key: cord-306411-dutbxfl4 authors: eifan, saleh a.; hanif, atif; aljohani, sameera mohammed; atif, muhammad title: