and velpatasvir for hcv genotype 1, 2, 4, 5, and 6 infection endpoints of therapy in chronic hepatitis b the global impact of covid-19 and strategies for mitigation and suppression tissue-resident memory cd8(+) t cells: from phenotype to function lung-resident gammadelta t cells and their roles in lung diseases influenza virus infection model with density dependence supports biphasic viral decay tissue-resident t cell derived cytokines eliminate herpes simplex virus-2 infected cells self-collection of foam nasal swabs for respiratory virus detection by pcr among immunocompetent subjects and hematopoietic cell transplant recipients a patient self-collection method for longitudinal monitoring of respiratory virus infection in solid organ transplant recipients comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children a concordance correlation coefficient to evaluate reproducibility a new method for determining the type of distribution of plant individuals validating clusters using the hopkins statistic r: a language and environment for statistical computing mucosal host immune response predicts the severity and duration of herpes simplex virus-2 genital tract shedding episodes we would like to thank our study participants. key: cord-345727-bcxkycjh authors: karimata, yosuke; kinjo, takeshi; parrott, gretchen; uehara, ayako; nabeya, daijiro; haranaga, shusaku; higa, futoshi; tateyama, masao; miyagawa, keiko; kishaba, tomoo; otani, kanako; okamoto, michiko; nishimura, hidekazu; fujita, jiro title: clinical features of human metapneumovirus pneumonia in non-immunocompromised patients: an investigation of three long-term care facility outbreaks date: 2018-09-15 journal: j infect dis doi: 10.1093/infdis/jiy261 sha: doc_id: 345727 cord_uid: bcxkycjh background: several studies have reported outbreaks due to human metapneumovirus (hmpv) in long-term care facilities (ltcf) for the elderly. however, most of these reports are epidemiological studies and do not investigate the clinical features of hmpv pneumonia. methods: three independent outbreaks of hmpv occurred at separate ltcf for intellectually challenged and elderly residents. a retrospective evaluation of hmpv pneumonia and its clinical and radiological features was conducted using available medical records and data. results: in 105 hmpv infections, 49% of patients developed pneumonia. the median age of pneumonia cases was significantly higher than non-pneumonia cases (p < .001). clinical manifestations of hmpv pneumonia included high fever, wheezing in 43%, and respiratory failure in 31% of patients. an elevated number of white blood cells as well as increased levels of c-reactive protein, creatine phosphokinase, and both aspartate and alanine transaminases was also observed among pneumonia cases. evaluation of chest imaging revealed proximal bronchial wall thickenings radiating outward from the hilum in most patients. conclusions: the aforementioned characteristics should be considered as representative of hmpv pneumonia. patients presenting with these features should have laboratory testing performed for prompt diagnosis. human metapneumovirus (hmpv) was discovered in 2001; however, seroprevalence studies indicate this virus has circulated among humans for at least 60 years [1] . although it is considered a community-acquired respiratory virus, children <5 years of age experience hmpv infection at least once, and reinfection is common [1] . seasonal patterns of infection have been observed in several regions, with the majority of cases occurring from late winter to early spring in european countries, the united states, and canada, and between spring and summer in asian countries, including japan [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . even though it is usually a mild and self-limiting disease, hmpv can potentially cause severe lower respiratory infections, especially in young children, the elderly, and immunocompromised patients [3] [4] [5] [12] [13] [14] . several studies have reported outbreaks due to hmpv in long-term care facilities (ltcf) for the elderly and described the high incidence of pneumonia [3] [4] [5] [14] [15] [16] [17] . however, most reports are epidemiological studies, and they often do not include the clinical and radiological features of hmpv pneumonia. three independent outbreaks of hmpv occurred in ltcf for intellectually challenged and elderly residents in okinawa, japan. as a result, approximately 50% of the symptomatic patients developed pneumonia during the outbreaks. the objective of the present study was to retrospectively evaluate the clinical features, laboratory, and radiological findings of hmpv pneumonia. patients with hmpv infection were identified during 3 independent ltcf outbreaks in okinawa, a subtropical region of japan. the outbreaks were designated as outbreak a, b, and c, and the facilities where those outbreaks occurred are also identified as facility a, b, and c, respectively. outbreak a occurred in an ltcf for intellectually challenged patients from march 28 to april 30, 2012. outbreak b occurred in a complex containing a nursing home and a hospital for the elderly from march 26 to may 1, 2013. outbreak c occurred in a different ltcf for intellectually challenged patients from may 1 to june 11, 2013. within their grounds, facilities a and b have the capacity and staff to provide primary care and routine check-ups; however, facility c does not. during the outbreaks, confirmed cases of hmpv were defined as having clinical respiratory samples positive for virus via an hmpv-specific polymerase chain reaction (pcr) and/or rapid antigen test (rat). probable cases had recently acquired clinical manifestations compatible with a respiratory infection (eg, respiratory symptoms and fever ≥37.5°c), within the appropriate time frame. cases of febrile disease without respiratory symptoms, or diagnosed with alternative causation, were excluded from the sample population. the medical records for both confirmed and probable cases were retrospectively evaluated. the institutional review board of the university of the ryukyus approved this study. informed consent from each patient was waived because the study was retrospective in approach and caused no additional adverse events for any subjects. during outbreak a, nasal swabs were collected from 14 patients and tested with a rat for influenza virus. after all patients returned negative results, the residual liquid underwent nucleic acid extraction using a commercially available extraction kit (ribospin vrd; geneall, seoul, korea). purified eluent from the sample was tested using a multiplex reverse-transcription pcr kit (seeplex rv15 onestep ace detection; seegene, seoul, korea), which can detect 15 respiratory viruses, including hmpv, influenza virus a/b, human adenovirus, coronavirus, parainfluenza virus 1/2/3, rhinovirus a/b/c, respiratory syncytial virus a/b, bocavirus 1/2/3/4, and enterovirus, simultaneously. the seeplex rv15 onestep ace detection kit targets the hmpv n and l genes. during outbreak b, nasal swabs from 11 patients were tested with an hmpv rat (check hmpv; sa scientific, san antonio, tx) targeting the hmpv n and f proteins. residual liquid from the hmpv rat was also tested using the multiplex pcr method described above. during outbreak c, hmpv rat and multiplex pcr were used for 4 patients each. overall, multiplex pcr and rat was performed in 29 and 15 patients and returned 27 and 10 positive results, respectively. serum antibodies against hmpv were also examined in a subset of patients (n = 11) from outbreak a. acute phase serum samples were collected within 1 week after the onset of symptoms, and convalescent phase samples were collected 1 month later. the hmpv antibody titers were determined by enzyme-linked immunosorbent assay (elisa) using purified virion as the antigen, a method described by okamoto et al [18] . in brief, 96 microwell plates (thermo fisher scientific, waltham, ma) were coated with a sendai-155-d06 (hmpv-a) and sendai-1311-04 (hmpv-b) antigen mixture and refrigerated at 4°c for 2 hours. plates were then blocked for 1 hour at room temperature and washed. starting at a 1:100 dilution, 50-μl serial 2-fold dilutions of samples were added to the prepared plates and incubated at 37°c for 1 hour. plates were labeled with horseradish peroxidase-labeled goat anti-human igg (millipore, billerica, ma) and detected using a peroxidase substrate kit (bio-rad laboratories, hercules, ca) at 415 nm. patients with a ≥4-fold increase of antibody titers between the acute and convalescent phase were regarded as having an acute hmpv infection [19] . chest x-rays and computed tomography (ct) images were analyzed by 3 pulmonologists. attention was focused on distribution, location, and pattern of the abnormal shadows. bronchial wall thickenings and pleural effusions were also assessed. final decisions were reached by consensus. for chest ct images, dense consolidation was considered to be present when vascular margins were obscured. ground-glass opacity (ggo) was defined as a hazy increase in attenuation without obscuring vascular markings. centrilobular nodule was defined as either a nodule recognized near the peripheral pulmonary artery branches or 3 to 5 mm away from the pleura, interlobular septa, or pulmonary veins. continuous variables, such as age, body temperature, and laboratory findings, between pneumonia and non-pneumonia cases were compared using the wilcoxon/kruskal-wallis test. categorical variables were evaluated using the pearson's χ 2 test or fisher's exact test, when appropriate. a two-sided p value of <.05 was considered to be statistically significant. all data were analyzed with jmp version 13 (sas institute inc., cary, nc). during the outbreaks, 105 patients with hmpv infections (30 confirmed cases and 75 probable cases) were identified. each facility experienced 63 (12 confirmed cases), 22 (11 confirmed cases), and 20 (7 confirmed cases) hmpv infections. the attack rates were 16% (63 of 406 residents) in outbreak a, 10% (22 of 220 residents) in outbreak b, and 20% (20 of 100 residents) in outbreak c. patient characteristics and clinical manifestations are shown in table 1 . the most frequently encountered disorders were intellectual disabilities, cerebral palsy, and schizophrenia. among 105 hmpv infections, 51 cases (49%) developed pneumonia as evident on chest x-rays and/or ct. for 49 of 51 (96%) cases, pneumonia was diagnosed within 5 days after the onset of symptoms. pneumonia patients were significantly older than non-pneumonia cases (median age, 58 vs 44; p < .001). it is interesting to note that almost all patients with dementia or in a bedridden state acquired pneumonia. however, these patients were frequently septuagenarians or older (age range, 70-100 and 83-100, respectively; data not shown). pneumonia patients also developed higher fever (p = .0001), wheezing (p < .0001), and needed oxygen therapy administered (p = .0002) more frequently. sputum culture for bacterial identification was performed for 26 pneumonia patients; 14 were positive for pathogenic bacteria and 12 were negative. streptococcus pneumoniae was the most commonly isolated (n = 5) followed by pseudomonas aeruginosa (n = 4) (supplementary table 1 ). moraxella catarrhalis and haemophilus influenzae were coinfected in 1 patient. all pneumonia patients were treated with empirical antibiotics regardless of the bacterial test results. seven pneumonia patients from facility c, where acute medical care could not be provided, required admittance to a local hospital for care. most patients improved within 1 week and none of the patients died. blood tests were performed in 81 patients including 49 pneumonia and 32 non-pneumonia patients. median white blood cell (wbc) count, c-reactive protein (crp), aspartate aminotransferase (ast), alanine aminotransferase (alt), and creatine phosphokinase (cpk) concentrations were significantly higher for pneumonia cases than non-pneumonia cases ( figure 1 ). more than a 4-fold increase in antibody titers was observed between the acute and convalescent phase of 5 patients. these patients were regarded as having acute hmpv infection. among 6 other patients, 5 had titers more than 12 800 at either the acute or convalescent phase. because 80% of healthy japanese adults do not have titers over 3200 [18] , these 5 symptomatic patients were assumed to have an acute hmpv infection during an hmpv outbreak, regardless of pcr results. one final patient had no significant elevation in antibody titers but was also regarded as having acute hmpv infection due to a positive pcr result (supplementary table 2 ). the chest x-ray findings of 41 pneumonia patients are summarized in table 2 . for these patients, abnormal shadows were primarily found in the proximal area of the lung (100%) and bilateral distribution was common (80%). bronchial wall thickenings were observed in all 41 pneumonia patients. of note, proximal bronchial wall thickenings radiating outward from the hilum were observed in most patients, and representative images are depicted in figure 2 . abnormal opacities were observed in a patchy pattern (90%) more frequently than confluent (12%). table 3 summarizes the chest ct findings of 24 pneumonia patients. multilobar distribution was observed in all cases and lower lobes were frequently involved. again, abnormal shadows were primarily found in the proximal rather than peripheral areas of the lung. lobular opacity was the most common shadow pattern (92%), in contrast to ggo (21%) and dense consolidation (13%). bronchial wall thickenings were also frequently seen on ct images (96%). representative ct images of selected pneumonia patients are shown in figures 3 and 4 . a follow-up chest x-ray or ct was performed in 47 of 51 pneumonia patients (92.2%), and abnormal shadows seen during the outbreaks were diminished, as demonstrated in figures 2 and 4 . to date, many outbreaks due to hmpv have been reported; however, few studies focus on the clinical features of hmpv pneumonia. our data shows older ltcf residents, especially those with dementia or in a bedridden state, frequently developed pneumonia due to hmpv when compared with other residents. moreover, high fever, respiratory failure, wheezing, and elevated wbc, crp, ast, alt, and cpk levels were frequently observed among hmpv pneumonia cases. typical hmpv pneumonia chest images exhibit proximal bronchial wall thickenings radiating outward from the hilum. although it is frequently the cause of a mild, self-limiting respiratory infection in healthy children and adults, hmpv infection can also induce a severe infection in the elderly. falsey et al [20] reported that older adults more frequently experience dyspnea and wheezing, during the course of the disease, compared with younger patients. although no fatal cases were recorded in this cohort, the mortality rate of elderly patients in ltcf during an hmpv outbreak has been reported to be approximately 10% . the box and whisker plots describe the 10th, 25th, 50th, 75th, and 90th percentiles. *, p < .05; **, p < .01. [3, 5] , demonstrating again that elderly patients are susceptible to more severe hmpv infections. darniot et al [21] also demonstrated that older mice develop a more severe hmpv infection compared with young mice. nevertheless, outbreaks of hmpv also occurred in ltcf for intellectually disabled patients (facilities a and c), where residents were not elderly. in general, the immune system of intellectually disabled patients is not weak; however, we speculate they may have been susceptible to hmpv due to isolation and lack of exposure to natural infection of hmpv for an extended time. therefore, it is possible their antibodies against hmpv might be unusually diminished. the present study demonstrated that wbc and crp, ast, alt, and cpk concentrations were higher in patients with pneumonia than non-pneumonia infections. although scheuerman et al [22] reported that ast and alt were slightly human immunodeficiency virus, and coxsackie virus are the most commonly documented [23] , whereas legionella species, francisella tularensis, and s. pneumoniae [23, 24] are common among bacterial pathogens. respiratory pathogens, such as chlamydophila psittaci and mycoplasma pneumoniae, have also been implicated [25] . however, neither rhabdomyolysis nor elevated levels of cpk have been reported in patients with hmpv pneumonia before. more importantly, these data indicate that hmpv infection should be included in the differential diagnosis when we treat pneumonia patients with elevated cpk. chest imaging revealed bronchial wall thickenings radiating out from the hilum were common for hmpv pneumonia. proximal tramlines arising out from the hilum seem like "spider legs" on the chest x-ray. although some studies report the radiological findings of hmpv pneumonia in immunocompromised patients, studies conducted within immunocompetent a á bb adults or the elderly are limited. previous studies have shown interstitial infiltrates, ggo, as well as centrilobular nodules suggestive of bronchitis and bronchiolitis are common radiological features for hmpv pneumonia in immunocompromised patients [26] [27] [28] [29] . however, only 1 report describes chest images among elderly, immunocompetant inpatients during an hmpv outbreak. four of the 8 affected patients had abnormal shadows in the chest x-ray, and 3 of the 4 had linear shadows and were diagnosed with hmpv-induced bronchitis or bronchiolitis [17] . because ciliated airway epithelial cells are the primary targets for hmpv infection [30] , it is easily justifiable for bronchial wall thickenings, evidence of bronchitis or bronchiolitis, to be common radiological features in hmpv pneumonia. centrilobular nodules, interstitial infiltrates, and ggo were not frequently observed in the present study. it is possible that an immunocompromised host's weaker immune response to hmpv may allow the virus to spread into the peripheral bronchiole and lung parenchyma. however, this cohort did not contain immunocompromised individuals, and radiological findings may reflect a more reasonable host response to hmpv infections. unfortunately, complete and detailed information regarding symptoms and physical examinations were not possible due to study design. in addition, this study may contain other limitations beyond those expected of a retrospective study. first, hmpv rat and/or pcr were not performed in all symptomatic patients during the outbreaks. due to the restrictions of the national health insurance system in japan, rat and pcr testing for hmpv is not approved for diagnostic decisions in adult patients. as such, attending physicians in each ltcf consulted the university of the ryukyus' department of infectious diseases, respiratory, and digestive medicine as each outbreak waned. as a result, only a subset of affected patients could be tested. other laboratory testing, bacterial cultures, and imaging tests (eg, chest x-ray and ct) were also only performed on subsets of patients. moreover, by including fever in the case definition, it is possible that the mildest forms of hmpv infection were overlooked. elderly patients do not always exhibit fever during infection. however, using respiratory symptoms alone may have broadened the case definition beyond usefulness. our cohort contained multiple, and in many cases severe, underlying disorders. therefore, patients were frequently unable to self-report andphysicians had to rely solely on observation. thus, bias should be considered when interpreting these data. in conclusion, we report the clinical and radiological features of hmpv pneumonia in non-immunocompromised patients collected from 3 outbreaks in ltcf in okinawa, japan. as a common virus, hmpv is capable of causing outbreaks in ltcf and causing pneumonia, especially in the elderly. when treating adult pneumonia patients that present with the features described, physicians should consider hmpv infection and perform laboratory testing for prompt diagnosis and adequate infection control, especially in isolated and at-risk populations. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. a newly discovered human pneumovirus isolated from young children with respiratory tract disease epidemiology of human metapneumovirus outbreaks of human metapneumovirus in two skilled nursing facilities -west virginia and idaho an outbreak of severe respiratory tract infection caused by human metapneumovirus in a residential care facility for elderly in an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility outbreak of human metapneumovirus infection in norwegian children performance of a rapid human metapneumovirus antigen test during an outbreak in a 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elderly inpatients in japan development and evaluation of a whole virus-based enzyme-linked immunosorbent assay for the detection of human metapneumovirus antibodies in human sera longitudinal course of human metapneumovirus antibody titers and reinfection in healthy adults human metapneumovirus infections in young and elderly adults age-associated aggravation of clinical disease after primary metapneumovirus infection of balb/c mice human metapneumovirus (hmpv) infection in immunocompromised children infectious etiologies of rhabdomyolysis: three case reports and review rhabdomyolysis and bacterial pneumonia communityacquired pneumonia with rhabdomyolysis human metapneumovirus infection in hematopoietic stem cell transplant recipients: high-resolution computed tomography findings clinical characterization of human metapneumovirus infection among patients with cancer the human metapneumovirus: a case series and review of the literature human metapneumovirus (hmpv) associated pulmonary infections in immunocompromised adults-initial ct findings, disease course and comparison to respiratory-syncytial-virus (rsv) induced pulmonary infections experimental human metapneumovirus infection of cynomolgus macaques (macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract we thank haley cash for help in laboratory testing. we also thank the physicians and other healthcare workers of the national hospital organization ryukyu hospital, hokuzan hospital, and okinawa ryouikuen.financial support. this work was funded by a research grant from okinawa prefectural government.potential conflicts of interest. all authors are without any conflict of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. key: cord-352526-t8odetzw authors: pinto, bruna g g; oliveira, antonio e r; singh, youvika; jimenez, leandro; gonçalves, andre n a; ogava, rodrigo l t; creighton, rachel; peron, jean pierre schatzmann; nakaya, helder i title: ace2 expression is increased in the lungs of patients with comorbidities associated with severe covid-19 date: 2020-06-11 journal: j infect dis doi: 10.1093/infdis/jiaa332 sha: doc_id: 352526 cord_uid: t8odetzw patients who died from covid-19 often had comorbidities, such as hypertension, diabetes, and chronic obstructive lung disease. although angiotensin-converting enzyme 2 (ace2) is crucial for sars-cov2 to bind and enter host cells, no study has systematically assessed the ace2 expression in the lungs of patients with these diseases. here, we analyzed over 700 lung transcriptome samples of patients with comorbidities associated with severe covid-19 and found that ace2 was highly expressed in these patients, compared to control individuals. this finding suggests that patients with such comorbidities may have higher chances of developing severe covid-19. correlation and network analyses revealed many potential regulators of ace2 in the human lung, including genes related to histone modifications, such as hat1, hdac2, and kdm5b. our systems biology approach offers a possible explanation for increase of covid-19 severity in patients with certain comorbidities. a c c e p t e d m a n u s c r i p t 3 recent studies of the epidemiological characteristics of covid-19 have revealed that severe infection is more likely in people with an existing chronic medical condition. two independent studies of infected populations in wuhan, china found that approximately half the subjects infected with covid-19 had an existing comorbidity (1, 2). in a study of 1099 patients across mainland china, 38.7% of patients with comorbidities progressed to severe infection (3) . 2020), and in a study of 52 inpatients in wuhan, 67% of patients with comorbidities died (2) . the most common comorbidities reported in these studies were hypertension, diabetes, cerebrovascular disease, chronic obstructive lung disease, and coronary heart disease (1-3). other comorbidities such as carcinoma, chronic kidney disease, chronic liver disease, digestive system disease, and nervous system disease have also been reported in patients with 2, 4) . a better understanding of the link between these conditions and covid-19 infection is required to inform better treatment and prevention interventions. the molecular mechanism responsible for the increased disease severity in patients with these comorbidities is not fully understood, but previous studies suggest a role for angiotensin-converting enzyme 2 (ace2) (5) . ace2 is a membrane protein required for sars-cov2 to bind and enter cells (6) (7) (8) . after binding, viral entry is facilitated by the activation of the viral spike glycoprotein and cleavage of the c-terminal segment of ace2 by proteases like tmprss2 and furin that are readily expressed in lung tissue (9) (10) (11) . ace2 is only moderately expressed in healthy lung tissue compared to the heart, kidneys, and testes (12) , but a c c e p t e d m a n u s c r i p t 4 staining of lung tissue sections from adults with pulmonary hypertension has revealed increased ace2 protein in the endothelium of pulmonary arteries, compared to healthy controls (13) . a comprehensive analysis of single-cell rna-seq datasets revealed that ace2 was co-expressed with tmprss2 within ileal absorptive enterocytes, nasal goblet secretory cells, and lung type ii pneumocytes (14) . ace2 upregulation has also been observed in animal models of liver fibrosis (15). however, the reason for this upregulation remains unclear, and a link to other covid-19 comorbidities has not been determined. here, we showed that the expression of the gene encoding the ace2 receptor in lung tissue is upregulated by diseases representing comorbidities along with covid-19. we also used systems biology approaches including co-expression analysis, meta-analysis, and network analysis to determine a potential cause of the ace2 upregulation. from this analysis, we found that ace2 expression could be regulated by enzymes that modify histones, including kdm5b. this identification of a common molecular mechanism of increased covid-19 severity in patients with diverse comorbidities could direct the development of interventions to reduce the infection risk and disease severity in this population. a c c e p t e d m a n u s c r i p t 5 relevant scientific literature related to key covid-19 morbidities was retrieved from pubmed on march 16, 2020 using the query terms "pulmonary hypertension," "chronic obstructive pulmonary disease," "hypertension," "smoking," "pulmonary fibrosis," and "asthma". for terms returning more than 100,000 papers ("hypertension," "smoking," and "asthma"), only the most recent 100,000 papers were analyzed. abstracts were annotated to identify all genes, diseases, and species appearing in the title or abstract using the pubtator central api (16) . this open source tool uses taggerone for disease annotations, gnormplus for gene annotations, and sr4gn for species annotations (16) . the data were filtered to retain only papers containing a human species annotation. annotation of the abstract text identified 6 relevant disease mesh terms: "autoimmune diseases," "cardiovascular diseases," "familial primary pulmonary hypertension," "hypertension," "hypertension, pulmonary," and "renal insufficiency, chronic", which were used in figure 1 . next, every possible combination of gene and disease annotation within the title and abstract of each paper was generated. only genedisease associations supported by at least four documents, and those with a proximity less than or equal to the median sentence length of the paper section were retained. gene ids were converted to gene symbols using the biomart r package (17, 18) , and disease ids were converted to disease mesh terms using the entrez programming utilities to query the entrez database provided by the national center for biotechnology information. the data was then further filtered to retain disease a c c e p t e d m a n u s c r i p t 6 mesh terms relevant to reported clinical covid-19 comorbidities (3). redundant terms were collapsed using fuzzy string matching. the final gene-disease data set was used to generate a network utilizing gephi software where the nodes were genes and diseases, and the edge weight was determined by the number of analyzed papers containing the gene-disease combination (19) . we manually curated gene expression omnibus (geo) repository (https://www.ncbi.nlm.nih.gov/geo/) on march 16, 2020 to find lung transcriptome datasets related to "pulmonary arterial hypertension" (pah), "chronic obstructive pulmonary disease" (copd), and "smoking". author-normalized expression values and metadata from these datasets were downloaded using the geoquery package (20) . we performed differential expression analyses between patients with a disease and the control individuals (see table s1 ) using the limma package (21) . the gene symbol for each probe was obtained from the annotation file (22) . probes that matched the same gene symbol were collapsed by taking the one with the lowest pvalue. meta-analysis was performed with the metavolcanor package (23) by combining the p-values using fisher's method. to adjusting for multiple comparisons, we calculated the false discovery rate (fdr) to identify the differentially expressed genes (fdr < 0.05). for enrichment analyses, we utilized the enrichr tool (24) with the "go biological process 2018" and "bioplanet 2019" databases. we then selected pathways with a p-value adjusted for multiple comparisons lower than 0.05. the network was created in cytoscape (25) . to identify the genes highly associated with key comorbidities of severe covid-19 (1, 3), we mined all relevant scientific literature of these human diseases. specifically, over 8,000 abstracts were gathered from pubmed by querying titles and abstracts for the terms "pulmonary hypertension," "chronic obstructive pulmonary disease," "hypertension," "smoking," "pulmonary fibrosis," or "asthma" (figure 1a ). several relevant terms, such as "autoimmune diseases" and "cardiovascular diseases" were excluded from the pubmed query because of the breadth of literature published in these fields. annotation of the abstract text identified 6 relevant disease mesh terms: "autoimmune diseases," "cardiovascular diseases," "familial primary pulmonary hypertension," "hypertension," "hypertension, pulmonary," and "renal insufficiency, chronic." (3) (figure 1a) . our text-mining analysis revealed 804 genes highly associated with one or more covid-19 morbidities (figure 1b) . among those genes, 26 were associated with four or more diseases (figure 1c) . although ace2 was known to be related to "cardiovascular diseases", "familial primary pulmonary hypertension", "hypertension, pulmonary", and "hypertension", none of the articles containing this gene-disease association studied how ace2 expression was altered in the lungs of patients with these diseases. based on the list of key comorbidities of severe covid -19 (1, 3) , we searched for lung transcriptome datasets available at geo repository. we identified seven lung transcriptome studies of patients with either chronic obstructive pulmonary disease (copd) or pulmonary arterial hypertension (pah), as well as smoking volunteers, compared to individuals who were non-smoking volunteers, were downloaded and used in our meta-analysis (table s1) . for each study, we performed differential expression analysis between patients and control individuals (table s1) . by we then decided to investigate whether the gene encoding the ace2 receptor was specifically up-regulated in the lungs of patients having one of these morbidities (figure 3a) . in a lung rna-seq dataset (table s1) , we compared ace2 expression between patients with copd and subjects with normal spirometry (30) . again, the expression of ace2 was significantly up-regulated in the disease compared to controls (figure 3b ). in fact, ace2 was significantly up-regulated in 6 out of 7 lung transcriptome studies (figure 3c (table s1 ), combined the p-values using fisher's method, and applied an fdr correction (figure 4a ). this approach identified 544 and 173 genes with positive and negative correlation with ace2, respectively (figure 4a) . several of these genes were related to histone modifications, such as hat1, hdac2, kdm5b, among others (figure 4a ). among the positively correlated genes, we found adam10 that regulates ace2 cleavage in human airway epithelia (32) and tlr3 that plays a key role in the innate response to sars-cov or mers-cov infection (33) . pathway enrichment analysis revealed that several of the genes positively associated with ace2 were regulated by kdm5b, and by specific histone acetylation (h3k27ac) and histone methylation (h3k4me1 and h3k4me3) (figure 4b ). in fact, kmd5b demethylates lysine 4 of histone h3 (i.e. h3k4) and is involved in transcriptional regulation and dna repair (34) . we then checked in the roadmap epigenomics project database (26) to see whether ace2 locus contained chip-seq information for these histone markers. in the human lung, peaks for h3k4me1 and h3k4me3, as well as h3k27ac, were identified in ace2 locus (figure 4c ), suggesting that ace2 may be epigenetically regulated in the lung. we showed here that patients with co-morbidities which have very distinct mechanisms have increased expression of ace2 in the lungs. although our findings did not include covid-19 infection data, we suggest that the higher expression of ace2 in the lungs is associated with higher chances of developing the severe form of covid19, by facilitating the sars-cov-2 entry into lung cells during the infection. in fact, covid19 patients classified as severe cases displayed higher viral loads in nasopharyngeal swab samples during the early stages of disease onset compared to mild patients (35) . the current diabetes pandemic (36) could be worsening the sars-cov-2 pandemic by increasing the comorbidities associated with severe covid-19. as we did not find lung transcriptome samples from patients with type 2 diabetes, we could not directly test whether ace2 expression was increased in patients with diabetes, compared to healthy controls. however, our text-mining approach revealed that il-6 and ins genes were associated with all the diseases we searched. the ins gene encodes the insulin hormone, and insulin is associated with the nad-dependent histone deacetylase sirtuin 1 (sirt1) (37) . we found that sirt1 was up-regulated in the lung of patients with severe covid-19 comorbidities in 4 out 7 studies (data not a c c e p t e d m a n u s c r i p t 12 shown). clarke et al (38) have demonstrated that, under conditions of cell energy stress, sirt1 can epigenetically regulate ace2. others too have shown that nonsteroidal anti-inflammatory drugs may inhibit the sirt1 deacetylase activity (39) , which in turn could impact ace2 expression. the "viral life cycle" pathway that was enriched with up-regulated genes in patients with severe covid-19 comorbidities contains several genes other than ace2 that can be potentially important for sars-cov-2 cell cycle and invasion/attachment. these include rab1a gene, whose product promotes the replication of vaccinia virus (40). also, rab1a is important for herpes simplex virus 1 secondary envelopment (41) , and is required for assembly of classical swine fever virus particle (42) . it is possible that sars-cov-2 utilizes rab1a as well. the fact that ace2 gene is located in the x chromosome, and the initial findings showing that older males with comorbidities are more likely to be have severe covid-19 compared to females (1), indicate that ace2 expression in the lung may be sex-biased. although no significant sexual differences was found in the activity of ace2 in mouse lung (43) , in rats, the levels of ace2 were dramatically reduced with aging in both genders, but with significantly higher ace2 expression in old female rats than male (44) . although the mechanisms by which ace2 is up-regulated in patients with severe covid-19 comorbidities were not addressed, our analysis may shed some light on the subject. among the genes whose expression was positively correlated with ace2, we detected genes associated with epigenetic regulation of gene transcription. for instance, hat and hdac modulate chromatin and dna condensation by changing histone acetylation status, thus permitting gene a c c e p t e d m a n u s c r i p t 13 transcription. this could be happening in lung tissue, facilitating ace2 expression, as observed during lung cancer and copd. kdm5b is associated with infection of hepatitis b virus (45) . in breast cancer cells, blockage of kdm5 triggers a robust interferon response that results in resistance to infection by dna and rna viruses (46) . this finding suggests that kdm5 demethylases are potential targets for preventing sars-cov-2 infection. covid-19 may kill between 5.6% and 15.2% of people infected with sars-cov-2 (47) . drug treatments that lower this mortality rate may save many thousands of lives. our systems biology approach offers putative gene targets for treating and preventing severe covid-19 cases. (table s1) the original studies are indicated and can be found in table s1 . the author reports no conflicts of interest in this work. not applicable as we utilized publicly available data. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirusinduced lung injury structural basis for the recognition of the sars-cov-2 by full-length human ace2 a pneumonia outbreak associated with a new coronavirus of probable bat origin expression of transmembrane serine protease tmprss2 in mouse and human tissues a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry different host cell proteases activate the sars-coronavirus spike-protein for cell-cell and virus-cell fusion a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase expression of pulmonary vascular angiotensin-converting enzyme in primary and secondary plexiform pulmonary hypertension sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues upregulation of angiotensin-converting enzyme (ace) 2 in hepatic fibrosis by ace inhibitors pubtator central: automated concept annotation for biomedical full text articles mapping identifiers for the integration of genomic datasets with the r/bioconductor package biomart biomart and bioconductor: a powerful link between biological databases and microarray data analysis the international aaai conference on weblogs and social media geoquery: a bridge between the gene expression omnibus (geo) and bioconductor limma powers differential expression analyses for rna-sequencing and microarray studies long noncoding rnas are involved in multiple immunological pathways in response to vaccination enrichr: interactive and collaborative html5 gene list enrichment analysis tool cytoscape: a software environment for integrated models of biomolecular interaction networks the human epigenome browser at washington university viral interactions with host cell rab gtpases structure, function, and antigenicity of the sars-cov-2 spike glycoprotein the transcriptional landscape and mutational profile of lung adenocarcinoma comprehensive analysis of transcriptome sequencing data in the lung tissues of copd subjects webcemitool: co-expression modular analysis made easy ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection small molecule inhibitors of kdm5 histone demethylases increase the radiosensitivity of breast cancer cells overexpressing jarid1b viral dynamics in mild and severe cases of covid-19 the diabetes pandemic and associated infections: suggestions for clinical microbiology epigenetic regulation of angiotensinconverting enzyme 2 (ace2) by sirt1 under conditions of cell energy stress inhibition of sirt1 deacetylase and p53 activation uncouples the antiinflammatory and chemopreventive actions of nsaids rab1a promotes vaccinia virus replication by facilitating the production of intracellular enveloped virions analysis of rab gtpase-activating proteins indicates that rab1a/b and rab43 are important for herpes simplex virus 1 secondary envelopment rab1a is required for assembly of classical swine fever virus particle sex differences in renal angiotensin converting enzyme 2 (ace2) activity are 17beta-oestradiol-dependent and sex chromosome-independent age-and gender-related difference of ace2 expression in rat lung hepatitis b virus x protein induces hepatic stem cell-like features in hepatocellular carcinoma by activating kdm5b kdm5 histone demethylases repress immune response via suppression of sting real estimates of mortality following covid-19 infection we would like to thank tiago lubiana for his valuable inputs. a c c e p t e d m a n u s c r i p t query: "pulmonary hypertension", "chronic obstructive pulmonary disease", "hypertension", "smoking", "pulmonary fibrosis", or "asthma" key: cord-345101-h0i5o0do authors: koo, bon-sang; oh, hanseul; kim, green; hwang, eun-ha; jung, hoyin; lee, youngjeon; kang, philyong; park, jae-hak; ryu, choong-min; hong, jung joo title: transient lymphopenia and interstitial pneumonia with endotheliitis in sars-cov-2-infected macaques date: 2020-08-03 journal: j infect dis doi: 10.1093/infdis/jiaa486 sha: doc_id: 345101 cord_uid: h0i5o0do using a reliable primate model is critical for developing therapeutic advances to treat humans infected with severe acute respiratory syndrome coronavirus-2 (sars-cov-2). here, we exposed macaques to high titres of sars-cov-2 via combined transmission routes. we observed acute interstitial pneumonia with endotheliitis in the lungs of all infected macaques. all macaques had a significant loss of total lymphocytes during infection, which were restored over time. these data show that sars-cov-2 causes a coronavirus disease 2019 (covid-19)-like disease in macaques. this new model could investigate the interaction between sars-cov-2 and the immune system to test therapeutic strategies. m a n u s c r i p t 3 in march 2020, the world health organization classified coronavirus disease 2019 (covid-19) as a pandemic. there is an urgent need for rapid diagnosis and development of therapeutic modalities. the absence of a reliable preclinical animal model that recapitulates patients with severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infection poses a major limitation to the development of improved diagnostics and therapeutics. the immune system of non-human primates (nhps) resembles that of humans, and the structure of the ace2 receptor is very similar [1] . however, current studies in nhps infected with human coronaviruses have shown inconsistent outcomes. in sars-covinfected macaques, previous studies reported significant lung lesions [2, 3] , while others found oedema and inflammation in alveoli, with mild clinical findings [4, 5] . studies reported that sars-cov-2 developed no severe clinical signs, but pulmonary pneumonia in 50% of cynomolgus macaques, recapitulating mild symptoms in humans [6] . however, others demonstrated that macaques showed pulmonary infiltrates and high viral loads in the lungs, suggesting moderate disease [7] . in this study, we establish a promising nhp model by addressing the following: 1) genetic and immunological variability in subspecies, 2) inoculum dose, 3) inoculum route, 4) virulent strain of virus isolated from patient, and 5) demographic background of nhps. a c c e p t e d m a n u s c r i p t 4 sixteen male and female macaques, including eight healthy cambodian-origin cynomolgus (macaca fascicularis) and chinese-origin rhesus macaques (macaca mulatta), aged 3-6 years, were selected by the institutional veterinary experts based on their general health. all animals reared in indoor cages in the animal biosecurity level 3 (abl-3) laboratory in the korea national primate research centre (knprc) at the korea research institute of bioscience and biotechnology (kribb). animals were anaesthetized with a combination of ketamine sodium (10 mg/kg) and tiletamine/zolazepam (5 mg/kg) for viral challenges, swabs, and blood collection. as illustrated in supplementary figure 1a , all animals were challenged with a total of 12.5 ml of virus (2.1 × 10 6 tcid50/ml) via intratracheal (4 ml), oral (5 ml), conjunctival (0.5 ml), intranasal (1 ml), and intravenous (2 ml) route. both cynomolgus and rhesus macaques (n= 4 each species, two males and two females) were euthanized and necropsied at 3 days post infection (dpi). after viral challenges, all live animals were subjected to swab sampling of nasopharyngeal, oropharyngeal, conjunctival, and rectal tissues, at 0, 1, and 3 dpi. all swab samples were collected in universal viral transport medium, centrifuged (1600 × g for 10 min), and filtered with 0.2 μm pore size syringe filters for further virus quantification. upon necropsy, tissue samples, including respiratory, immune, intestinal, cardiovascular, and reproductive organs were grossly examined, and collected for viral detection and microscopic examination. all animal procedures were approved by the kribb institutional animal care and use committee (permit no. a c c e p t e d m a n u s c r i p t 5 a sars-cov-2 virus (accession no. 43326) isolated from a korean patient was obtained from the national culture collection for pathogens (cheongju, korea). this pathogen was passaged three times in vero cells. the virus titre expressed as 50% tissue culture infectious doses/ml (tcid50/ml), was measured in vero cells and determined using the reed and muench method. all procedures were performed in a biosafety cabinet class ⅱ in the abl-3 facility in the knprc at the kribb (permit no. kribb-ibc-20200206). all tissue samples were diluted ten-fold (w/v) with sterile phosphate buffered solution (pbs, ph 7.4) and homogenized using precellys homogenizer (bertin instruments). after centrifugation, the supernatants were directly inoculated into vero cells and incubated for 3 days at 37℃, for virus isolation to calculate the values of tcid50/ml. the viral rna genome was extracted from the supernatant using qiaamp viral rna mini kit (qiagen) and stored at -80℃ in the abl-3 facility until use. rt-qpcr was performed with a primer set targeting partial regions of the orf1b gene in the sars-cov-2 virus using the qiagen onestep rt-pcr kit (qiagen) as previously reported [8] . for histopathological examination, all tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and 4-to 5-μm sections were stained with haematoxylin and eosin. for the staining of sars-cov-2 antigens, the immunohistochemistry assay was performed (see the supplemental materials for more detailed information). haematological evaluation was conducted using an auto-haematology analyser (mindray bc-5000) on exam days. whole blood samples were stained with antibodies. after using facs tm lysing solution (bd biosciences), blood cells were washed with pbs containing 2% foetal bovine serum. group differences were determined using one-way analysis of variance (anova) with tukey's posthoc test in prism version 8.4.2 (graphpad software). significance was set at p< 0.05. to determine whether animals had depressed behaviour during wake/sleep states after sars-cov-2 infection, the average mean locomotion activity (mla) was measured by acticals attached to collars for 3 days before and after infection. we observed decreased activity in some animals post infection (supplementary figure 1b) . no changes in weight and respiratory rate were observed, but most animals (13/16) showed an increase in temperature 1 dpi and returned to baseline thereafter (supplementary figure 1c) . post-mortem examination of all animals at 3 dpi showed multifocal, bright red lesions in the upper, middle, and lower lobes of the lungs ( figure 1a ). in these pulmonary lesions, acute interstitial pneumonia (thickening of alveolar wall with type-ii pneumocyte hyperplasia, mononuclear and polymorphonuclear (pmn) leukocytes infiltration) was observed ( figure 1b) and were similar to previous data in humans [9] and monkeys [6] . additionally, endotheliitis was observed in the lungs of all animals (mononuclear and pmn leukocyte infiltrates within the intima of many vessels, oedema of vessel, and focal haemorrhage) ( figure 1b and c) , which is in line with a recent report [10] . the viral rna was highest in the upper respiratory swab samples and lung tissues at the earliest phase of infection, and the viral antigen was present in the lungs ( figure 1c and d) , suggesting the predominant site of the virus. however, only low viral levels (below 10 2 ) were present in most other organs and not detected in plasma at any time point (supplementary figure 2) . this low viral rna lacking replication in tissues could be detected by the presence of virus inoculated via multiple routes. our rt-qpcr, with a primer and probe set specific for orf1b, could not differentiate the reproducible viral rna a c c e p t e d m a n u s c r i p t 8 from inoculated viral rna. without exception, all macaques had a significant decrease in total lymphocytes counts, including cd4 + and cd8 + t cells, b cells, and nk cells at 1 dpi (figure 2a and b) . this loss was observed across naïve and memory subpopulations of t and b cells (supplementary figure 3) . however, our nhp model showed that these decreased values gradually recovered and returned to baseline at 7 dpi (figure 2a and b) . rnaseq analyses revealed that genes relevant to the viral response (such as those associated with cytokine/chemokine signalling) were significantly elevated in pbmcs at 3 dpi, but no viral copies were detected ( figure 2c and d) . here, we exposed nhps to a high viral titre via combined routes, including the ocular route, nasal cavity, trachea, oral route, and blood, which have been reported as the possible routes of transmission [11] [12] [13] , regardless of it not being a physiological route. using a high viral titre administered through combined routes, virus assays, and histopathological changes suggests that both cynomolgus and rhesus macaques are permissive to infection of sars-cov-2 and recapitulate covid-19-like disease in human. during early infection, acute interstitial pneumonia with endotheliitis was observed in the lungs of all infected macaques. upper and lower respiratory tracts were the predominant sites of virus replication. this nhp model may also be suitable for investigating interactions between sars-cov-2 and the a c c e p t e d m a n u s c r i p t 9 human immune system. all macaques had a significant loss of total lymphocytes, including cd4 + and cd8 + t cells, b cells, and nk cells during early infection. similarly, lymphopenia was reported in 83.2% of hospitalized sars-cov-2 patients [14] . the alteration of peripheral lymphocyte subsets seems to be correlated with severe clinical cases [15] . therefore, the nhp model during early infection may be used to validate the effect of immune modulators (e.g. il-7) in combination with therapeutic approaches to improve lymphopenia. receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome cynomolgus macaque as an animal model for severe acute respiratory syndrome macaque model for severe acute respiratory syndrome comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model respiratory disease in rhesus macaques inoculated with sars-cov-2 molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia pathological study of the 2019 novel coronavirus disease (covid-19) through postmortem core biopsies endothelial cell infection and endotheliitis in covid-19 detection of sars-cov-2 in different types of clinical specimens evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov-2 infection evidence for gastrointestinal infection of sars-cov-2 clinical characteristics of coronavirus disease 2019 in china characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia the authors would like to thank the staff of the korea national primate research centre for their the authors declare no competing financial interests.a c c e p t e d m a n u s c r i p t 11 key: cord-350737-nrtrhq1f authors: chen, xinchun; zhou, boping; li, meizhong; liang, xiaorong; wang, huosheng; yang, guilin; wang, hui; le, xiaohua title: serology of severe acute respiratory syndrome: implications for surveillance and outcome date: 2004-04-01 journal: j infect dis doi: 10.1086/380397 sha: doc_id: 350737 cord_uid: nrtrhq1f background. severe acute respiratory syndrome (sars) is a novel infectious disease. no information is currently available on host-specific immunity against the sars coronavirus (cov), and detailed characteristics of the epidemiology of sars cov infection have not been identified. methods. elisa was used to detect antibody to sars cov. reverse-transcriptase polymerase chain reaction was used to detect sars cov rna. t cells in peripheral blood of patients were quantified by flow cytometry. results. of 36 patients with probable sars cov infection, 30 (83.3%) were positive for igg antibody to sars cov; in contrast, only 3 of 48 patients with suspected sars cov infection, 0 of 112 patients with fever but without sars, and 0 of 96 healthy control individuals were positive for it. igg antibody to sars cov was first detected between day 5 and day 47 after onset of illness (mean ± sd, 18.7±10.4). conclusion. detection of antibody to sars cov is useful in the diagnosis of sars; however, at the incubation and initial phases of the illness, serological assay is of little value, because of late seroconversion in most patients. severe acute respiratory syndrome (sars) is a novel infectious disease with global impact. since november 2002, an outbreak of sars has affected 33 countries on 5 continents, with 8435 reported cases and 789 deaths at the time when a world health organization (who) report was published in 2003 [1] . a virus from the family coronaviridae, termed "sars coronavirus" (sars cov), has been identified as the cause [2] [3] [4] [5] [6] [7] , and criteria for laboratory confirmation of sars cov infection have been provided by who, on the basis of the following methods: (1) detection of sars cov rna by reversetranscription polymerase chain reaction (rt-pcr); (2) serological detection of sars cov-related antibody; and (3) isolation of sars cov by cell culture [4] . thus, an investigation of the profile and implications of the presence of specific anti-sars cov antibody would be likely to provide information that would be beneficial in diagnostics (confirmation and exclusion of sars cases) and that could function as a valuable indicator to be used in the analysis of host-specific immunity against sars cov and of the character of sars cov infection. the immunology and characteristics of sars cov infection have not yet been fully understood, because there has been such a short span of time since the outbreak of sars and because of the unavailability of such tools as a detection reagent. using an indirect immunofluorescence assay and parallel acute and convalescent serum samples obtained from patients with sars, tested for igg antibody to sars cov, peiris et al. recently documented seroconversion of igg antibody in 93% of patients, at a mean of 20 days [5] . the results of peiris et al.'s study prompted us to study the serology and humoral immunity of sars cov infection. to gain a comprehensive understanding of antibody to sars cov, additional clarification, such as that which would be gained by moredetailed profiles of igg and igm antibodies to sars cov (by such convenient assays as elisa), was required. we also sought to determine the implications of these antibody profiles. included in the present study were 36 patients with probable sars cov infection and 48 patients with suspected sars cov infection. sars was diagnosed on the basis of the case definition provided by who [5] . also included in the study were 112 patients with fever but without sars who were admitted to our hospital during the study's time frame; in addition, 96 healthy individuals, all health-care workers, were included as controls. of the 112 patients with fever but without sars, 35 had an upper-respiratory-tract infection, 46 had pneumonia, 22 had influenza a, and 9 had pulmonary tuberculosis. the 96 individuals in the control group consisted of 36 physicians and 60 nurses, all of whom came into close contact with patients with sars and routinely underwent isolation procedures prior to contact. all of the patients and control individuals who participated in the present study were negative for hiv. the characteristics of the study participants are shown in table 1. all patients and control individuals were informed of the purposes, procedures, and content of our clinical study, and all clinical samples were obtained from them after they had given written informed consent. we obtained clinical specimens of serum, nasopharyngeal aspirate, feces, and whole blood from all patients with probable sars cov infection and all of the patients with suspected sars cov. serum samples were obtained every 3-4 days during the first month after the patients' admission to the hospital and, thereafter, every week until 60 days after the onset of fever. samples of nasopharyngeal aspirate and of feces were obtained on day 0 (i.e., the day of admission) and on day 7 after the patients' admission. whole blood for the measurement of cd4 + and cd8 + t cells was obtained on day 0. for patients with fever but without sars, serum samples were obtained on days 0 and 21, samples of nasopharyngeal aspirate and of feces on days 0 and 7, and samples of whole blood on day 0. for the control group, serum samples were obtained on days 7, 21, and 90 after their first contact with patients with sars; and samples from nasal swabs and samples of feces were obtained on days 7 and 90. complete sars cov particles purified from the supernatant of sars cov-infected vero e6 cell cultures were used as antigen in the detection of igg and igm antibodies to sars cov, by use of an elisa kit (jibiai biotech). all serum samples were stored at ϫ30њc, and the elisas for all samples were performed in parallel. the procedures and the interpretation of the results strictly followed the elisa supplier's instructions. total rna was extracted from the clinical samples, as described elsewhere [6] , by use of a commercial rna-extraction kit. in brief, a qiaamp viral rna mini kit (qiagen) was used for samples from nasal swabs and for samples of nasopharyngeal aspirate, and a qiaamp stool kit (qiagen) was used for samples of feces. for nested rt-pcr, 5 ml of total rna obtained from each clinical sample was reverse transcribed by use of the oligonucleotide 5 -aatgtttacgcaggtaagcg-3 (nt 15627-15608); then the cdna was amplified by use of the outer primers 5 -cagag-ccatgcctaacatg-3 (nt 15239-15257) and 5 -aatgttt-acgcaggtaagcg-3 (nt 15608-15627) and, subsequently, by use of the inner primers 5 -tgttaaaccaggtggaac-3 (nt 15376-15393) and 5 -cctgtgttgtagattgcg-3 (nt 15515-15532) [7] . real-time quantitative rt-pcr assays were performed as described elsewhere [6] , by use of a commercial kit (realart hpa-coronavirus lc rt-pcr reagents; roche biomedical laboratories). rt-pcr was performed on a lightcycler (roche biomedical laboratories), in accordance with the manufacturer's instructions. all samples positive for sars cov rna were confirmed when direct dna sequencing, by use of a dna sequence analyzer (abi 3100; applied biosystems), indicated 99.4% nucleotide-sequence alignment with the sars cov sequence published by genbank (accession numbers gi29826276, gi30027610, and gi30027610). in brief, samples of whole blood were collected in edta and were immunolabeled with monoclonal antibodies cd4-fitc, cd8-pe, and cd3-cy5 (immunotech) at room temperature; mouse igg1-fitc, mouse igg1-pe, and mouse igg1-cy5 (immunotech) were incubated as isotype controls, to allow for subtraction of nonspecific staining. red blood cells were lysed, for 10 min at room temperature, by use of 0.5 ml optilyse c (immunotech), and the cells were then washed 2 times with 0.5 ml of pbs and were resuspended for flow-cytometry analysis by use of a coulter epics xl (beckman-coulter). statistical analysis. continuous variables were compared by student's t test, and correlation was assessed by pearson correlation analysis, both by use of the software program spss version 10.0; was considered to be statistically significant. p ! .05 the production of igg and igm antibodies to sars cov were seen as early as day 3 and day 5, respectively, after the onset of fever. of the 36 patients with probable sars cov infection, 9 (25.0%) had not produced anti-sars cov antibody by day 21, and 6 (16.7%) had not produced it by day 60 (figure 1). of 48 patients with suspected sars cov infection, 3 (6.3%) were positive for igg antibody to sars cov and 2 (4.2%) were positive for igm antibody to sars cov. sars cov sequence published by genbank (accession numbers gi29826276, gi30027610, and gi30027610). moreover, all samples positive for sars cov rna were further confirmed when real-time pcr indicated a viral load of 110 5 copies/ml. no patients with suspected sars cov infection, no patients with fever but without sars, and no control individuals were positive for sars cov rna. in addition, there was no difference between the amount of anti-sars cov antibody produced by patients positive for sars cov rna and that produced by patients negative for sars cov rna. the relationship between the production of igg antibody to sars cov and t cell immunity. we quantified cd3 + cd4 + t cells and cd3 + cd8 + t cells in the peripheral blood of the patients with probable sars cov infection and of those with suspected sars cov infection. our results indicated that all of these patients experienced a dramatic decrease in cd3 + cd4 + t cell percentage (mean ‫ע‬ sd, 19.72% ‫ע‬ 7.78%) and cd3 + cd8 + t cell percentage (mean ‫ע‬ sd, 23.7% ‫ע‬ 6.35%), compared with patients with fever but without sars (p ! .05), whose mean ‫ע‬ sd cd3 + cd4 + t cell percentage and cd3 + cd8 + t cell percentage were and 28.7% ‫ע‬ 7.1%, respectively. in pa-40.6% ‫ע‬ 7.8% tients with probable sars cov infection, there was a significant difference between the cd3 + cd4 + t cell percentage in those positive for igg antibody to sars cov (mean ‫ע‬ sd, ) and that in those negative for it (mean ‫ע‬ 24.07% ‫ע‬ 7.95% sd, ) ( ); however, no significant dif-13.60% ‫ע‬ 10.19% p ! .05 ference was found between the cd3 + cd8 + t cell percentages in these 2 groups (table 2) . also in patients with probable sars cov infection, there was no significant difference, in either the cd3 + cd4 + or the cd3 + cd8 + t cell percentage, between those positive for igm antibody to sars cov and those negative for it ( ). a correlation was found between the day of igg p 1 .05 antibody seroconversion and the cd3 + cd4 + t cell percentages measured on the day of the patients' admission to our hospital ( , ), if we assume day 60 to be the day of r p ϫ0.543 p ! .05 seroconversion for those patients who had not actually seroconverted by then. much progress has been made in sars research, including the identification of the etiologic agent [2] [3] [4] [5] [6] [7] , the complete sequencing of the sars cov genome [8] , the establishment of such diagnostic laboratory methods as rt-pcr and indirect immunofluorescence assay [5, 6] , and the establishment of measures for the prevention of sars and for the management of probable sars cases. however, it is still uncertain whether sars is recurrent and what the impact of recurrence might be, because there is no information on the status of host-specific immunity against sars cov and because the detailed characteristics of the epidemiology of sars cov infection are not known [9, 10] . given this uncertain background, we investigated, for its potentially significant clinical implications, the profile of anti-sars cov antibody in different populations, including patients with probable sars cov infection, patients with suspected sars cov infection, patients with fever but without sars, and healthy control individuals (health-care workers) who came into close contact with patients with sars. the results of our investigations of the presence of anti-sars cov antibody in patients with either probable or suspected sars cov infection show that the use of elisa to detect igg antibody and/or igm antibody to sars cov is a specific and useful method for the diagnosis of sars, especially given that elisa's sensitivity in the detection of anti-sars cov antibody (83.3%) is much better than rt-pcr's sensitivity in the detection of sars cov rna (∼25% in our study). serological assay, however, is of little value during the incubation and initial phases of the illness, because of late seroconversion in most patients ( figure 1a ). in addition, our results indicated that 9 (25.0%) of 36 patients with probable sars cov infection had not produced detectable anti-sars cov antibody by day 21 after the onset of fever; this implies that 25.0% of patients with sars might be misdiagnosed by the laboratory confirmation guidelines that who currently recommends [5] . however, this was not the case in our study, because (1) 3 of these 9 patients were later confirmed, by detection of seroconversion before day 47, to be infected by sars cov and (2) the 6 other patients, who had remained negative for anti-sars cov antibody until day 60, were later confirmed, by fulfillment of who criteria and by exclusion of other pathogenic infections, to be infected by sars cov. specifically, all of the patients with probable sars cov infection came into contact with someone with sars, had documented persistent fever (138њc), showed a consistent clinical course of the illness, and showed evidence of pneumonia, by plain radiography and/or computed tomography; in addition, there was no evidence of infection by other pathogens (including influenza viruses a and b, human parainfluenza viruses 1-3, respiratory syncytial virus, adenovirus, chlamdia pneumoniae, c. psittaci, mycoplasma pneumoniae, and mycobacterium tuberculosis), and there was no conventional pathogenic bacterial infection and no response to antibiotic treatment (0.3 g of levofloxacin/day for 48-72 h). the discrepancy between our diagnosis of sars in these patients and diagnosis on the basis of who guidelines likely reflects the evolution of the identification of definitive sars cases, as more data on the disease accumulate; the current who guidelines are probably not 100% accurate. it is notable that, on 16 july 2003, the centers for disease control and prevention revised its laboratory criteria in its definition of sars, to require that convalescent serum be collected 128 days, instead of 121 days, after the onset of symptoms [11] . the choice of day 28 presumably reflects data indicating that 195% of patients with sars mount a detectable convalescent antibody response [11] . a more technical explanation for our identification of late-seroconverting patients may involve differences between the sensitivity of the elisa used in the present study and the sensitivity of other serological assays, on which the who guidelines are based; this issue should be further clarified as more data are collected. in 6 of our patients with a late antibody response, there may have been other mitigating factors that could have led to a decrease in basic immunity. of these 6 patients, 2 (who were 165 old) had emphysema, 1 had diabetes, 1 was hypertensive and had coronary artery disease, 1 had tetanus, and 1 was pregnant; these conditions may be associated with late seroconversion in these patients. it is notable that a few (2 of 36 [5.6%]) patients with probable sars cov infection were positive for igg antibody to sars cov but, until day 60, were negative for igm antibody to sars cov; the reason for this is uncertain, but one possible explanation is that, in these 2 cases, igm antibody to sars cov persisted for a short time and had disappeared before the patients were admitted to the hospital. we found production of igg antibody to sars cov to be associated with host t cell immunity, because patients who did not produce igg antibody to sars cov had significantly lower cd3 + cd4 + t cell percentages, compared with those in patients who seroconverted. in addition, the day of igg antibody seroconversion correlated with cd3 + cd4 + t cell percentages taken on the day of the patient's admission ( ; ). r p ϫ0.543 p ! .05 these results suggest that the production of igg antibody to sars cov is dependent on cd4 + t cells and that the appearance of igg antibody to sars cov might be an indicator of the production of protective immunity against sars cov. recently, we treated 1 pregnant patient with severe sars cov infection by using convalescent plasma in which the titer of igg antibody to sars cov was 11:500; the igg antibody could be detected until 60 days after the infusion, 2 days before her own igg antibody to sars cov appeared. with combined treatment with the antiviral drug methylprednisolone and convalescent serum, she recovered fully. however, whether the igg antibody to sars cov is itself a neutralizing antibody needs further study. recently, krokhin et al. reported that acute and early convalescent serum, obtained from several patients recovering from sars, can react only with the 46-kda nucleoprotein (which appears to be the major antigen) of sars cov [12] . their results suggest that immune response to this nucleoprotein could serve as an early diagnostic indicator for infection; however, it is unlikely that immune response to this protein offers protection, because it is an internal protein and because neutralizing antibodies are more likely to target cellsurface proteins [13] . nevertheless, it has been shown, for other covs, that some antigenic peptides of the nucleoprotein can be recognized on the surface of infected cells by host t cells [13] ; thus, the appearance of igg antibody in patients with sars will more likely be an indicator of the production of protective immunity against sars cov. our results also indicate that all individuals positive for anti-sars cov antibody should present symptoms, whether severe or not. from 9 feb 2003 to the time when this article was written, our hospital admitted 52 patients with probable sars cov infection and 48 patients with suspected sars cov infection; however, at our hospital there is not a single health-care worker infected with sars cov-a situation that is in stark contrast to that at other hospitals in china, where nearly one-third of patients with sars are health-care workers [14] . that our hospital has no health-care workers infected by sars cov was confirmed by both serological assay and lack of detection of sars cov rna. the 96 health-care workers who participated in our study all came into close contact with patients with sars, for a period of 3 months, but igg and igm antibodies to sars cov were not detected in the serum of the health-care workers. these results may be somewhat difficult to interpret-and it was certainly not the point of our study to investigate this matter-but their implications may be very important; they suggest that the possibility that people can be asymptomatically infected by sars cov-and that such individuals (if they do exist) can transmit the virus-might be very small. the preventive measures taken at our hospital were almost exactly the same as those taken at other hospitals, with the only known difference being that, for prophylaxis, the health-care workers at our hospital took 400 mg of ribavirin/ day. we must caution, however, that these are only anecdotal observations; further study is necessary to determine whether ribavirin has a prophylactic effect against sars cov infection. world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) koch's postulates fulfilled for sars virus coronavirus confirmed as cause of sars updated interim surveillance case definition for severe acute respiratory syndrome (sars)-united states clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study identification of a novel coronavirus in patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection sars coronavirus: a new challenge for prevention and therapy modeling the sars epidemic updated interim us case definition for severe acute respiratory syndrome (sars) mass spectrometric characterization of proteins from the sars virus: a preliminary report coronaviridae: the viruses and their replication world health organization. china daily report of sars cases we thank the staff of shenzhen municipal hospital of infectious disease and the staff of shenzhen institute of hepatology, for their technical assistance and management of patients; all of the patients and, especially, the health-care workers who participated in the present study, for their great cooperation; and michael graner (dept. of pediatrics, university of arizona, tuscon), for his helpful comments. key: cord-353495-c3s5n5vo authors: yao, yanfeng; bao, linlin; deng, wei; xu, lili; li, fengdi; lv, qi; yu, pin; chen, ting; xu, yanfeng; zhu, hua; yuan, jing; gu, songzhi; wei, qiang; chen, honglin; yuen, kwok-yung; qin, chuan title: an animal model of mers produced by infection of rhesus macaques with mers coronavirus date: 2014-01-15 journal: j infect dis doi: 10.1093/infdis/jit590 sha: doc_id: 353495 cord_uid: c3s5n5vo in 2012, a novel coronavirus (cov) associated with severe respiratory disease, middle east respiratory syndrome (mers-cov; previously known as human coronavirus–erasmus medical center or hcov-emc), emerged in the arabian peninsula. to date, 114 human cases of mers-cov have been reported, with 54 fatalities. animal models for mers-cov infection of humans are needed to elucidate mers pathogenesis and to develop vaccines and antivirals. in this study, we developed rhesus macaques as a model for mers-cov using intratracheal inoculation. the infected monkeys showed clinical signs of disease, virus replication, histological lesions, and neutralizing antibody production, indicating that this monkey model is suitable for studies of mers-cov infection. coronaviruses (covs) can infect humans and a wide variety of animals, causing respiratory, enteric, hepatic, and neurological diseases of varying clinical severity. based on their genotypic and serological characteristics, they have been classified into 3 genera: alphacoronavirus, betacoronavirus, and gammacoronavirus [1] . coronaviruses are well known for their high frequency of recombination and high mutation rates, which may allow them to adapt to new hosts and ecological niches. this is best exemplified by the severe acute respiratory syndrome (sars) epidemic, which was caused by sars-cov [2] . sars-cov was shown to have originated from animals, with horseshoe bats (rhinolophus sinicus) as the natural reservoir and the palm civet (paguma larvata) as the intermediate host, allowing animal-to-human transmission [3] . since the sars epidemic, many other novel covs have been discovered in both humans and animals. a novel betacoronavirus lineage c, including the tylonycteris bat cov hku4 (ty-batcov hku4) and pipistrellus bat cov hku5 (pi-batcov hku5), was discovered in the lesser bamboo bat (t. pachypus) and the japanese pipistrelle (p. abramus) in 2007 in hong kong, china. these viruses were found to be closely related to a novel strain of human cov, referred to as middle east respiratory syndrome cov (mers-cov), which was identified in saudi arabia [4] [5] [6] . this novel human virus is now classified as a lineage c betacoronavirus that differs from other covs previously found in humans, including sars [7] . to date, 114 human cases of mers-cov have been reported, with 54 fatalities [8] . mers-cov is associated with severe respiratorytract infection, renal failure, and fatalities. similar to sars-cov, mers-cov is closely related to bat covs, suggesting that bats may be the natural reservoir of this family of viruses [9, 10] . specifically, a virus from egyptian tomb bats showed 100% nucleotide identity to virus from the human index case-patient [11] . it is currently unclear whether the human cases were a result of direct zoonotic transmission from bats to humans or whether an intermediate host was involved. however, a recent study showed mers-cov reactive antibodies in retired racing camels in oman, which neighbors saudi arabia [12] . in middle eastern nations, large amounts of camel meat were consumed every year, much of which was imported from african countries like egypt. this epidemiological evidence may suggest a bat-camel-human viral linkage, while more investigations are needed to define the source of mers-cov. currently, there is no evidence of efficient transmission between humans by mers-cov. however, the occurrence of some clusters of cases suggests that human-to-human transmission is possible and has raised concerns regarding the potential of this virus to cause a pandemic similar to that caused by sars-cov in 2002/ 2003 [13, 14] . the pathogenic mechanism of mers-cov is not clear, and neither an effective vaccine nor therapeutic drugs are available for prevention and treatment. the development of animal models for mers-cov infection of humans is of utmost importance to study the pathogenesis of this virus and to test the efficacy of potential therapeutic or prophylactic intervention strategies. at present, there are few reports of animal model for mers-cov infection, thus limiting further study [15, 16] . nonhuman primates have played an essential role in our understanding of the various forms of the pathogen, which could reflect variable clinical symptoms and pathology in humans. previous studies have reported nonhuman primate disease models for influenza, sars, and other viruses [17] [18] [19] . therefore, in the present study, we explored the suitability of the rhesus monkey as an animal model for mers-cov isolate human coronavirus-erasmus medical center (hcov-emc) infection or disease. the research on mers-cov virus was discussed among the staff members of the department of pathogen biology at the institute of laboratory animal science (ilas) of the chinese academy of medical sciences and peking union medical college (pumc). the mers-cov animal model experiments and protocols were discussed explicitly and extensively among the staff members of the department of pathogen biology. these discussions were followed by additional discussions with biosafety officers and facility managers at the ilas of pumc, as well as with numerous specialists from the sars-cov and general infectious disease fields throughout china. all research procedures were approved by the ilas institutional animal care and use committee and laboratory safety committee (lsc). the committee recommended that the number of animals be reduced to comply with the 3r (reduction, replacement, refinement) principles; we therefore designed the experiments to include 6 animals to test the animal model of mers-cov, 4 monkeys infected with virus and 2 uninfected monkeys as controls. the approved registration number is ilas-pc-2013-004. all experiments were conducted within the animal biosafety level 3 (absl-3) facility, which was constructed and accredited based on national standard gb19489 at the ilas of pumc, beijing, china. mers-cov strain hcov-emc was a kind gift from professor fouchier [4] . seed stocks of hcov-emc were propagated in vero cells. the seed stocks were diluted to the designated titer and used for determining the hcov-emc 50% tissue culture infection dose (tcid 50 ) and performing the neutralizing antibody assays. vero cells were maintained in dulbecco's modified eagle medium (dmem; invitrogen) supplemented with 10% fetal bovine serum (fbs), 100 international units (iu)/ml penicillin, and 100 µg/ml streptomycin and cultured at 37°c in 5% co 2 . four monkeys, aged 2-3 years, were inoculated intratracheally with hcov-emc (6.5 × 10 7 tcid 50 /1 ml) diluted in dmem. the monkeys were anesthetized, and 1 ml of the inoculum was administered intratracheally. mock-infected monkeys (2 monkeys) intratracheally inoculated with dmem were included as controls. the monkeys were observed twice daily, with detailed recording of clinical signs, symptoms, morbidity, and mortality, including the nature, onset, severity, and duration of all gross or visible changes. chest x-rays were performed before inoculation and 3 and 5 days postinoculation (d.p.i.) with mers-cov. two infected monkeys and a control monkey were sacrificed at 3 d.p.i. tissue specimens, including lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. for the virological and immunological tests, swab samples of the oropharyngeal, nasal turbinates, and cloacal regions were collected at 1, 3, 5, 7, 9, 11, 14, 21, and 28 d.p.i., and blood was collected at 7, 14, 21, and 28 d.p.i. total rna was isolated from individual samples using the rneasy mini kit (qiagen) according to the manufacturer's instructions. reverse transcription (rt) reactions were performed using the superscript iii first strand synthesis kit (invitrogen) according to the manufacturer's instructions. all complementary dna (cdna) samples were stored at −20°c until use in the polymerase chain reaction (pcr). for most experimental samples and the positive control (hcov-emc), duplicate cdna samples were obtained from each total rna sample. the primers used for hcov-emc were described previously [20] and are specific for the rna-dependent rna polymerase (rdrp) gene. the sequences of the primers used for pcr detection were as follows: rdrpseq-fwd (tgctat wagtgctaagaatagrgc; r = a/g, w = a/t) and rdrpseq-rev (gcatwgcncwgtcacacttagg; w = a/ t, n = a/c/t/g). the amplification protocol was as follows: 95°c for 3 minutes, followed by 45 cycles of 95°c for 15 seconds, 56°c for 15 seconds, and 72°c for 30 seconds, with a terminal elongation step of 72°c for 2 minutes. for cases in which no amplification products were obtained with the pcr assay, a 50-µl second-round reaction was set up containing 2 µl of the reaction mixture from the first round, the primer rdrpseq-fwd (the same as that used in the first round), and rdrpseq-rnest (cacttaggrtartcccawccca). thermal cycling was performed as follows: 95°c for 3 minutes, followed by 45 cycles of 95°c for 15 seconds, 56°c for 15 seconds, and 72°c for 30 seconds, followed by a 2-minute extension step at 72°c. tissue samples were homogenized to a final 10% (weight per volume) suspension in dmem and clarified by low-speed centrifugation at 4500 g for 30 minutes at 4°c. swab samples were immersed in 1 ml dmem, vortexed, and clarified by low-speed centrifugation at 5000 g for 10 minutes at 4°c. virus titers were determined in vero cells monolayers grown in 96-well plates. vero cells were seeded (1.5 × 10 4 /well) in a 96-well plate and incubated overnight at 37°c in a co 2 incubator. then, 100 µl of 10-fold serially diluted suspension was added to each well in quadruplicate. the virus was allowed to adsorb to the cells at 37°c for 1 hour. after adsorption, the viral inocula were removed, and 0.1 ml medium (dmem, 2% fbs) was added to each well. the plates were incubated in a co 2 incubator at 37°c for 3 days, after which the cytopathic effects (cpes) were observed microscopically at 40× magnification. the virus titer of each specimen, expressed as the tcid 50 , was calculated by the reed-muench method. for the neutralization test, serum samples were diluted 2-fold from an initial 1:10 dilution to a final dilution of 1:1280, mixed with 50 µl of 2000 tcid 50 /ml virus, and incubated at 37°c for 1 hour; this step was performed in quadruplicate. thereafter, 100 µl virus-serum mixture was added to vero cells previously seeded at 1.5 × 10 4 /well. the inoculated plates were incubated in a co 2 incubator at 37°c for 3 days, after which the cpes of the virus were observed microscopically at 40× magnification. animal necropsies were performed according to a standard protocol. samples for histological examination were stored in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin prior to examination by light microscopy. alternatively, the sections were immunohistochemically stained using a polyclonal antibody against the nucleoprotein of hcov-emc. the monkeys were observed daily for clinical signs of disease for 28 d.p.i., and body weight and temperature were recorded. the rectal temperature was obtained daily in the first week after challenge, after which the temperature was measured at specific time points; graphs of the temperature profiles (figure 1 ) depict the mean temperatures for the mock-infected and mers-cov-infected monkeys. the temperature of the infected monkeys increased at 1-2 d.p.i, and returned to normal thereafter. moreover, within the first few days, the infected monkeys drastically decreased their water intake. none of the infected monkeys showed overt weight loss when compared with the mock-infected monkeys. radiographic imaging revealed varying degrees of localized infiltration and interstitial markings ( figure 2 ). none of the infected monkeys died during the experiment. total rna was isolated from tissue homogenates and oropharyngeal, nasal turbinates, and cloacal swab samples and analyzed using primers specific for the hcov-emc rdrp gene. viral rna was detected in lung homogenates obtained from the infected monkey at 3 d.p.i.; however, the other organs remained rt-pcr negative (table 1) . no viral rna was detected in any of the swab samples (table 2) . nasal turbinates, oropharyngeal, and cloacal swabs were collected from inoculated animals at 1, 3, 5, 7, 11, 14, 21, and 28 d. p.i., and virus titers were determined by end-point titration in vero cells. the oropharyngeal, nasal turbinates, and cloacal swabs contained no detectable virus titers during the experiment, which was in agreement with the rt-pcr results. samples of the tissues described above were homogenized, and virus titers were determined. for the animals inoculated with mers-cov, virus could be detected in the lung (10 1.67 tcid 50 / ml); however, samples from the kidney, trachea, brain, heart, liver, spleen, and intestine had no detectable virus titers. the levels of hcov-emc-neutralizing antibody were evaluated. no neutralizing antibodies were detected in mock-infected monkeys. by contrast, in the infected monkeys, neutralizing antibodies were detected beginning at 7 d.p.i., reaching a peak titer of 1:320 at 14 d.p.i., and decreasing slightly to 1:160 at 28 d.p.i. (figure 3 ). two mers-cov-infected animals were euthanized at 3 d.p.i., and the trachea, lung, brain, heart, liver, spleen, kidney, and intestine were histologically and virologically examined. lung lesions were present in the inoculated monkeys. grossly, there was congestion, and palpable nodules, which showed as being scattered in distribution, were particularly evident in the posterior regions of the lobes (figure 4) . microscopically, lung tissue showed multifocal mild-to-moderate interstitial pneumonia and exudative pathological changes ( figure 5) . immunohistochemistry was also performed to assess the presence of mers-cov-infected cells in tissues collected from the infected animals and control animals ( figure 5 ). in monkeys inoculated with the mers-cov, infected cells were identified in the lung and in the same regional area as the pathological changes. types i and ii pneumocytes and alveolar macrophages were found to contain viral antigen. no vascular endothelium was observed ( figure 5 ). in addition, infected cells were not observed in other organs. in the control group, no positive areas were identified in any organs. currently, humans infected with mers-cov have been reported to develop a severe acute respiratory illness with symptoms of fever, cough, shortness of breath, and often, renal failure. more than half of the reported patients have died. some laboratory-confirmed cases reportedly experience a mild respiratory illness. the development of animal models that reflect these variations in the human population is challenging. in this study, we sought to develop an animal model that displays these symptoms. our studies have tested mouse, ferret, and guinea pig models and found these animals are not susceptible to mers-cov (unpublished data). nonhuman primates have been useful for evaluating vaccines and studying disease pathogenesis for several respiratory viruses, including influenza, sars-cov, respiratory syncytial virus, and human parainfluenza viruses. in this study, the rhesus monkey was explored as a model for mers-cov. not only did this monkey model support viral growth, it also manifested respiratory and generalized illness along with tissue pathology. the mers-cov dose used in this study was 6.5 × 10 7 tcid 50 by intratracheal infection. after infection, we observed a temperature increase in the infected animals. fever is one of the markers of mers-cov infection in humans [21] , and in the included monkeys, the temperature increase was significant from 1-2 d.p.i. the body weight of each monkey was also recorded prior to viral infection, but no significant change in mean weight was observed. virus replication was detected in the lungs of the infected animals. however, we did not detect virus rna or measurable titers in the nasal turbinate or oropharyngeal samples, suggesting that the monkeys did not shed virus in the upper-respiratory tract. in humans, mers-cov was detected in lower-respiratory-tract specimens with a high viral load, whereas nasopharyngeal samples were weakly positive or inconclusive [22] . this monkey model showed similar results. the primary mers-cov infection-related pathology in humans was acute pneumonia accompanied by multiorgan dysfunction and acute renal failure, which is compatible with a broad range of tissue tropisms in cell culture susceptibility testing [23] . the pathological presentation of pneumonia was also observed in the infected monkeys ( figure 3) ; however, no extrapulmonary lesions were found. these findings are consistent with the report by vincent et al, who reported similar findings of acute localized-to-widespread pneumonia that resulted in mild-to-moderate clinical disease [15] . in humans, renal failure is a major symptom of mers; however, we observed no pathological changes in the kidneys. the reasons for this discrepancy are not clear and deserve further study. it is possible that dissemination of the virus may not yet have occurred and the virus was limited to the lung, as we did not detect the virus in the blood. to determine whether the animals were infected, we developed a neutralizing antibody assay against mers-cov. there was evidence of seroconversion in all the inoculated animals. mers-cov-specific antibody was detected in the monkey serum samples beginning at 7 d.p.i., and the titer increased gradually. neutralizing antibody protection assays showed that the infected monkeys produced neutralizing antibodies that could prevent infection due to rechallenge by mers-cov. the results also indicate the potent immunogenicity of mers-cov, suggesting the possibility of using the virus as an inactivated or attenuated vaccine. in summary, the viral detection assays, immunological response, and pathological changes in the lungs of the infected animals suggest that mers-cov was able to establish an infection in the inoculated monkeys. additionally, the rhesus macaque appears to be a suitable model for studies of mers-cov pathogenesis or potential prophylactic or therapeutic intervention strategies. up to this stage, pathogenesis of mers-cov in human is still not clear. rhesus macaques can be infected by mers-cov in the laboratory, but we recognize that it is necessary to search for more suitable animal model that will have similar disease presentations as that observed among those laboratoryconfirmed human cases. however, before another animal model is defined, rhesus macaques may be used as an infection model for the evaluation of vaccine and antiviral drugs as stated in a recent paper [24] . is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? coronavirus as a possible cause of severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation of a novel coronavirus from a man with pneumonia in saudi arabia comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features genetic characterization of vetacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans world health organization. global alert and response: novel coronavirus infection-update human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe coronaviruses in bats from mexico middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence of person-to-person transmission within a family cluster of novel coronavirus infections family cluster of middle east respiratory syndrome coronavirus infections pneumonia from human coronavirus in a macaque model the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters cross-reactive t cells are involved in rapid clearance of 2009 pandemic h1n1 influenza virus in nonhuman primates an animal model of sars produced by infection of macaca mulatta with sars coronavirus persistent pneumocystis colonization leads to the development of chronic obstructive pulmonary disease in a nonhuman primate model of aids assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques key: cord-344954-gpb25fga authors: hashem, anwar m; algaissi, abdullah; agrawal, anurodh shankar; al-amri, sawsan s; alhabbab, rowa y; sohrab, sayed s; s. almasoud, abdulrahman; alharbi, naif khalaf; peng, bi-hung; russell, marsha; li, xuguang; tseng, chien-te k title: a highly immunogenic, protective, and safe adenovirus-based vaccine expressing middle east respiratory syndrome coronavirus s1-cd40l fusion protein in a transgenic human dipeptidyl peptidase 4 mouse model date: 2019-11-15 journal: j infect dis doi: 10.1093/infdis/jiz137 sha: doc_id: 344954 cord_uid: gpb25fga background: infection control measures have played a major role in limiting human/camel-to-human transmission of middle east respiratory syndrome coronavirus (mers-cov); however, development of effective and safe human or camel vaccines is warranted. methods: we extended and optimized our previous recombinant adenovirus 5 (rad5)–based vaccine platform characterized by in vivo amplified and cd40-mediated specific responses to generate mers-cov s1 subunit-based vaccine. we generated rad5 constructs expressing cd40-targeted s1 fusion protein (rad5-s1/f/cd40l), untargeted s1 (rad5-s1), and green fluorescent protein (rad5-gfp), and evaluated their efficacy and safety in human dipeptidyl peptidase 4 transgenic (hdpp4 tg(+)) mice. results: immunization of hdpp4 tg(+) mice with a single dose of rad5-s1/f/cd40l elicited as robust and significant specific immunoglobulin g and neutralizing antibodies as those induced with 2 doses of rad5-s1. after mers-cov challenge, both vaccines conferred complete protection against morbidity and mortality, as evidenced by significantly undetectable/reduced pulmonary viral loads compared to the control group. however, rad5-s1– but not rad5-s1/f/cd40l–immunized mice exhibited marked pulmonary perivascular hemorrhage post–mers-cov challenge despite the observed protection. conclusions: incorporation of cd40l into rad5-based mers-cov s1 vaccine targeting molecule and molecular adjuvants not only enhances immunogenicity and efficacy but also prevents inadvertent pulmonary pathology after viral challenge, thereby offering a promising strategy to enhance safety and potency of vaccines. the middle east respiratory syndrome coronavirus (mers-cov) is a novel zoonotic virus that was first isolated from a fatal human case in saudi arabia in 2012 [1] . the virus can cause severe acute and fatal respiratory symptoms associated with systemic infection and occasional multiorgan failure [1] . as of october 2018, mers-cov has caused >2250 laboratoryconfirmed infections with an approximately 35% mortality rate in 27 countries, with saudi arabia being the most affected country [2] . most global cases are linked to the arabian peninsula, where mers-cov continues to be endemic for >6 years with the potential to spread globally, as seen in the 2015 outbreak in south korea [3] . the spike (s) glycoprotein of mers-cov (1353 aa) is a type i trimeric membrane protein expressed on the virus surface, and binds to dipeptidyl peptidase 4 (dpp4) on target cells [4] . it is comprised of an n-terminal globular s1 subunit (aa 1-751) containing the receptor-binding domain (rbd) and a membraneproximal s2 subunit (aa 752-1353) consisting of a transmembrane domain and an intracellular cytoplasmic domain (cd) involved in viral fusion with target cells [4, 5] . given its critical role in viral replication, the s protein has been the focus for mers-cov vaccine development similar to severe acute respiratory syndrome coronavirus (sars-cov), where it has been the main target for vaccines that led to robust induction of neutralizing antibody (nab)-mediated protection in immunized animals [6] [7] [8] . several vaccine candidates based on full-length or truncated s protein including vectored, dna, and nanoparticle vaccines, as well as s or rbd protein-based subunit vaccines, have been developed and investigated in animal models [9] [10] [11] [12] [13] [14] [15] [16] [17] . although some of these candidates can induce high levels of nabs in vaccinated animals and have entered phase 1 clinical trials [15] [16] [17] , most of these candidates are associated with low immunogenicity requiring multiple doses [18] . importantly, vaccine research on other members of the coronaviruses including sars-cov [19] [20] [21] [22] [23] [24] [25] [26] [27] revealed several safety concerns associated with the use of s-based vaccines, including inflammatory and immunopathological effects such as pulmonary eosinophilic infiltration and antibody (ab)-mediated disease enhancement following subsequent viral challenge of vaccinated animals. recently, we also showed that whole inactivated mers-cov vaccine (wiv) is associated with significantly increased pulmonary eosinophilic infiltration accompanied by elevated levels of th2 cytokines in vaccinated human dpp4 transgenic (hdpp4 tg + ) mice in response to virus challenge [28] . given these previous observations, a mers-cov vaccine based on the full-length s protein could pose potential safety concerns similar to those associated with sars-cov vaccines. while the neutralizing epitope-rich s1 region could be considered as an alternative target for an effective and safe mers vaccine, s1-based protection could clearly be enhanced through appropriate immunological modulation. cd40 ligand (cd40l), a type ii membrane protein, is a key co-stimulatory molecule and an essential regulator of the immune system. it is expressed transiently on activated cd4 + t cells mainly [29, 30] . cd40l and its receptor cd40, which is constitutively expressed on all antigen-presenting cells (apcs) [29] [30] [31] , represent a crucial link between innate and adaptive immunity [31, 32] . the potential of cd40l as a molecular adjuvant has been investigated by several groups using multiple strategies [33] [34] [35] [36] . we have previously developed a nonreplicating recombinant adenovirus 5 (rad5) vectored prototype vaccine in which secreted viral proteins are targeted to cd40-expressing apcs using cd40l [37, 38] . such studies provided a proof of concept for a vaccine platform characterized by enhanced durability, breadth, potency, and universal protection against influenza viruses in different mouse models. here, we further extended and optimized our previous platform using rad5 expressing a secreted and cd40-targeted fusion protein comprised of head globular ectodomains of both mers-cov s1 protein and murine cd40l (rad5-s1/f/cd40l) using a trimerization motif to express the fusion protein as a trimer and a nonpolar linker to provide flexibility. the vaccine was then subjected to systematic analyses of protection and safety in the highly mers-cov permissive hdpp4 tg + mouse model. the fusion gene was designed and codon-optimized to express a secreted and cd40-targeted consensus mers-cov s1 subunit (amino acids 1-747). in brief, all available mers-cov s sequences were downloaded from the genbank database, and the dataset was filtered by removing sequences containing ambiguous amino acid codes (bjouxz). the final dataset was multiply aligned using clustalw, the shannon entropy for each position was determined, and the consensus s1 subunit sequence was obtained. the signal peptide from the mers-cov s protein was maintained at the n-terminus to secrete the fusion protein from rad5-infected cells. the synthetic fusion gene was synthesized by linking the coding region of mers-cov s1 to a histidine tag coding sequence, followed by coding region for the fibritin trimerization motif [37] connected via a nonpolar amino acid linker to the ectodomain of cd40l (amino acids 117-260) coding region to express the fusion protein s1/f/cd40l. the fusion gene was then used to generate the proposed rad5 construct (rad5-s1/f/cd40l) in addition to rad5 vaccines expressing secreted and consensus s1 protein alone (rad5-s1) and a vector control expressing green fluorescent protein (rad5-gfp) as shown in figure 1 . vero e6 and huh7.5 cells were cultured and maintained in complete dulbecco's modified eagle's medium (dmem) supplemented with 10% heat-inactivated fetal bovine serum. mers-cov-emc/2012 was provided by heinz feldmann (national institutes poly a p s1 f tag cd40l l poly a p s1 poly a p gfp rad5-s1/f/cd40l rad5-s1 rad5-gfp figure 1 . schematic representation of middle east respiratory syndrome coronavirus vaccine candidates. the proposed recombinant adenovirus 5 (rad5) construct (rad5-s1/f/cd40l) was engineered to express a fusion protein with the s1 subunit containing the s protein signal peptide at the n-terminal, followed by a trimerization motif derived from t4 bacteriophage fibritin (f) fused with the ectodomain of murine cd40 ligand at the c-terminus (cd40l). thus, the s1 and cd40l are expressed as a trimerized fusion protein via f (s1/f/cd40l). the construct was placed under the control of the cytomegalovirus promoter (cmv) and in front of the bovine growth hormone-poly a site (poly a). other control constructs included rad5-s1 and a control rad5 vector expressing green fluorescent protein (rad5-gfp). 50 ) assay as previously described [11] . all work involving infectious mers-cov was conducted within approved biosafety level 3 (bsl-3) at the galveston national laboratory (gnl) at the university of texas medical branch (utmb) in galveston. the immunogenicity and the protective efficacy of the rad5based vaccine candidates were evaluated against mers-cov infection in the hdpp4 tg + mouse model. a total of 45 hdpp4 tg + mice aged 4-5 months were used (15 mice/group). mice were intramuscularly immunized twice, 28 days apart, with 10 9 of rad5 vaccine candidates ( figure 2 ). blood samples were collected 3 weeks after each immunization via retro-orbital bleeding. four weeks after the second immunization, mice were intranasally challenged with 10 3 tcid 50 (~100 median lethal dose [ld 50 ]) of mers-cov. on days 3 and 6 postinfection, 4 mice from each group were killed to assess the virus titer and lung pathology. the remaining mice were monitored for morbidity and mortality on a daily basis for up to 18 days. all animal studies involving infectious mers-cov were conducted within approved animal bsl-3 laboratories at gnl in accordance with the guide for the care and use of laboratory animals of the nih and association for assessment and accreditation of laboratory animal care, and with institutional animal protocol approval from utmb. animals were housed in on-site animal facilities under a 12:12 light:dark cycle with room temperature and humidity kept between 21°c and 25°c and 31%-47%, respectively, with ad libitum access to food and water. at day 3 and 6 after challenge, 4 mice in each group were euthanized and their lungs were collected and examined for pathological changes after immunization and viral challenge. in brief, lung tissues were fixed in 10% buffered formalin for 72 hours, transferred to 70% ethanol, and later paraffin embedded. histopathological evaluation was performed on deparaffinized sections stained by routine hematoxylin and eosin staining. evaluations for histopathology were done by pathologists who were blinded to each specimen source. numeric scores were assigned to assess the extent of pathological damage as follows: 0, no observed pathology (undetectable infiltration); 1, mild pathology (up to 5% infiltration); 2, moderate pathology (up to 20% infiltration); 3, severe pathology (>20% infiltration). lungs collected from challenged mice on day 3 postchallenge were used to determine viral titer by tcid 50 . in brief, pieces of collected lung tissue were weighed and homogenized in phosphate-buffered saline containing 2% fetal calf serum with a tissuelyser (qiagen). after clarification of the cellular and tissue debris by centrifugation, the titers of infectious virus in the suspensions of infected tissues were determined using tcid 50 assay. the virus titers of individual samples were expressed as log 10 tcid 50 per gram of tissue and the minimal detectable level of virus was 2.5 log 10 tcid 50 as determined by lung size. lung samples from each group of mice (n = 4 per group) were transferred to individual vials containing rnalater solution and subsequently homogenized and subjected to total rna isolation using trizol reagent. to determine the viral titer in the lung, mers-cov-specific upstream e gene (upe) and endogenous control gene (mouse β-actin) were quantified using 1-step reverse-transcription quantitative polymerase chain reaction (rt-qpcr monitoring for morbidity and mortality upe mrna expression value was calculated for each replicate and expressed as the equivalent of log 10 equivalents per gram (tcid eq/g) of the tissue by the standard ct (∆∆ct) method using bio-rad cfx manager 3.0 software. the end-point titers of anti-s1 total immunoglobulin g (igg) as well as its isotypes including igg1, igg2a, and igg2b from immunized mice were determined by enzyme-linked immunosorbent assay as described previously [11] . serum samples were tested in a 2-fold serial dilution starting from 1:100. end-point titers were determined and expressed as the reciprocals of the highest dilution that gave an optical density signal higher than the cutoff value, defined as the mean of prebleed samples plus 3 standard deviations (sd). mers-pseudotyped viral particles (merspp) were produced and titrated using the huh7.5 cell line as described previously [39] . plasmids for generating merspp were kindly provided by dr nigel temperton at the university of kent, united kingdom. heat-inactivated serum samples were prepared in a 3-fold serial dilution starting from 1:20 and tested in duplicates in 96-well nunc white plates. a standard amount of merspp (~200 000 relative light units) was added to each well and plates were incubated for 1 hour at 37°c. then, approximately 10 000 huh7.5 cells were added to each well and incubated for 48 hours. cells only and cells with merspp only were included in quadruplicate as controls in all plates. following incubation, cells were lysed and luciferase activity was developed and measured using the bright-glo luciferase assay system (promega) according to the manufacturer's instructions. log 10 median inhibitory concentration (ic 50 ) neutralization titers were calculated for each serum sample using graphpad prism software. the standard live virus microneutralization (mn) assay was used as previously described [11] . in brief, starting at a dilution of 1:10, 60 μl volumes of serial 2-fold dilutions of heat-inactivated sera were mixed with equal volume of media containing 120 tcid 50 of mers-cov. each dilution was tested in duplicate in 96-well plates. the antibody-virus mixtures were incubated for 1 hour at 37°c before transfer of 100 μl into confluent vero e6 cell monolayers in 96-well plates. vero e6 cells cultured with dmem medium with or without virus were included as positive and negative controls, respectively. after 72 hours of incubation, the nab titer of each serum sample was determined as the reciprocal of the highest dilution capable of completely preventing virus-induced cytopathic effect in 50% of the wells (mn 50 ). statistical analysis was conducted using 1-way or 2-way analysis of variance with bonferroni posttest to adjust for multiple comparisons between groups using graphpad prism software. to evaluate the immunogenicity of the generated rad5 vectorbased mers vaccines, we immunized mice intramuscularly with 2 doses of individual vaccines and measured the levels of circulating s1-specific igg and its isotypes before and at 3 weeks after each immunization. here, we observed that both rad5-s1/f/cd40l and rad5-s1 induced s1-specific abs from all isotypes in hdpp4 tg + mice after primary or secondary immunization ( figure 3 ). as expected, control vector (rad5-gfp) failed to elicit any s1-specific ab response. notably, a single dose or 2 doses of rad5-s1/f/cd40l consistently elicited significantly higher levels of total igg as well as igg1, igg2a, and igg2b isotypes compared with the control group immunized with rad5-gfp (figure 3 ), whereas immunization with a single dose of rad5-s1 induced significant titers of total igg, igg1, and igg2a but not igg2b compared to the control group, with a second dose of rad5-s1 eventually inducing significantly higher levels of igg2b compared to the control group. interestingly, immunizing mice with a second dose of rad5-s1/f/cd40l enhanced the levels of circulatory igg and all tested isotypes compared with the rad5-s1-vaccinated group. furthermore, 2 doses of rad5-s1 only enhanced igg2b significantly compared to 1 dose, with mean igg1:igg2a ratios being 3.8 (sd, 3.2) and 3.9 (sd, 3.2) after priming and boosting, respectively. in contrast, boosting with a second dose of rad5-s1/f/cd40l resulted in a significant increase in total igg, particularly in igg2a and igg2b isotypes but not igg1, compared with a single dose of rad5-s1/f/cd40l, suggesting a bias toward th1 response as demonstrated by mean igg1:igg2a ratios of 3.4 (sd, 2.4) and 1.8 (sd, 1.4) after priming and boosting, respectively. together, these data showed that targeting the secreted s1 to cd40-expressing apcs via cd40l (rad5-s1/f/ cd40l) not only enhanced s1-specific igg abs levels but also boosted th1 responses, as demonstrated by markedly elevated titers of s1-specific igg2a and igg2b antibodies, especially after boosting. to extend our analysis and to evaluate the effector function of induced abs, we measured their neutralizing activities before and after each immunization. as expected, all immunized mice from all groups showed no detectable levels of nabs in samples collected before immunization. as shown in figure 4 , a single dose of either rad5-s1/f/cd40l or rad5-s1 elicited high levels of nabs compared to the control group (rad5-gfp), with rad5-s1/f/cd40l, but not rad5-s1, being consistently capable of inducing significantly higher levels of nabs against both live and pseudotyped mers-cov virus, suggesting that rad5-s1/f/cd40l is better than rad-s1 vaccine in promoting strong humoral response and neutralizing activity against mers-cov. however, after boosting once, all rad5-s1/f/ cd40l-and rad5-s1-immunized animals elicited robust and significant levels of nabs, indicating that at least 2 doses of rad-s1 are required to induce nab levels similar to those obtained by a single dose of rad5-s1/f/cd40l. these findings clearly confirm that incorporation of cd40l as molecular adjuvant could effectively enhance the immunogenicity of s1-based vaccines and may represent a very promising vaccine platform to induce protective immunity with a single dose. having demonstrated that cd40-targeted vaccine was superior in eliciting immune responses in hdpp4 tg + mice, we next investigated if these s1-based vaccines could effectively protect these highly mers-cov permissive mice from viral challenge. to this end, the vaccinated mice were challenged with 100 ld 50 of mers-cov and subsequently monitored for 3 weeks. it was clear that mice immunized twice with either rad5-s1 or rad5-s1/f/cd40l were completely protected based on clinical signs of disease (weight loss) and mortality ( figure 5 ), whereas rad5-gfp-immunized animals expectedly succumbed to lethal infection within days, likely due to encephalitis [28, 40] . these findings suggest that both rad5-s1 and rad5-s1/f/cd40l vaccines are protective in this mouse model. to further confirm that immunized hdpp4 tg + mice were protected from mers-cov infection after challenge, we measured lung viral titer, both infectious progeny virus and viral rna, on day 3 postchallenge by vero e6-based infectivity assay and rt-qpcr, respectively. analysis of infectious viral titer revealed that immunization with both vaccines significantly protected against viral replication to undetectable levels compared with rad5-gfp-immunized and -challenged mice ( figure 6 ). these results were further confirmed by an average of 3-to 4-log reduction in pulmonary viral rna, compared to that of the rad5-gfp-immunized group ( figure 6 ). it should be mentioned that, albeit not statistically significant, rad5-s1/f/cd40l-immunized animals had a viral rna load lower by 1 log compared with the rad5-s1 group, consistent with the overall stronger immune response we observed in this group. finally, investigations were carried out to assess safety of these vaccine candidates to rule out any vaccine-associated pulmonary injuries as observed with some vaccines against other coronaviruses. evaluation of lung pathology in immunized and challenged mice showed up to 15% multiple monocytic and lymphocytic infiltrations (moderate pathology) in the lungs of the rad5-gfp group 3 and 6 days postchallenge compared to other groups, which showed minimal to no inflammatory infiltration ( figure 7) . surprisingly, although rad5-s1 immunization protected animals from death and weight loss and prevented the lung infiltration similar to rad5-s1/f/cd40l, we observed perivascular hemorrhage in the whole lung of all examined mice immunized with rad5-s1 but not those vaccinated with rad5-s1/f/cd40l (figure 7) . together, these results suggest that although s1 alone could protect the animals from viral infection, it may inadvertently induce lung pathology, and that using cd40l as targeting molecule and molecular adjuvant does not only enhance immunogenicity and protective efficacy but also prevents vaccine-associated pulmonary pathology. the rapid spread and persistence of mers-cov in the arabian peninsula, in addition to the associated high mortality rates, represent a serious global public health concern, particularly that the elimination of the zoonotic reservoir is extremely unlikely at the current setting. this threat is further complicated by the absence of prophylactic or therapeutic measures. therefore, development of an effective and safe vaccine is urgently needed to prevent or contain mers-cov. several groups have investigated various mers-cov vaccine platforms in several animal models [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 28] . however, serious safety concerns are associated with vaccines for several coronaviruses including mers-cov and need to be elucidated and better understood [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . we showed in this study that although rad5 expressing s1 or cd40-targeted s1 were both capable of inducing significant levels of igg and nabs specific to mers-cov in immunized mice, incorporation of cd40l substantially enhances the immunogenicity of s1, as demonstrated by the effectiveness of a single immunization dose, which was sufficient to elicit stronger and robust immune responses compared to control groups, consistent with our previous reports [37, 38] . importantly, both s1 and cd40-targeted s1 vaccines provided complete protection, prevented virus replication significantly, and prevented monocytic and lymphocytic lung infiltration as compared to control group. however, the non-cd40-targeted vaccine (rad5-s1) uniformly induced varying degrees of severe perivascular hemorrhage within the lungs of all examined mice following viral challenge, regardless of the exhibited full protection against lethal mers-cov challenge. while these data suggest that there might be yet to be explained lung pathology associated with rad5 vector-and/or mers-cov s1-based vaccines, our results clearly show that including cd40l as a fusion protein of s1-based mers-cov vaccine could prevent or at least mitigate the safety concern of undesirable immunopathology while affording complete protection in hdpp4 tg + mice. at least 3 groups have developed different rad-vectored mers-cov vaccines [14, 41, 42] . however, these studies have mainly focused on the immunogenicity and/or the protective efficacy, and may have overlooked any possible vaccine-associated pathology, especially after viral challenge. our finding that rad5-s1 immunized animals were completely protected despite the observed pathology is reminiscent of the immunopathology documented using wiv for mers-cov, sars-cov, and respiratory syncytial virus after viral challenge [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [43] [44] [45] . it is of note that lung pathology was also observed in some previous studies involving rhesus macaques immunized with high doses of dna vaccines expressing mers-cov s protein [16] , as well as in mouse models vaccinated with rbd or vectored vaccines encoding s protein [46] [47] [48] . nevertheless, this is the first report of vaccine-associated severe pulmonary perivascular hemorrhage after viral challenge. although the exact molecular mechanisms underlying such vaccine-induced pulmonary injury associated with rad5-s1 vaccination remain to be determined, several factors could have been involved. specifically, the likely higher levels of residual infectious viruses in rad5-s1-immunized mice as detected by rt-qpcr, and the quantitative and perhaps qualitative difference of specific antibody and t-cell responses, as evidenced by the lower levels of ig2a and igg2b found in this group, could have played a role. . pulmonary viral load. viral load was determined in the lungs of immunized and challenged mice with middle east respiratory syndrome coronavirus (mers-cov). viral load was determined as median tissue culture infectious dose per gram (tcid 50 /g) and tcid 50 equivalent per gram (tcid 50 eq/g) by tcid 50 and reverse-transcription quantitative polymerase chain reaction (rt-qpcr) assays, respectively. limit of detection in the tcid 50 assay was 2.5 log 10 (dashed line) . tcid 50 eq/g was determined using a standard curve generated from dilutions of rna from a mers-cov with known virus titer. titers are shown as mean with standard deviation from 4 mice per group from 1 experiment. ***p < .0002 (1-way analysis of variance with bonferroni posttest); ns, not significant. see the figure 1 legend for explanation of the recombinant adenovirus 5 (rad5) constructs. rad5-gfp rad5-s1 rad5-s1/f/cd40l 100 μm therefore, future studies should include systematic analyses of t-cell responses as we have previously conducted [37, 38] , to fully explore the mechanisms of immunization-induced hemorrhage of the lungs. collectively, these previous data, along with our report here, indicate that viral components (ie, within the s protein) could be involved in inadvertent adverse reactions similar to those associated with the different s-based sars vaccines [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . our study suggests that although rad5-s1 could be a potential vaccine candidate, it might be associated with severe lung pathology upon viral challenge or infection. these findings are of great importance in order to develop safe and effective human mers-cov vaccine. moreover, there is a huge gap in our understanding of the correlates of protection in camels, and the waning nature of nabs in these animals [49, 50] might hinder the "one health" approach in tackling the mers epidemic. therefore, it is critical to better understand and elucidate any safety concerns that might be associated with mers-cov vaccine to develop a safe and effective human vaccine. our previous studies have mainly focused on the development of candidate universal influenza vaccines using vectored vaccines by targeting antigens to cd40-expressing cells via cd40l. this platform was modified, optimized, and utilized to generate an immunogenic, protective, and safe mers-cov vaccine based on the s1 subunit of the mers-cov s protein, thus representing a promising platform for vaccine development against a broad range of pathogens. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia disease outbreak news outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine identification and characterization of novel neutralizing epitopes in the receptor-binding domain of sars-cov spike protein: revealing the critical antigenic determinants in inactivated sars-cov vaccine the spike protein of sars-cov-a target for vaccine and therapeutic development evaluation of candidate vaccine approaches for mers-cov purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice immunogenicity of candidate mers-cov dna vaccines based on the spike protein searching for an ideal vaccine candidate among different mers coronavirus receptorbinding fragments-the importance of immunofocusing in subunit vaccine design middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates chadox1 and mva based vaccine candidates against 1566 • jid 2019:220 (15 november) • hashem et al mers-cov elicit neutralising antibodies and cellular immune responses in mice prospects for a mers-cov spike vaccine immunization with modified 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with an inactivated rs virus vaccine respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study key: cord-352433-sts48u9i authors: galanti, marta; shaman, jeffrey title: direct observation of repeated infections with endemic coronaviruses date: 2020-07-07 journal: j infect dis doi: 10.1093/infdis/jiaa392 sha: doc_id: 352433 cord_uid: sts48u9i background: although the mechanisms of adaptive immunity to pandemic severe acute respiratory syndrome coronavirus 2 (sars-cov-2) are still unknown, the immune response to the widespread endemic coronaviruses hku1, 229e, nl63, and oc43 provide a useful reference for understanding repeat infection risk. methods: here we used data from proactive sampling carried out in new york city from fall 2016 to spring 2018. we combined weekly nasal swab collection with self-reports of respiratory symptoms from 191 participants to investigate the profile of recurring infections with endemic coronaviruses. results: during the study, 12 individuals tested positive multiple times for the same coronavirus. we found no significant difference between the probability of testing positive at least once and the probability of a recurrence for the betacoronaviruses hku1 and oc43 at 34 weeks after enrollment/first infection. we also found no significant association between repeat infections and symptom severity, but found strong association between symptom severity and belonging to the same family. conclusions: this study provides evidence that reinfections with the same endemic coronavirus are not atypical in a time window shorter than 1 year and that the genetic basis of innate immune response may be a greater determinant of infection severity than immune memory acquired after a previous infection. the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) appears to have emerged in humans in the hubei province of china during november 2019 [1] . human-to-human transmission was confirmed in early january, and since then the virus has rapidly spread to all continents except antarctica. the outbreak was declared a pandemic by the world health organization on 11 march 2020. as of 4 july 2020, it has spread to >180 countries, with 10 922 324 confirmed cases and 523 011 deaths reported [2] . symptoms associated with sars-cov-2 vary from none to extremely severe, with elder adults and people with underlying medical conditions more at risk for developing severe and potentially fatal disease [3] . at present, there is no vaccine or approved antiviral treatment for sars-cov-2, and therapies rely principally on symptom management. many institutions across the world are working to develop a sars-cov-2 vaccine, and clinical trials with some vaccine candidates have already begun [4] . as the pandemic progresses, infecting millions of people across the world, a key question is whether individuals upon recovery are prone to repeat infection. there have been reports of individuals again testing positive by polymerase chain reaction (pcr) weeks after recovering from a sars-cov-2 infection. however, in korea, as reported by the korean centers for disease control and prevention, viable sars-cov-2 was not isolated in cell culture of respiratory samples from potentially reinfected individuals [5] ; thus, these subsequent positive results may have been due to inactive genetic material detected by molecular testing. a recent animal challenge study showed evidence of (at least) short-term protection against reinfections in rhesus macaques experimentally reinfected 4 weeks after first infection [6] . the immune response following reexposure to a virus depends highly on the pathogen and on host-pathogen interactions. some viruses such as measles elicit lifelong immunity; others, like influenza, do not. moreover, when a reinfection is prone to occur, there can be differences in symptom severity. reinfection with the same virus may be less symptomatic, as shown for subsequent influenza infections among children [7] . however, reinfection can also result in a more severe disease via antibody-dependent enhancement, a phenomenon in which antibodies raised against a virus bind with but are unable to neutralize a different strain of the same virus [8] two main processes appear to be responsible for the shortlived immunity engendered against some pathogens: (1) waning of antibodies and memory cells in the host system; and (2) antigenic drift of the pathogen that enables escape from the immunity built against previous strains. reinfections with the respiratory viruses have been reported in previous studies, in which individuals were infected in 2 sequential challenges with the same h1n1 virus [7, 9] . studies focusing on respiratory syncytial virus have provided evidence of subsequent reinfection with very similar strains or with the same strain in <1 year [10, 11] . serological studies have documented subsequent infections with endemic coronaviruses [12] . sequential rhinovirus infections have also been reported in a number of studies; however, this finding could be due to the multitude (>150) of antigenically distinct types of rhinovirus in circulation [13] . to contextualize the issue of protective immunity to sars-cov-2, we here present findings from a recent proactive sampling project carried out in new york city that documented rates of infection and reinfection among individuals shedding seasonal cov (types: hku1, 229e, nl63, and oc43). the results are discussed and analyzed in the broader context of coronavirus infections. data are derived from sampling performed between october 2016 and april 2018 as part of the virome project, a proactive sampling of respiratory virus infection rates, associated symptom self-reports, and rates of seeking clinical care. we enrolled 214 healthy individuals from multiple locations in the manhattan borough of new york city. cohort composition is described in [14] and includes children attending 2 daycares, along with their siblings and parents; teenagers and teachers from a high school; adults working at 2 emergency departments (a pediatric and an adult hospital); and adults working at a university medical center. the cohort was obtained using convenience sampling, and all participants were <65 years of age. while the study period spanned 19 months from october 2016 to april 2018, some individuals enrolled for a single cold and flu season (october-april) and others for the entire study period. participants (or their guardians, if minors) provided informed consent after reading a detailed description of the study (columbia university medical center institutional review board aaaq4358). nasopharyngeal samples were collected by study coordinators once a week irrespective of participant symptoms. samples were screened using the genmark esensor respiratory viral panel (rvp) system for 18 different respiratory viruses, including coronavirus 229e, nl63, oc43, and hku1. sample collection and extraction followed the same protocol as shown in [15] . in addition, participants completed daily self-reports rating 9 respiratory illness-related symptoms (fever, chills, muscle pain, watery eyes, runny nose, sneezing, sore throat, cough, chest pain), each of which was recorded on a likert scale (0 = none, 1 = mild, 2 = moderate, 3 = severe); see supplementary text 1, supplementary tables 1 and 2, and galanti et al [14] for further survey details. for this analysis, only the 191 participants who contributed at least 6 separate pairs of nasopharyngeal samples in the same season were included. we defined an infection (or viral) episode as a group of consecutive weekly specimens from a given individual that were positive for the same virus (allowing for a 1-week gap to account for false negatives and temporary low shedding). we classified all infection episodes as symptomatic or asymptomatic according to individual symptom scores in the days surrounding the date of the first positive swab of an episode. we considered multiple criteria for discriminating between symptomatic and asymptomatic episodes, as a standard definition for symptomatic infection does not exist in the literature. table 1 reports the 5 symptomatic thresholds used; all of the symptom scores are described in reference to a -3 to +7-day window around the date of the initial positive swab for each infection episode. the daily symptom score is defined as the sum of the 9 individual symptoms (range, 0-27) on a given day. total symptom score is the daily symptom score summed over the -3 to +7-day window. see supplementary text 1 for details and examples on how symptom scores were calculated (supplementary tables 1 and 2) per the definitions in table 1 . we applied standard methods of survival analysis to our longitudinal records of infections to estimate (1) the probability of infection with each endemic coronavirus type; and (2) the probability of being reinfected with the same coronavirus type following a previous documented infection. more specifically, the probability of infection and reinfection by time t, i (t),was estimated as: is the standard kaplan-meier estimator and time t is measured in either weeks from enrollment in the cohort, for the first analysis, or weeks from the previous documented infection (with a specific coronavirus type), for the second analysis. here d i are the participants testing positive exactly i weeks after enrollment (after first infection) and n i are the participants who are still enrolled i weeks after enrollment (after first infection). the denominator n i corrects for participants withdrawing from the study at different times by right censoring. the kaplan-meier estimators are compared statistically using the log-rank test. we used fisher exact test to analyze the difference between symptoms developed during subsequent infections and analysis of variance (anova) comparison to test differences in symptom scores reported by different family clusters. we restricted the last analysis to the family clusters within the cohort that presented at least 3 coronavirus infections during the study. among all participants enrolled, 86 individuals tested positive at least once during the study for any coronavirus infection. fortyeight individuals tested positive at least once for oc43, 31 tested positive for 229e, 15 tested positive for nl63, and 28 tested positive for hku1. figure 1 shows a kaplan-meier plot estimating the probability of becoming infected with each coronavirus within x weeks following enrollment (see supplementary table 3 for the number of individuals infected and censored at each time point). oc43 was the most widely diffused virus; the probability of testing positive following 80 weeks in the study was 0.47. in contrast, nl63 was the least frequently isolated coronavirus type; the probability of testing positive after 80 weeks was 0.17. among the study participants, 12 individuals tested positive multiple times during the study for the same coronavirus: 9 tested positive multiple times for oc43, 2 tested positive twice for hku1, 1 tested positive twice for 229e, and none tested positive multiple times for nl63. among the 9 participants with multiple oc43 infections, 3 individuals experienced 3 separate infection episodes, and the other 6 experienced 2 separate episodes. the median time between reinfection events was 37 weeks. the shortest time for a reoccurrence of infection was 4 weeks (oc43), and the longest was 48 weeks (oc43). among the 12 individuals testing positive multiple times for the same coronavirus, 9 were children aged between 1 and 9 years at enrollment, and 3 were adults aged between 25 and 34 years (see supplementary table 4 and supplementary figure 1 for characteristics and timelines of the repeated infections). figure 2 shows a kaplan-meier plot estimating the probability of becoming reinfected with the same betacoronavirus (oc43 and hku1) within x weeks after a previously documented infection (see supplementary table 5 for the number of individuals infected and censored at each time point). a comparison between the data shown in figure 2 and figure 1 found no significant differences between the probability of testing positive at least once and the probability of a recurrence for both hku1 and oc43 at 34 weeks after enrollment/first infection. to control for false-positive pcr results, we tested the sensitivity of the findings to different choices of the positivity threshold used in rvp testing (see supplementary text 2 and supplementary figures 2-5) . the probability of reinfection with betacoronaviruses at >38 weeks after prior infection was robust across different thresholds, whereas short-term reinfection signals could be an artifact due to pcr amplification. this shifted threshold also yields a statistically significant difference between the probability of testing positive at least once and the probability of a recurrence after first infection until week 43 (p = .04). there was no significant difference in the likelihood of experiencing symptomatic infection between the first and subsequent infection episodes by any of the 5 definitions provided in table 1 ; that is, severity of symptoms neither increased nor decreased significantly upon second infection. in particular, all of the individuals who were completely asymptomatic during the first recorded occurrence did not report any symptoms during subsequent infection(s) with the same coronavirus type. however, there was a significant association between severity of symptoms associated with any coronavirus infection and belonging to the same family cluster (p < .0001, 1-way anova). figure 3 shows the total symptom score associated with any coronavirus infection for infections grouped by family cluster. as the sars-cov-2 pandemic spreads to millions of individuals worldwide, it is extremely important to understand the mechanisms of protective immunity elicited by infection. until direct observations of adaptive immune response to sars-cov-2 become available, analyses of protective immunity elicited by other coronaviruses may offer useful insights. several studies in the last 4 decades have shown that infections with the 4 endemic coronaviruses 229e, oc43, nl63, and hku are common in the general population [12, 16] . infection with these viruses generally produces mild and even asymptomatic infection [17] . serological studies have shown that >90% of the population presents a baseline level of antibodies against these endemic coronaviruses, with first seroconversion occurring at a young age [16, 18] . shortly after infection, baseline antibody titers increase sharply; this response has been demonstrated for both natural and experimentally induced infections [12, 19, 20] . antibody titers start increasing roughly 1 week following infection, reach a peak after about 2 weeks [20] , and by 4 months to 1 year have returned to baseline levels [12, 20] . a challenge study [20] showed that the likelihood of developing an infection after inoculation correlated with participants' concentration of antibodies at enrollment. moreover, a positive correlation has been shown between antibody rise after infection, severity of clinical manifestation, and viral shedding [19] , with milder cases linked to less substantial postinfection antibody rises. instances of natural reinfections with the same virus type have been documented previously [12] in which repeated infections with oc43 and 229e were recorded by serological testing. subsequent infections were separated by at least 8 months, though study participants were tested every 4 months. participants in a separate challenge study were inoculated with coronavirus 229e and then rechallenged with the same virus after 1 year [20] . in most cases, reinfection occurred, though it presented with decreased symptom severity and shortened duration of shedding. the adaptive immune response to coronavirus is mainly directed toward the most variable part of the virus, a region that is not conserved across types; consequently, cross-reactive protection between different types does not appear to be an important factor [21, 22] . in addition, the effects of antigenic drift on reinfection have not been elucidated [23] , and more studies are warranted to understand whether repeat infections are ascribable to rapid virus evolution rather than a decline in antibody titers. the mild pathogenicity of seasonal coronavirus infection (with immune response often localized to the upper respiratory trait) is also often regarded as the reason for short-lived immunity. coronavirus infections, and the adaptive immunity acquired toward them, have also been studied in animals. in a study on porcine respiratory coronavirus, which causes subclinical infections in pigs, antibody titers waned approximately 1 year after experimental infection [24] . in contrast, an experimental study on murine coronavirus, which produces severe, systemic infections in mice, has shown an interplay between virus-specific antibodies and t cells, that upon survival in the host lead to lifelong protection against reinfection [25] . similarly, a longer immunity profile has been hypothesized for severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) due to their increased severity and to the systemic response that infection induces [21] . specific antibodies were detectable for at least 2 years in sars and mers survivors [26, 27] . although longitudinal studies on sars survivors have not detected specific sars immunoglobulin g antibody persistence 5 years after infection, they have found that specific memory t cells persist in the peripheral blood of recovered sars patients, and at higher levels in patients who experienced severe disease [28] . whether the presence of these memory t cells would be enough to induce a fast, protective response upon reinfection with sars has not been assessed. our study confirms that seasonal coronaviruses are widespread in the general population, with infections directly documented for a large fraction of the participants in our study. the methods for our analysis are based on the hypothesis that infection probabilities are comparable among participants enrolled at different times in the study. however, the seasonality of endemic coronaviruses, which are mostly absent during the summer months, and the relative magnitude across years of seasonal coronavirus epidemics are limitations. in the united states, the prevalence of oc43 during the 2016-2017 season was much higher than during the 2017-2018 season, whereas the opposite trend was observed for hku1 [29] . moreover, our estimates of infection and reinfection probabilities must be considered as a lower bound, due to the occurrence of weekly swabs missed by the participants and due to the design of the study itself, which may have missed infections of short duration in between consecutive weekly tests. nevertheless, this study confirms that reinfections with the same coronavirus type occur in a time window shorter than 1 year, and finds no significant association between repeat infections and symptom severity. instead, it suggests the effect of possible genetic determinants of innate immune response, as individuals asymptomatic during first infection did not experience symptoms during subsequent infections, and members of the same families reported similar symptom severity. genetic variations associated with immune responses have been associated with increased severity and exacerbation of symptoms due to respiratory infections [30, 31] . . total symptom score associated with infections by any coronavirus type. the score is calculated as the sum of daily symptom scores for the -3/+7 day window around the test date, as indicated for definition 3 in table 1 . each point represents an infection event, and each cluster represents a family group. each family group 1 to 9 is composed of a parent and 1-4 children. for each box, the red line indicates the median, and the bottom and top edges of the blue box are the 25th and 75th percentiles. the dashed lines extend to the most extreme data points that are not outliers, whereas the outliers are indicated by the red "+" symbol. we recognize that the self-reporting of symptoms is an important limitation in this analysis and that parents reported symptoms for their dependents, which possibly introduced bias. moreover, the majority of the repeated coronavirus infections were found in children, a cohort more vulnerable to infection because of their immature immune system [32] , and 26% of the episodes in the repeated infections were coinfections with other respiratory viruses (see supplementary table 2 ). another potential limitation of our study is the high sensitivity of pcr tests, which can amplify very small amounts of genetic material, possibly not ascribable to active infections. however, the occurrence of repeated infections separated by at least 38 weeks was corroborated by repeating the analysis with different positivity thresholds for the rvp. still, without virus sequencing, we cannot exclude the possibility that subsequent positives are the resurgence of the same infection rather than new infections, especially for infections reoccurring within a short time window. additional analyses involving viral sequencing and serological testing would be necessary to confirm repeated infections and to help disentangle the effects of antigenic drift and antibody waning. more studies analyzing the genetic basis of individual response to coronavirus infections are also warranted. even though endemic coronaviruses are very rarely associated with severe disease, their widespread diffusion, together with the fact that oc43 and hku1 belong to the same betacoronavirus genus as sars-cov-2, offers important opportunities for investigation. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. disclaimer. the funders had no role in study design, data collection and analysis, preparation of the manuscript, or decision to submit the manuscript for publication. financial support. this work was supported by the defense advanced research projects agency (contract number w911nf-16-2-0035). potential conflicts of interest. j. s. and columbia university disclose partial ownership of sk analytics. j. s. also discloses consulting for bni. m. g. reports no potential conflicts of interest. both authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. the proximal origin of sars-cov-2 centers for disease control and prevention. coronavirus disease 2019: symptoms. 2020 national institutes of health. nih clinical trials of investigational vaccine for covid-19 begins reinfection could not occur in sars-cov-2 infected rhesus macaques. biorxiv influenza a: infection and reinfection antibody-dependent enhancement of virus infection and disease influenza a reinfection in sequential human challenge: implications for protective immunity and "universal" vaccine development immunity to and frequency of reinfection with respiratory syncytial virus molecular analysis of respiratory syncytial virus reinfections in infants from coastal kenya rises in titers of antibody to human coronaviruses oc43 and 229e in seattle families during 1975-1979 prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness longitudinal active sampling for respiratory viral infections across age groups asymptomatic summertime shedding of respiratory viruses repeated endemic coronavirus infection â�¢ jid 2020:xx (xx xxxx first infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood rates of asymptomatic respiratory virus infection across age groups development of a nucleocapsid-based human coronavirus immunoassay and estimates of individuals exposed to coronavirus in a u.s. metropolitan population enzymelinked immunosorbent assay for detection of antibody in volunteers experimentally infected with human coronavirus strain 229 e the time course of the immune response to experimental coronavirus infection of man middle east respiratory syndrome vaccines antibody to virus components in volunteers experimentally infected with human coronavirus 229e group viruses viral infections of humans neutralizing antibody decay and lack of contact transmission after inoculation of 3-and 4-day-old piglets with porcine respiratory coronavirus effective clearance of mouse hepatitis virus from the central nervous system requires both cd4+ and cd8+ t cells longitudinal profile of antibodies against sars-coronavirus in sars patients and their clinical significance persistence of antibodies against middle east respiratory syndrome coronavirus lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study national respiratory and enteric virus surveillance system genetic susceptibility to respiratory syncytial virus bronchiolitis is predominantly associated with innate immune genes genetic associations with viral respiratory illnesses and asthma control in children evolution of the immune system in humans from infancy to old age key: cord-351571-gwtkrt5u authors: mackay, ian m.; lambert, stephen b.; faux, cassandra e.; arden, katherine e.; nissen, michael d.; sloots, theo p.; nolan, terence m. title: community-wide, contemporaneous circulation of a broad spectrum of human rhinoviruses in healthy australian preschool-aged children during a 12-month period date: 2013-05-01 journal: j infect dis doi: 10.1093/infdis/jis476 sha: doc_id: 351571 cord_uid: gwtkrt5u human rhinovirus (hrv) replication triggers exacerbation of asthma and causes most acute respiratory illnesses (aris), which may manifest as influenza-like illness. the recent assignment of 60 previously unknown hrv types to a third hrv species, human rhinovirus c, raised questions about the prevalence of these picornavirus types in the community, the extent of hrv diversity at a single site, and whether the hrvs have an equally diverse clinical impact on their hosts. we quantified hrv diversity, and there was no clinical impact attributable to hrv species and genotypes among a community population of preschool-aged children with ari who provided respiratory samples during 2003. all hrv species were represented among 138 children with ari, and 74 distinct hrv types were cocirculating. fever accompanied 32.8% of hrv-positive ari cases. hrvs were less likely than dna viruses to be codetected with another virus, suggesting virus interference at the community level, demonstrated by the inverse correlation between influenza virus detection and hrv detection. human rhinovirus (hrv) infections trigger exacerbations of asthma and chronic obstructive pulmonary disease and the majority of acute respiratory illnesses (aris), some of which meet criteria for influenza-like illness. these upper and lower respiratory tract illnesses are associated with considerable direct healthcare costs and indirect costs due to time lost from and reduced performance of regular duties [1] . there are 100 known hrv serotypes, with 15-30 circulating simultaneously at a given site [2] [3] [4] [5] [6] [7] . recently, 60 distinct, molecularly defined hrv genotypes were formally assigned to a third species, human rhinovirus c (hrv-c) [8, 9] . the contribution of hrv species and individual types to the annual burden of circulating hrv is poorly defined. there are 2 apparently distinct phylogenetic clades of hrv-c when typed using the 5′ untranslated region (5′utr) and adjoining encoding region [10] . it is hypothesized that these clades evolved with or without genetic recombination with human rhinovirus a (hrv-a) types [10] . nevertheless, each available majority sequence of an hrv-c type has to date represented a genetically unique, phylogenetically distinct, and globally distributed virus detected in patients with aris. there are specific seasonal and annual variations in respiratory virus circulation and interactions [11] [12] [13] . in a retrospective pediatric hospital-based study, hrvs were found to be statistically least likely of 17 examined viruses to be codetected with another virus during 2003 [14] . this aspect of virus-to-virus interaction is described as virus interference and is hypothesized to be the result of the host's response to one virus diminishing the likelihood of infection by another virus [15, 16] . for hrvs, virus-to-virus interaction was once exploited as an in vitro diagnostic tool, whereby successful experimental hrv infection of organ cultures was indicated by blockading the replication of another, superinfecting respiratory virus [17] . we sought to quantify the genetic diversity, epidemiology, and impact of hrv and enterovirus species, conjointly referred to hereafter as picornaviruses, circulating among a community cohort of preschool-aged children who provided respiratory samples over a 1-year period. we also sought to build on our hospital-based virus-to-virus interaction analyses [14] by seeking preliminary observational evidence of virus interference within the community. after receipt of informed consent from the parent or guardian of a potential study subject, we enrolled 234 healthy children <5 years of age into a community-based dynamic cohort study conducted over 12 months in melbourne, australia [1] . the study commenced in january 2003, and enrollment was progressive and continued until november 2003, with children observed until january 2004. parents monitored a set of symptoms in the study child each day, and when the definition of ari was met we asked parents to collect a combined nosethroat swab specimen [18] . the specimen was couriered to the victorian infectious diseases reference laboratory, where it underwent conventional polymerase chain reaction (pcr) testing, with reverse-transcription pcr performed for rna viruses, including influenza a and b viruses, respiratory syncytial virus, parainfluenza viruses, hrvs, and enteroviruses [19] . pcr was conducted for adenoviruses [19] . illnesses were classified as aris uncomplicated or complicated by fever and/ or otitis media. at the completion of data collection, we transported all available specimens (original nose-throat swab specimens and complementary dna) on dry ice to the queensland paediatric infectious diseases (qpid) laboratory, where they were tested for metapneumovirus and coronavirus nl63 (hereafter, "coronavirus"), using real-time pcr [19] . hrv templates for pcr amplification at the qpid laboratory were complementary dna, created at the victorian infectious diseases reference laboratory during the original studies [19] , or fresh rna extracted by means of the corbett x-tractor gene system (corbett research, australia) from the original nose-throat swab specimen. all extracts were amplified using a broadly reactive screening assay that targets the 5′utr [20] , yielding amplicons (length, approximately 380 base pairs) for sequencing by the prism bigdye sequencing kit v3.1 (applied biosystems). sequences generated by this study and submitted to genbank included accession numbers jn861783-92 and jq406000-184. sequence analysis was conducted in geneious pro [21] . the picornavirus species was determined by best match, using the following algorithm: when a basic local alignment search tool (blast) comparison returned matches with ≥95% sequence identity in the 5′utr, with characterized members assigned to a given species, our sequence was assigned to that species. for viruses not classified at this stage, we used definitive 5′utr-derived data from lee et al [22] as a guide to classify any query sequence that shared >96% nucleotide identity in the 5′utr with a blast match as a variant of that hrv type. counts and proportions were recorded for descriptive analyses. similar to methods we described previously [14] , univariate analysis involving the χ 2 test or the fisher exact test, using 2 × 2 contingency tables, was used to evaluate the relationships between picornavirus type, season, and demographic variables, such as age and sex, as well as to study virus codetection with another virus. a p value of <.05 was considered to indicate a statistically significant association. we observed 56 397 child-days in just over 12 study months. there were 730 aris identified; in 563 cases (74%), at least 1 parent-collected combined nose-throat swab specimen was returned [18] . for 269 aris (274 specimens; 47.8% of specimens), hrv or enterovirus was identified by conventional pcr at victorian infectious diseases reference laboratory [19] . of 274 specimens previously positive for a picornavirus and shipped to qpid laboratory, 238 (86.9%) yielded a genotypable sequence (figure 1 ), 14 (5.1%) yielded uninterpretable sequences, and 22 (8.0%) yielded sequences that were not amplifiable [20] . the picornavirus-positive specimens originated from 138 children (mean age, 25.7 months), including 48 from infants (age, 1-12 months), 107 from toddlers (age, 12-24 months), and 119 from older children (age, 2-5 years). a mean of 1.8 picornaviruses (range, 0-6) were detected per child. sixty picornavirus-positive children (43.5%) had ≥2 picornaviruses detected during study participation, and 32 had ≥3 (table 1 and figure 2 ). no child was positive for the same hrv type during different ari episodes, although the same species might have been detected. of the genotyped viruses, 99 (41.5%) were hrv-a, 13 (5.5%) were human rhinovirus b (hrv-b), 113 (47.5%) were hrv-c, and 13 (5.5%) were enteroviruses ( figure 1 ). picornavirus species distributions were not associated with sex (table 1 ). there was a decreased likelihood of identifying any of the picornavirus species in infants and an increased risk of identifying hrv-b, hrv-c, and enteroviruses in older children (p < .05 for these associations). the proportion of ari episodes in which hrv-b and hrv-c were detected was lowest during summer, while the proportion of ari episodes in which hrv-a was detected was lowest in both summer and winter (figure 3 ). detection of each hrv species declined dramatically when detection of respiratory syncytial virus and metapneumovirus increased and detection of influenza a virus peaked (in august [19] ). hrv-a and hrv-c could be detected in subsequent ari specimens from the same child, but hrv-b positivity did not recur for any child. peak picornavirus activity occurred in may and october, approximately 2 weeks after school terms commenced. activity of the species were observed to peak distinctly (species exchange) during the course of the year. hrv-positive ari cases were accompanied by fever in 84 instances (14.9% of all aris; 30.7% of hrv detections; table 2 ), whereas the majority of cases (9 [69.2%] of 13) in which enterovirus types (including coxsackievirus a1, a2, a4, and b5; echovirus 3, 11, 18, 21, and 27; and poliovirus 1) were identified had fever reported. only 5 cases of wheeze were reported: 2 were associated with hrv-c, 1 was associated with enterovirus, and 2 were associated with untypable picornaviruses. otitis media without fever accompanied ari in 4 picornavirus-positive instances (0.7%), and fever and otitis media co-occurred with ari in 14 instances (2.5%). hrv-a, hrv-b, and hrv-c were detected in similar proportions of ari cases with complications (42.0%, 46.2%, and 36.8% respectively) and those without complications (58.0%, 53.8%, and 63.2% respectively). of 395 samples with any virus present, 47 (11.9%) included a second virus, whereas 3 (0.8%) were positive for 3 viruses. among 252 picornavirus-positive specimens, 39 (15.5%) were pcr positive for another virus; 213 (84.5%) were not detected with another virus. only respiratory syncytial virus was codetected with another virus on fewer occasions (9.8% of respiratory syncytial virus detections). despite this, among children with ≥1 hrv detection during enrollment (28 children had 2 pvs; 17 had 3 pvs; 10 had 4 pvs, 3 had 5 pvs and 2 had 6 pvs), there was a consistent pattern of a reduced likelihood of codetection with a number of other viruses. for example, an hrv detection was associated with a reduced likelihood of codetecting any of 5 viruses or virus groups (adenovirus, parainfluenza virus, respiratory syncytial virus, influenza a virus, metapneumovirus, or coronavirus), as reflected by an odds ratio of <1 (table 2 ). among picornaviruses, enterovirus was most frequently associated with codetection (31% of enterovirus detections), and hrv-b was least frequently associated with codetection (8% of hrv-b detections). there was an average of 50 days between consecutive aris in study children. after each hrv, influenza a virus, respiratory syncytial virus, or adenovirus detection, there was a mean interval of 46, 51, 60, and 45 days, respectively, before the next ari episode. the evolutionary history was inferred using the neighbor-joining method in mega5 [37, 38] . the optimal tree is shown drawn to scale, with branch lengths in the same units as those of the evolutionary distances (base substitutions per site; maximum composite likelihood method [39] ) the analysis involved 401 nucleotide sequences, including completely sequenced referenced types [40] . our analysis of aris among children in the community identified 74 named or uncharacterized rhinovirus types among 138 children followed during this 12-month study. three-tofive times more distinct hrv types were identified in our single-year cohort than would have been expected from the literature. recently, van der zalm et al identified 27 different subtypes among a cohort of 18 children sampled fortnightly during a six-month period [7] . because a lower sequence threshold was used to identify those viruses, it is likely that the true number of hrv types was underestimated as compared to the higher threshold (96% vs 90%) we applied, which we considered essential when using the highly conserved 5′ utr region. recent cohort studies that also used the 5′utr for hrv typing identified 56 [23] distinct hrv types, including 32 new types [23] , whereas other cohort studies have not conducted detailed typing analyses [24, 25] . cohorts with longer follow-up [26] or more-frequent sampling than ours [27] unsurprisingly find greater numbers of hrv types since they span multiple seasons or have a better likelihood of sampling so-called "asymptomatic" infections. frequently, sampling in longitudinal cohort studies improves our understanding of the role of respiratory viruses in illness because it allows temporal association with signs and symptoms. despite the dominance of hrv-c types here and their reported links with asthma elsewhere [28] , few new cases of wheezing were reported in our cohort, most likely because of exclusion of children with chronic pulmonary disorders (including diagnosed asthma or frequent use of asthma medication). a frequently sampled, well-controlled longitudinal cohort of children with asthma will be required to answer whether there is a species-specific hrv impact in an asthmatic population. all hrv species were represented among ari cases with fever, providing further evidence of the confounding capacity of hrv infections during an influenza epidemic or pandemic unless specific diagnostics are included. most respiratory viruses were detected in toddlers and children, from whom the majority of specimens were obtained. in our study, the respiratory syncytial virus and metapneumovirus ari with fever and om 9 (1. season preceded the influenza a virus season. it was also noteworthy that hrv prevalence declined sharply as respiratory syncytial virus, metapneumovirus, and, particularly, influenza a virus cases peaked. this mirrored the findings from our hospital studies, conducted on specimens from brisbane, queensland, australia, that were also collected during 2003 [14] . we observed that viruses with an rna genome, in particular the hrvs, were less frequently involved in codetections than those with a dna genome in this community cohort from victoria. these numbers might have been different if other virus, such as bocavirus, parainfluenza virus-4, and influenza c virus, had been included, but our previous findings suggest the adjustment would not have changed the significance of the virus-to-virus interaction [14] . our observation most likely reflects the capacity of rna viruses to efficiently trigger the early innate interferon response through interactions with a range of pattern-recognition receptors, including toll-like receptors 3 and 8, mda5, and rig-i. whether dna viruses are better adapted to interfere with a host interferon response or are better at exploiting the opportunities provided by rna viruses remains to be defined. it has been previously suggested that the 5′utr is unsuitable for hrv genotyping because in silico signs suggest that the region has, at some point, been a site for recombination between some hrv types [29] . however, we have not found, nor has there been published evidence for, false genotyping (a 5′utr erroneously representing a polyprotein sequence from a different virus) of the ≥100 hrv-a, hrv-b, and hrv-c genomes fully sequenced to date. even when an hrv-c type falls into the hrv-a clade phylogenetically, to date it exists within a distinct space in that clade. carefully chosen sequence identity thresholds and experience can successfully produce robust genotyping results, as has been shown here and elsewhere in the literature [23, 26, 27, [30] [31] [32] . in this study, we found that each 5′utr sequence was a unique identifier of hrv type. others suggest the hrvs are under positive selective pressure to remain conserved [33] . while this may not remain evident as more completed hrv genomes become available, no proof exists to support a theory that the 5′utr is an unsatisfactory target for genotyping, and it continues to be used successfully elsewhere [34] . nevertheless, phylogeny based on the 5′utr region, whether partial or complete, does not accurately discriminate all members of the hrv-a species from those currently considered to be hrv-c but harboring elements of an hrv-a utr [10] . this is similar to the phylogenetic patterns of enterovirus types derived from use of the 5′utr, in which the 4 known species are represented by only 2 clades. a longer target encompassing a 5′utr-vp2 stretch improves phylogenetic tree construction, and these sequences are becoming well represented in the genbank sequence database [35] . however, until the hrv virome is completely characterized, it is also premature to assume that the 5′utr-vp2 region's apparent congruity with the rest of an hrv's polyprotein sequence will be maintained by the genomes of future distinct hrv strains. a vp1 target is less sensitive when used directly on clinical specimens [22] and is likely to underrepresent enterovirus diversity [36] . in summary, our screening and 5′utr-based genotyping study identified extensive hrv genetic diversity as compared to data generated from culture-based diagnostics. there were no associations between sex or clinical outcome and hrv species, but hrvs were less often present in codetections than would have been expected by chance. in particular, the influenza a virus season preceded a precipitous decline in hrv detections. whether a reduction in hrvs allows an increase in other viruses during the winter months is unknown, and studies to define the immunobiological mechanisms behind these observations are required. the cost of communitymanaged viral respiratory illnesses in a cohort of healthy preschool-aged children rhinovirus infections in an industrial population. 3. number and prevalence of serotypes screening and comprehensive 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airway rhinovirus burden and the seasonal risk of asthma exacerbation proposals for the classification of human rhinovirus species c into genotypically-assigned types new respiratory enterovirus and recombinant rhinoviruses among circulating strains mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods the neighbor-joining method: a new method for reconstructing phylogenetic trees prospects for inferring very large phylogenies by using the neighbor-joining method sequencing and analyses of all known human rhinovirus genomes reveals structure and evolution acknowledgments. we thank the families and children who participated in this study; kelly allen, other virgo staff, maternal key: cord-350201-tluc2ck7 authors: kuiken, thijs; breitbart, mya; beer, martin; grund, christian; höper, dirk; van den hoogen, bernadette; kerkhoffs, jean-louis h; kroes, aloys c m; rosario, karyna; van run, peter; schwarz, matthias; svraka, sanela; teifke, jens; koopmans, marion title: zoonotic infection with pigeon paramyxovirus type 1 linked to fatal pneumonia date: 2018-10-01 journal: j infect dis doi: 10.1093/infdis/jiy036 sha: doc_id: 350201 cord_uid: tluc2ck7 the characteristics and risk factors of pigeon paramyxovirus type 1 (ppmv-1) infection in humans are poorly known. we performed virological, pathological, and epidemiological analyses of a dutch case, and compared the results with those of a us case. both infections occurred in transplant patients under immunosuppressive therapy and caused fatal respiratory failure. both virus isolates clustered with ppmv-1, which has pigeons and doves as reservoir. experimentally inoculated pigeons became infected and transmitted the virus to naive pigeons. both patients were likely infected by contact with infected pigeons or doves. given the large populations of feral pigeons with ppmv-1 infection in cities, increasing urbanization, and a higher proportion of immunocompromised individuals, the risk of severe human ppmv-1 infections may increase. we recommend testing for avian paramyxovirus type 1, including ppmv-1, in respiratory disease cases where common respiratory pathogens cannot be identified. changes in socioeconomic, environmental, and ecological factors in recent decades are probably responsible for the significant increase of emerging infectious diseases, the majority of which are zoonotic [1] . of particular concern are emerging respiratory viruses of the families orthomyxoviridae [2, 3] , coronaviridae [4, 5] , and paramyxoviridae [6, 7] . the use of novel molecular techniques has made it possible to identify previously unsuspected or unknown viruses [8] . however, it is important to assess the clinical and public health relevance of these viruses by determining their origin and establishing their impact in the human population. the impetus for the current study was the identification of a virus related to avian paramyxovirus type 1 (apmv-1) from a fatal human case of unknown cause in the netherlands by viral metagenomics analysis [8] . apmv-1 is classified in the genus avulavirus, family paramyxoviridae [9] . infection with apmv-1 is mainly restricted to birds, but occasionally causes mild disease-usually transient conjunctivitis-in humans [10] . so far, there is only one case report, from new york state, of a person who died with apmv-1 infection [11] . in this study, we fully characterized the dutch clinical isolate of apmv-1-like virus, determined its phylogenetic relationship to other apmv-1 strains, and correlated presence of this virus with lesions in tissues obtained from the patient at autopsy. we also examined virulence and excretion pattern of this virus in chickens and domestic pigeons. a 54-year-old woman with a history of multiple myeloma received an allogenic bone marrow transplant in september 2002, following preparation with a nonmyeloablative regimen (cyclosporin) and while on antifungal therapy (intravenous itraconazole). she initially did well, but was hospitalized 8 weeks after transplantation with symptoms of malaise, anorexia, fever, and abdominal complaints (including abdominal pain, diarrhea, and food intolerance). she lived in the country near to a city, but was not employed in agriculture, and there was no evidence of contact with animals in her clinical history. gastroscopic examination showed no abnormalities. infiltrative lesions in the thorax were detected by radiological and computed tomographic examinations, and there was progressive swelling of cervical lymph nodes. no pathogens were detected in bronchoalveolar lavage (bal) specimens (virus culture on hel, llc-mk2, hep-2, and human rhabdomyosarcoma cells; bacterial culture on blood agar, chocolate agar, columbia blood agar containing colistin and nalidixic acid, and sabouraud agar; cytological examination for bacteria, fungi, and pneumocystis carinii), and a biopsy of the peripheral right lower lung lobe was histologically normal. epstein-barr virus (ebv) antigen expression and lymphoproliferation were detected in a biopsy of the right jugular lymph node, and a blood sample was positive for ebv dna by polymerase chain reaction (pcr). reactivation of ebv and associated lymphoproliferation were treated successfully with rituximab (anti-cd20) and discontinuation of cyclosporin therapy, but other clinical signs remained. enteroscopic examination of the small intestine showed no abnormalities, and no pathogens were detected in fecal samples by bacterial culture. five weeks after hospitalization, the patient developed high fever and progressive worsening of respiratory signs. treatment with cotrimoxazole was initiated for suspected p. carinii infection, and with prednisone for suspected bronchiolitis obliterans organizing pneumonia or graft-vs-host disease of the lung. two weeks later, the patient developed respiratory failure. there was crepitation over both lung fields and bilateral, progressively expanding, blotchy infiltrates in the thorax by radiological examination. gram-negative, rod-shaped bacteria (pseudomonas species) were detected by microscopic examination of bal fluid, and treatment with ceftazidime and tobramycin was initiated. despite intubation and artificial respiration at increasing pressure, respiratory function deteriorated further and circulatory failure developed. artificial respiration was stopped after 3 days, after which the patient died. autopsy was performed 1 day after death. there were extensive areas of bronchopneumonia associated with pseudomonas species infection in both lungs by bacterial culture. in addition, there was marked diffuse alveolar damage, characterized by abundant hyaline membranes lining the alveolar septa. the mucosa of the colon ascendens was lined by a hemorrhagic exudate, but autolysis was too advanced to confirm or rule out graft-vs-host disease, and no satisfactory explanation was found for the abdominal complaints. in conclusion, the progressive respiratory failure was explained by severe diffuse alveolar damage of undetermined cause, and extensive bilateral bronchopneumonia caused by terminal pseudomonas species infection. as part of an in-depth investigation, archived cell culture samples with an unexplained cytopathic effect were subjected to metagenomic analysis as described previously [8] . these samples included human rhabdomyosarcoma cells inoculated with a bal specimen collected in 2002 from the above patient. metagenomic analysis revealed the presence of short nucleotide fragments with homology to apmv-1. to complete the genome of the clinical isolate, fragments obtained through whole transcriptome amplification were assembled against the apmv-1 genome with the highest similarity (supplementary methods). in brief, the assembly was performed using sequencher software (version 4.1; gene codes). because the whole-transcriptome amplification fragments did not overlap into a complete genome, the assembly was used to design primers for genome walking, pcr assays were performed with these primers, and products were sequenced. these efforts resulted in the genome completion of the dutch clinical virus isolate. nucleotide sequences were aligned with the clustal w program running within the bioedit software package, version 5.0.9 [12] . with the nucleotide sequence alignment, the best-fit model of nucleotide substitution was determined by jmodeltest [13] . maximum-likelihood (ml) phylogenetic trees were generated using the gtr+γ4+i model of nucleotide substitution and the phyml package version 3.0 [14] . the reliability of all phylogenetic groupings was determined through a bootstrap resampling analysis of a 1000 replicates with phyml. trees were visualized through the figtree program version 1.4. (http://tree.bio.ed.ac. uk/software/figtree/) and rooted on the apmv-1 with the highest blast similarity in genbank (apmv-1/nl/152608/1993). the following tissue biopsies and autopsy tissue specimens had been fixed in 10% neutral-buffered formalin and embedded in paraffin blocks: heart, lung, jugular lymph node, esophagus, thyroid gland, stomach, pancreas, liver, kidney, skin, diaphragm, and muscle. these specimens were used for detection of apmv-1 rna by real-time reverse-transcription pcr (rt-pcr), of apmv-1 antigen by immunoperoxidase method, and of histological lesions by hematoxylin and eosin stain (supplementary methods). chickens were inoculated intracerebrally with the dutch clinical virus isolate to determine the intracerebral pathogenicity index (supplementary methods). domestic pigeons were inoculated intratracheally with the dutch clinical virus isolate to determine infectivity and transmissibility, clinical signs, and pathological changes (supplementary methods). the sequence of the full-length genome of the dutch clinical virus isolate displayed 97% nucleotide sequence homology with an apmv-1 isolated from pigeons in belgium (apmv-1/ belgium/98-238/1998; jx901109). phylogenetic analysis of fulllength genomic sequences of closely related apmv-1 isolates demonstrated that the dutch clinical isolate clustered among belgian and chinese avian strains in genotype vib/1 ( figure 1 ). viruses in this genotype are also called pigeon paramyxovirus type 1 (ppmv-1), because pigeons and doves are the animal reservoir. therefore, the dutch clinical virus isolate was named human ppmv-1 (hppmv-1/nl/579/2003; accession number kj544861). comparison of predicted amino acid sequences revealed that all of the hppmv-1/nl/579/2003 encoded proteins displayed >95% identity with those of other ppmv-1. in general, the fusion protein (f) cleavage site of medium/high virulent apmv-1s contain the sequence 113 rq(k/r)r*f 117 , while those of low virulent apmv-1s usually have the sequence 113 (k/r) q(g/e)r*l 117 . the cleavage site of hppmv-1/nl/579/2003 ( 113 rqkr*f 117 ) was consistent with a virulent pathotype, similar to that observed in all ppmv-1 ( 113 r(q/k) kr*f 117 ) [15] [16] [17] . based on phylogenetic analysis of sequences of partial f protein sequences, ujvári et al identified 4 sublineages of ppmv-1 (ie, apmv-1 genotype vib/1) strains. these subgroups, designated iraqi (iq), early european (eu/ea), north american (na), with related avian paramyxovirus type 1 (apmv-1) strains based on analysis of full-length genomes. assignment of apmv-1 genotypes (vib/1 or vii) and sublineages (eure1, eure2, euea1, or euea2) was based on the studies of ujvári et al [16] and czegledi et al [35] and the sequence comparison shown in table 1 and recent european (eu/re), corresponded partly to the times of isolation and/or geographical origin [16] . alignment of the f protein amino acid sequences from hppmv-1/nl/579/2003 with those of ujvári and others revealed that it had identical amino acids substitutions as isolates belonging to sublineage eur/re2 (table 1) . no sequence information was available for this partial f protein sequence of the only other apmv-1 clinical isolate, from a fatal human case in new york state [11] . the available partial sequence for this new york clinical virus isolate demonstrated the closest relationship with a ppmv-1 isolated from pigeons in new york in 2006 (kc013033.1), and clustering in sublineage vib/1 na1 (data not shown). real-time rt-pcr analysis revealed the presence of high levels of ppmv-1 rna in the left and right lung ( table 2) . lower levels were detected in liver, kidney, and bone marrow, while the remaining tissues tested negative. immunohistochemistry analysis revealed the expression of ppmv-1 antigen, visible as red-brown granular staining, in the same tissues that tested positive with real-time rt-pcr ( table 2 ). the exact localization of these granules was not clear, although some aggregates of granules appeared to be located in the cytoplasm of degenerate cells (figure 2 ). ppmv-1 antigen expression was present in the positive control tissue and absent in the isotype control and negative control tissues. histopathological examination demonstrated that ppmv-1 antigen-expressing sections of lung had diffuse alveolar damage, characterized by loss of alveolar epithelium, rare hypertrophic type ii pneumocytes, widened alveolar septa, and fibrin thrombi in alveolar capillaries ( figure 2 ). no histological lesions were apparent in other tissues that expressed ppmv-1 antigen, or in ppmv-1-negative tissues. based on intracerebral inoculation into 1-day-old chickens, the intracerebral pathogenicity index of hppmv-1/nl/579/2003 was 0.164 (supplementary table 2 ). this corresponds with a lentogenic pathotype. in pigeons, all 5 intratracheally inoculated animals developed a productive infection, with shedding from the pharynx between 2 and 10 dpi, and from the cloaca between 7 and 17 dpi (supplementary figure 1) . virus was transmitted to 1 of the 2 naive pigeons. none of the pigeons displayed clinical signs at any time point during the 3-week observation period. no gross lesions were observed in any pigeons at autopsy at 21 dpi. however, histopathological analysis showed that all 5 intratracheally inoculated pigeons plus the 1 infected naive pigeon had diffuse interstitial nonpurulent nephritis, which was absent in 2 negative control pigeons from the same flock (supplementary figure 2) . also, 1 inoculated pigeon had multifocal nonpurulent pancreatitis. real-time rt-pcr analysis revealed that hppmv-1/nl/579/2003 was present in the kidney sample of 3 inoculated pigeons with nephritis, and in the pancreas sample of 2 inoculated pigeons, including the 1 with pancreatitis (supplementary figure 3) . discussion we diagnosed ppmv-1 infection as the cause of diffuse alveolar damage in the dutch female patient, based on colocalization of ppmv-1 antigen, high loads of ppmv-1 rna, and characteristic histological lesions in autopsy specimens ( figure 2 and table 2 ). the ppmv-1-associated diffuse alveolar damage was exacerbated by bronchopneumonia due to pseudomonas species infection, probably from intravenous catheterization. the combination of these 2 pulmonary diseases likely explains the severe respiratory failure leading to the death of this immunosuppressed patient. it is rare to diagnose ppmv-1 infection as the cause of severe respiratory disease in humans. to our knowledge, the only other known case was reported by the new york state department of health in 2007 [11] . it is not clear whether this paucity of reports is because the disease is rare or because apmv-1, including ppmv-1, is not suspected in such cases. we suggest that apmv-1, including ppmv-1, should be included in the differential diagnosis of cases of respiratory disease that test negative for more common respiratory pathogens. samples could be tested by various molecular methods, such as a family-wide rt-pcr assay for paramyxoviruses or metagenomics approaches [18, 19] . when they became infected with ppmv-1, the dutch and new york patients were receiving immunosuppressive therapy to improve the success of peripheral blood stem cell or bone marrow transplantation. there is an increase in the proportion of immunocompromised people in the population [20] . as these immunocompromised people are at high risk of severe disease from other opportunistic infections [20] , they also may be at risk of severe disease from infection with apmv-1, including ppmv-1. ppmv-1 appears to target the lower respiratory tract epithelium in humans. this is based on the demonstration of ppmv-1 antigen expression in the alveolar epithelium of autopsy lung samples from both human cases (figure 2 ) [11] . the tissue tropism of ppmv-1 in these 2 human cases resembles that of other emerging zoonotic respiratory viruses-h5n1 influenza virus and the severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses-that also target the lower respiratory tract [20, [22] [23] [24] . there was evidence of extrarespiratory spread of ppmv-1 in the dutch case. this was based on evidence of ppmv-1 infection in liver, kidney, and bone marrow ( table 2 ). this is consistent with the new york case, where evidence of ppmv-1 infection in feces and urine also suggested extrarespiratory pigeon paramyxovirus-linked pneumonia • jid 2018:218 (1 october) • 1041 table 1 spread [11] . the immunosuppressive state of the dutch and new york patients may have allowed spread of ppmv-1 beyond the respiratory tract. pigeons and doves are the animal reservoir of ppmv-1 ( figure 1 and table 1 ). in europe, these viruses are circulating predominantly in domestic pigeons (columba livia domestica) or their wild relatives (rock pigeons, c. livia), and other members of the family columbidae, especially eurasian collared doves (streptopelia decaocto) and european turtle doves (streptopelia turtur) [15] . the tropism of hppmv-1/ nl/579/2003 for the kidney and pancreas of experimentally infected pigeons ( supplementary figures 2 and 3 ) matches the tissue tropism of ppmv-1 in naturally infected pigeons [25] . the low pathogenicity of this isolate in chickens fits with the phenotype of other ppmv-1 strains [26] . the number of feral pigeons-free-living birds that have descended from domestic pigeons-in european and north american cities increased substantially from the 1940s to the 1970s as a result of changes in agricultural practices and the rapid human population increase after world war ii. feral pigeon numbers then stabilized, likely due to reaching the carrying capacity of the urban environment. however, population growth of feral pigeons is still likely to occur in recently colonized cities and at the newly built outskirts of cities [27] . first reported in italy in 1981 [28] , ppmv-1 subsequently spread across europe and has become endemic in feral pigeons, with regular spread to wild pigeons and doves [15] . in north america, ppmv-1 was introduced in the 1980s and now is maintained endemically in pigeons and doves [29] , including the eurasian collared dove [29, 30] . this invasive species, first reported in florida in the 1980s, has rapidly spread across most of north america (http://www.audubon.org/field-guide/bird/ eurasian-collared-dove), and could facilitate virus dispersal throughout north america [29, 30] . the clinical histories of the dutch and new york cases do not readily explain how they became infected. the dutch patient lived in a rural area near a city, but was not employed in agriculture, and there was no evidence of contact with animals (this study). the new york patient was an urban dweller, but it is unknown whether he had pets or was exposed to birds in other settings [11] . based on phylogenetic analysis of the viruses, the most probable route of transmission was contact with infected pigeons or doves. ppmv-1 is environmentally stable in bird feces and can be spread by direct contact or by windborne dust [31] . the route of transmission of other zoonotic pathogens from pigeons to humans may be instructive. five pathogen species (chlamydia psittaci, histoplasma capsulatum, aspergillus species, candida parapsilosis, and cryptococcus neoformans) have been reported to be routinely transmitted from feral pigeons to people [32] , mostly by inhalation of airborne excreta, including dried feces, ocular discharges, and crop milk. contact was sometimes brief, and patients did not always recall any bone marrow (n = 1) 37 + heart, esophagus, stomach, pancreas, right kidney, skin, diaphragm, and muscle tested negative for ppmv-1 rna by real-time reverse-transcription polymerase chain reaction and for ppmv-1 antigen by immunohistochemistry; lung biopsy and lymph node biopsy tested negative for ppmv-1 antigen by immunohistochemistry. abbreviations: +, rare positive granules; ++, occasional positive granules; ct, cycle threshold; ppmv-1, pigeon paramyxovirus type 1. encounters with birds. it is relevant for these ppmv-1 cases that the risk of 2 pigeon-associated diseases-chlamydiosis and cryptococcosis-was largely a function of the immune status of patients, rather than contact with infected birds [32, 33] . close contact between feral pigeons and humans commonly occurs in squares, public gardens, parks, markets, and railway stations in urban areas [33] . therefore, people in urban areas are likely to have a higher rate of contact with feral pigeons than in rural areas. the proportion of the global human population living in urban areas is increasing. in 1950, 30% of the world's population lived in urban areas; this increased to 54% in 2014, and by 2050, 66% of the world's population is projected to be urban [34] . this suggests that the number of people at risk of contracting zoonotic infections, including ppmv-1, from feral pigeons will increase in coming decades. the combination of the above factors-large populations of feral pigeons with endemic ppmv-1 infection, increasing urbanization, and a higher proportion of immunocompromised individuals-may increase the risk of severe human cases of ppmv-1 infection. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. global trends in emerging infectious diseases adaptive pathways of zoonotic influenza viruses: from exposure to establishment in humans limited airborne transmission of h7n9 influenza a virus between ferrets the severe acute respiratory syndrome cross host transmission in the emergence of mers coronavirus nipah virus: transmission of a zoonotic paramyxovirus hendra virus ecology and transmission metagenomic sequencing for virus identification in a public-health setting fields virology. philadelphia: wolters kluwer avian influenza and newcastle disease isolation of avian paramyxovirus 1 from a patient with a lethal case of pneumonia bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt jmodeltest: phylogenetic model averaging a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood newcastle disease in the european union phylogenetic analysis reveals extensive evolution of avian paramyxovirus type 1 strains of pigeons (columba livia) and suggests multiple species transmission virulence of pigeon paramyxovirus type 1 does not always correlate with the cleavability of its fusion protein a family-wide rt-pcr assay for detection of paramyxoviruses and application to a large-scale surveillance study virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis pathogenesis of middle east respiratory syndrome coronavirus epidemiology of invasive mycoses in north america newly discovered coronavirus as the primary cause of severe acute respiratory syndrome pathology of human influenza revisited pathogenesis of influenza-induced acute respiratory distress syndrome avian paramyxovirus type 1 infections in racing pigeons in california. i. clinical signs, pathology, and serology partial characterisation of five cloned viruses differing in pathogenicity, obtained from a single isolate of pigeon paramyxovirus type 1 (ppmv-1) following passage in fowls' eggs feral pigeons: problems, dynamics and control methods. integrated pest management and pest control: current and future tactics successful establishment and global dispersal of genotype vi avian paramyxovirus serotype 1 after cross species transmission expansion of an exotic species and concomitant disease outbreaks: pigeon paramyxovirus in free-ranging eurasian collared doves newcastle disease: methods of spread health hazards posed by feral pigeons chlamydial infections in feral pigeons in europe: review of data and focus on public health implications world urbanization prospects: 2014 revision, highlights (st/esa/ser. a/352) the occurrence of five major newcastle disease virus genotypes we thank the department of pathology, leiden university medical center, the netherlands, for providing biopsies and autopsy specimens from the dutch patient.financial support. this work was supported by european fp7 programme antigone (anticipating the global onset of novel epidemics, project number 278976) and by the european commission h2020 programme under contract number 643476 (www.compare_europe.eu).potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-346411-d2re00r9 authors: boonyaratanakornkit, jim; vivek, meghana; xie, hu; pergam, steven a; cheng, guang-shing; mielcarek, marco; hill, joshua a; jerome, keith r; limaye, ajit p; leisenring, wendy; boeckh, michael j; waghmare, alpana title: predictive value of respiratory viral detection in the upper respiratory tract for infection of the lower respiratory tract with hematopoietic stem cell transplantation date: 2020-02-01 journal: j infect dis doi: 10.1093/infdis/jiz470 sha: doc_id: 346411 cord_uid: d2re00r9 background: hematopoietic cell transplant (hct) recipients are frequently infected with respiratory viruses (rvs) in the upper respiratory tract (urt), but the concordance between urt and lower respiratory tract (lrt) rv detection is not well characterized. methods: hematopoietic cell transplant candidates and recipients with respiratory symptoms and lrt and urt rv testing via multiplex pcr from 2009 to 2016 were included. logistic regression models were used to analyze risk factors for lrt rv detection. results: two-hundred thirty-five hct candidates or recipients had urt and lrt rv testing within 3 days. among 115 subjects (49%) positive for a rv, 37% (42 of 115) had discordant sample pairs. forty percent (17 of 42) of discordant pairs were positive in the lrt but negative in the urt. discordance was common for adenovirus (100%), metapneumovirus (44%), rhinovirus (34%), and parainfluenza virus type 3 (28%); respiratory syncytial virus was highly concordant (92%). likelihood of lrt detection was increased with urt detection (oods ratio [or] = 73.7; 95% confidence interval [ci], 26.7–204) and in cytomegalovirus-positive recipients (or = 3.70; 95% ci, 1.30–10.0). conclusions: high rates of discordance were observed for certain rvs. bronchoalveolar lavage sampling may provide useful diagnostic information to guide management in symptomatic hct candidates and recipients. respiratory virus infections are a major cause of mortality and morbidity in hematopoietic cell transplant (hct) recipients [1] [2] [3] . although symptomatic patients are frequently tested for viruses in the upper respiratory tract (urt), lower respiratory tract (lrt) testing with bronchoscopy and/or bronchoalveolar lavage (bal) is done less frequently, often only when prompted by clinical deterioration or for further evaluation of findings on radiologic imaging. our study assessed the correlation between concurrent urt and lrt testing for respiratory viruses in hct pretransplant candidates and posttransplant recipients. few studies have examined differences in respiratory viral detection by polymerase chain reaction (pcr) between upper and lower tract samples. in immunocompetent children with chronic respiratory symptoms, paired nasopharyngeal (np) aspirate and bal samples showed discordance in approximately one third of patients, with positive np aspirate/negative bal discordance being most common [4] . in a small study of adults, the majority of which were hct recipients or had a hematologic malignancy, with matched np and bal specimens, pcr-based np testing for respiratory viruses in patients with clinical evidence of lrt disease had a high negative predictive value (npv) and a lower positive predictive value (ppv) [5] . a larger study that included mostly immunocompromised patients concluded that if a pathogen (a respiratory virus or 1 of 3 bacterial pathogens detected by a multiplex pcr panel) was already identified from an np sample, bal testing is unlikely to provide additional information; however, a significant number (20%) of subjects had a pathogen detected in the bal without a positive np sample [6] . likewise, in a study of lung transplant recipients, viral detection exclusively in the lrt was reported, and thus the authors caution on the use of urt sampling alone to rule out lrt infection in this population [7] . our study aimed (1) to describe discordance in hct candidates and recipients and (2) to characterize specific viral, patient, and treatment risk factors that are associated with lrt detection. in addition, using quantitative pcr methodology, we aimed to define the role of viral load in respiratory virus lrt detection. we retrospectively identified hct pretransplant candidates (within 90 days of hct) and posttransplant recipients who underwent a bal ±1 and ±3 days from an np aspirate with testing of respiratory specimens by multiplex pcr testing for 11 [8] . in total, exactly 1000 hct recipients underwent bal respiratory virus testing during the study period. clinically indicated bronchoscopy for lrt symptoms and/or radiographic abnormalities was determined by a pulmonologist. a bal was generally performed to either rule-in respiratory viral involvement of the lrt and/or to rule out alternative causes for a lrt process. the bals were collected per institutional standard practice using up to three 30-ml aliquots of normal saline. from this cohort, we then identified subjects who had urt viral pcr testing (from nasal wash or np swab) within 3 days or within 1 day of the bal. only the first bal per subject was included in the analysis. cycle thresholds (ct) were used as a proxy for viral load and were compared between upper and lower respiratory sample pairs that were positive for the same virus for a given subject. patient charts were reviewed for steroid use, radiology results, presence of copathogens, and potential alternate diagnoses. steroid dose was defined as the highest dose of steroid in milligrams/kilogram per day expressed in equivalent doses of prednisone in the 2 weeks preceding the bal. computed tomography of the chest obtained within 1 week of the bal were reviewed. we chose to categorize imaging results for the presence versus absence of a solitary nodule because patients with a solitary nodule may carry an alternative diagnosis and be at lower risk for viral lrt involvement. copathogens were defined as follows: (1) bacterial ->10 000 colony-forming units of a gram-positive organism or any gram-negative organism on bal; (2) viral -any other respiratory virus detected in bal by multiplex pcr or cytomegalovirus (cmv) shell vial positivity; and (3) fungal -positive serum or bal galactomannan, bal fungal pcr positivity, or bal fungal culture positivity (excluding candida species) [9, 10] . alternate diagnoses included diffuse alveolar hemorrhage based on finding progressive bloody fluid return on bal [11] . discordance was defined as either (1) a urt sample positive for a respiratory virus with the paired lrt sample negative for the same virus (termed positive/negative [p/n]) or (2) a urt sample negative for a respiratory virus with the paired lrt sample positive for the same virus (termed negative/positive [n/p]). positive concordance was defined as both urt and lrt paired samples being positive for the same virus (p/p), and negative concordance was defined as both urt and lrt paired samples being negative for the same virus (n/n). the sensitivity and specificity for lrt detection by ct value in the urt was plotted by generating a receiver operating characteristic (roc) curve. the ct value for subjects with negative testing in the urt was set at 40, above the upper limit of assay detection, to allow inclusion of these subjects in the roc analysis. positive and negative predictive values for lrt infection were plotted as a function of all possible ct value cutpoints. logistic regression models were used to evaluate odds ratios (or) for the association between candidate risk factors and viral lrt detection. patients with more than 1 virus detected in the urt (n = 12) were excluded from the logistic regression analysis. all patients in the cohort with lrt detection of adenovirus also had adenovirus testing of the plasma by pcr for viremia. patients with disseminated adenovirus as evidenced by a positive plasma pcr at the time of lrt detection (n = 4) were excluded from the logistic regression and roc analysis. variables with p ≤ .2 in univariable analysis were candidates for multivariable models and were retained in the models if they remained significant themselves or modified the effect of another factor (confounder). covariates evaluated as candidate risk factors for inclusion in multivariable models are listed in table 1 . statistical significance was defined as 2-sided p < .05. sas version 9.4 ts1m3 (sas institute inc., cary, nc) was used for all statistical analyses. we identified 235 subjects with a bal performed during the study period who had urt rv testing within 3 days of the bal. table 1 shows the demographic characteristics of the cohort. the majority of patients (63%) were between 21 and 60 years of age, and 60% were male. the majority of patients (84%) underwent allogeneic transplant. only 14% had a bal before hct. the median number of days between bal and nasal swab was 1 day. table 1 also shows the demographics for a subset of 131 subjects in this cohort with urt and lrt testing within 1 day of each other; the subset is largely representative of the whole cohort. among the 235 sample pairs in the overall cohort, 49% (115 of 235) were positive for a respiratory virus in either the urt or lrt. of these, 63% (73 of 115) were concordant positive for the same virus in both upper and lower tracts (p/p). discordance was noted in 37% (42 of 115) of sample pairs. among the discordant pairs, 60% (25 of 42) were positive in the urt but negative in the lrt (p/n), and 40% (17 of 42) were negative in the urt but positive in the lrt (n/p). figure 1 shows the distribution of concordance/discordance for pairs with at least 1 test positive for individual viruses. in patients who underwent bal within 3 days of an np aspirate, discordance between urt and lrt results was observed at the highest rate for hmpv (9 positive pairs, 33% n/p and 11% p/n), hrv (44 positive pairs, 7% n/p and 27% p/n), piv2 (2 positive pairs, 50% n/p), and piv3 (25 positive pairs, 12% n/p and 16% p/n) ( figure 1a ). all 9 pairs with a positive adenovirus result were discordant (56% n/p and 44% p/n). respiratory syncytial virus had the highest frequency and percentage of concordant results (13 positive pairs, 92% concordant with 8% p/n and no n/p pairs). similar patterns of discordance were observed when analyzing patients who underwent bal within 1 day of an np aspirate ( figure 1b ) and when analyzing the subset of patients who underwent bal after a positive np aspirate (supplemental figure 1) . the ct values of all viruses grouped together between the urt and lrt in concordant pairs were not significantly different when the bal was performed either ±3 days (δct urt-lrt = −0.1, p = .90) or ±1 day (δct urt-lrt = −0.57, p = .56) from collection of the urt specimen. the distribution of copathogens and alternate diagnoses was examined for concordant positive pairs and discordant pairs ( figure 2 ). aspergillus fumigatus, another respiratory virus, or bacteria were the most commonly identified copathogens. no significant differences were observed in the proportion of copathogens or alternate diagnoses in subjects with discordant testing versus concordant positive results by fisher's exact test. in a univariable analysis of risk factors for lrt detection, detection of virus in the urt (or = 54.9; 95% confidence interval [ci], 22.4-135) and recipient cmv seropositivity (or = 2.44; 95% ci, 1.20-4.76) were associated with an increased risk for lrt detection (table 2 ). of note, factors not found to be associated with lrt detection included conditioning regimen, transplant type (allogeneic versus autologous), steroid use, presence of a solitary nodule on imaging, or lymphocyte, neutrophil, monocyte, or overall white blood cell counts. in a multivariable analysis, detection of virus in the urt (or = 73.7; 95% ci, 26.7-204) and recipient cmv seropositivity (or = 3.70; 95% ci, 1.30-10.0) remained strongly associated with an increased risk for lrt detection ( table 2 ). an analysis of the subset of patients who underwent bal within 1 day of the np aspirate and a separate analysis of the subset of only hct recipients who underwent bal after transplantation yielded similar results ( supplementary tables 1 and 2) . the proportion of patients with a positive lrt sample was similar between patients with urt testing before lrt testing (28% or 57 of 201) versus after lrt testing (28% or 5 of 18). thirty-day mortality in patients with positive respiratory viral testing in the lrt was 23.0% (14 of 61) compared with 15.2% (24 of 158) in those with negative testing, although this difference was not statistically significant (p = .23). a roc curve was generated to summarize sensitivity and specificity of varying ct cutpoints in the urt for the presence of lrt infection. a ct cut point of 27.5 in the urt had a sensitivity of 70% and a specificity of 98%, whereas a cutpoint of 32.0 had a sensitivity of 80% and a specificity of 96% ( figure 3a ). using a ct cutoff of 27.5 in the urt with a prevalence of lrt infection of 28% (61 of 219) in the cohort, the ppv was 94% and the npv was 90% for lrt infection ( figure 3b ). with higher ct values in the urt, the ppv declined whereas the npv for lrt infection increased. for comparison, any positive test in the urt had a ppv of 76% and a npv of 95%. in the present study, we characterized the rates of discordance in respiratory viral detection between matched urt and lrt samples in a large cohort of hct candidates/recipients who underwent bal for suspected lrti and had concomitant urt testing. we demonstrate high discordance rates for hrv, hmpv, piv3, and adenovirus between the urt and lrt. furthermore, we identified risk factors for detection of respiratory viruses in the lungs of hct candidates and recipients, including viral detection in the np and recipient cmv seropositivity. before the advent of molecular testing, a high rate of discordance was observed with rapid antigen detection assays which had a sensitivity of 15% in urt specimens and 89% in lrt specimens from immunocompromised adults [12] . discordance between molecular diagnostic testing of urt and lrt specimens has also been reported in other studies, particularly in immunocompromised populations, where 79%-86% of patients with a positive lrt specimen had a concordant urt specimen [6, 13, 14] . the positive and npvs of urt testing were 86%-88% and 89%-94%, respectively. in a different study of hct recipients with hmpv or rsv detected in the lrt, 33% had a discordant negative test for hmpv, whereas no patients had a discordant negative test for rsv [2] . in the present study, we found high levels of discordance with 37% of sample pairs among subjects with positive testing showing discordance between urt and lrt testing for any virus. although the sample sizes were too small for each virus to perform statistical testing, discordance rates were highest for hmpv, hrv, adenovirus, and piv3. of note, this discrepancy was present also among patients with urt specimens obtained within 1 day of the bal. human metapneumovirus lrt disease is associated with high mortality in hct recipients, and our results suggest that a negative urt specimen may not be sufficient to rule out lrt infection [2] . we noted that adenovirus testing showed discordance in every subject with the virus. reactivation in other tissues followed by viremia and dissemination to the lungs may have contributed to this finding. in contrast, rsv testing showed an approximately 100% concordance, suggesting that patients with rsv detected in the urt and clinical/radiographic evidence of lrt involvement may be presumed to have rsv in the lrt. these patients could be treated accordingly for rsv pneumonia and also be enrolled for clinical trials. we have previously categorized lrt infection with respiratory viruses into groups depending on viral detection in the lrt, where proven lrti is defined as a positive lrt sample (bal, lung biopsy, or autopsy specimen) with radiographic abnormality, probable lrti is defined as a positive lrt sample without radiographic abnormality, and possible lrti is defined as a positive urt sample with radiographic abnormality but no lrt sampling. we have shown that subjects with possible lrti have outcomes more similar to urt infection for both piv and rsv, and that subjects with proven/probable lrti have worse outcomes including need for oxygen, oxygen-free days, and mortality [15, 16] . these results suggest that lrt testing to stratify patients into possible versus proven/probable lrti can provide useful prognostic information in hct recipients. this may become increasingly important because new antivirals are in development, many of which are being evaluated depending on the site of infection (upper versus lower tract). our study included 17 patients positive for a respiratory virus in the lrt but negative in the urt. five viruses, namely, adenovirus influenza a, piv2, piv3, hmpv, and hrv, had n/p discordance. thus, proximal urt testing did not identify the lrt pathogen in these cases, including viruses that may warrant treatment with current antivirals (influenza a and adenovirus) as well as viruses for which specific antiviral therapy is being developed (piv and hmpv). the probability of detecting virus in the lrt was increased in patients with virus detected in the urt. other studies have found an association between higher respiratory viral loads and more severe disease manifestations [17] [18] [19] . we found that lower ct values in the urt were associated with higher ppvs for lrt infection. however, the npv appeared to plateau such that the npv at a ct value of 27.5 was similar to the npv of a negative test in the urt (90% vs 95%, respectively). it is important to note that because even a negative pcr in the urt is not fully predictive, ct values for viruses detected in the urt cannot be used on their own to rule out lrt involvement. we also found that cmv-seropositive hct recipients had an increased risk for lrt respiratory virus detection. we have previously reported recipient cmv seropositivity as a risk factor for respiratory virus acquisition and progression to lrt infection after hct [20, 21] . another study reported an association between cmv reactivation and rsv infection after hct with the development of severe pneumonia [22] . cytomegalovirus seropositivity and reactivation have been associated with increased morbidity, mortality, and graft-versus-host disease after hct [23] [24] [25] . the pathogenesis of cmv infection and disease is complex with several immunomodulating interactions between cmv and the immune system, including effects on human leukocyte antigen expression and cytokine production (reviewed in reference [26] ). our findings here suggest that increased risk of lrt infection may be another indirect effect of cmv. furthermore, we observed a trend towards decreased risk of detecting virus in the lrt in patients transplanted between 2014 and 2016 and a trend towards decreased risk in patients transplanted between 2012 and 2013 compared to 2009 and 2011. it is possible this could have been secondary to a decrease in virus detected in the urt in the later years (31% between 2009 and 2011, 24% between 2012 and 2013, and 17% between 2014 and 2016) because a negative result in the urt was strongly associated with a lower risk for lrt detection. this reduction in urt respiratory viral detection may also have been secondary to improvements in infection control practices. alternatively, the trend towards reduced risk of lrt detection in later years may be a reflection of practice changes either with (1) delaying transplants in patients with positive testing for respiratory viruses in the urt or (2) fewer bronchoscopies being performed now compared to in the past [27] . this could have led to a sampling bias in more recent years in which a bronchoscopy was more often performed when lung disease due to an alternative, nonrespiratory viral, process was suspected. we also sought to understand the differences in viral load observed in concordant upper and lower samples. there was no significant difference between viral loads in the upper versus lower tract when analyzing all viruses together in patients with a bal within 3 days or 1 day of urt testing. one argument against early bronchoscopy to test for viral lrt involvement includes the notion that viruses in the urt may be "pushed" into the lrt during the procedure itself. lower respiratory tract positivity could also simply reflect upper tract secretions that are aspirated during the procedure, similar to the finding of oral flora in bacterial cultures of a bal. the finding of several cases of positive testing in the nose yet negative testing in the lungs argues against the "pushing down" of viruses from the urt to the lrt during bronchoscopy and against the detection of viruses in the lrt as an artifact of contamination from the urt during bronchoscopy. the sample size was too small to support analysis of ct values for individual viruses. our study has several limitations. first, even though our sample size was relatively larger than other studies of immunocompromised patients with paired urt and lrt sampling, we could not evaluate risk factors for discordance for individual viruses. second, data were collected retrospectively, and, therefore, need for sampling of the urt and lrt was determined by the clinician. the patient population was limited to those who underwent bal within 1 or 3 days of an np aspirate because viral detection in the lrt was an outcome measure, and therefore risk factors may differ with patients who undergo only urt testing. third, our primary analysis focused on hct recipients who had undergone bal within 3 days from an np aspirate. this could be considered too long a time period. more important, however, the results from this analysis were similar to that for patients who had undergone bal within 1 day of an np aspirate. fourth, differences in sampling from the lrt by bal and urt by swabbing may contribute to differences in ct values. the higher collection volume for bals compared with np aspirates (approximately 30 vs 5 ml) would generally lead to higher ct values in lrt specimens due to greater dilution. fifth, antiviral therapy in patients with influenza or rsv detected in np aspirates may have affected the detection of these viruses in the bal. however, we found that almost every case of rsv infection was concordant and only 1 case of influenza was discordant p/n. sixth, because copathogens and alternative diagnoses were identified in many patients, we cannot definitively conclude whether lrt symptoms, signs, or radiographic abnormalities were caused specifically by the respiratory virus, the copathogen, and/or a concomitant noninfectious process. finally, ct values may not be as generalizable between different assays compared with a true viral load measured in copies/milliliter. even though most currently available commercial pcr assays do not include ct values, our study shows the predictive value of these results for lrt infection. in summary, our data demonstrate discordance between urt and lrt respiratory virus detection for several common respiratory viruses. we suggest that early lrt viral testing could provide useful diagnostic information that may affect management of respiratory viral infections in certain hct candidates and recipients. the challenge of respiratory virus infections in hematopoietic cell transplant recipients mortality rates of human metapneumovirus and respiratory syncytial virus lower respiratory tract infections in hematopoietic cell transplantation recipients significant transplantation-related mortality from respiratory virus infections within the first one hundred days in children after hematopoietic stem cell transplantation respiratory virus detection in nasopharyngeal aspirate versus bronchoalveolar lavage is dependent on virus type in children with chronic 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thank jane kuypers for help with quantitative polymerase chain reaction analysis and zach stednick and chris davis for help with data collection and management.author contributions. j. b. and m. v. designed and performed the research, collected data, analyzed data, and wrote the manuscript; h. x. and w. l. performed statistical analyses, generated tables and figures, and critically reviewed the manuscript; s. a. p. and g. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.supplemental figure 1 . results of upper respiratory tract (urt) and lower respiratory tract (lrt) sample testing among patients with positive urt testing before undergoing bronchoalveolar lavage (bal). concordance or discordance is shown by specific virus (represented as result from urt/ lrt with n = negative and p = positive). data are shown for subjects with a bal ±3 days from the urt test. adeno, adenovirus; flua, influenza a; flub, influenza b; hcov, human coronavirus; hmpv, human metapneumovirus; piv, parainfluenza viruses 1-4; hrv, human rhinovirus; rsv, respiratory syncytial virus.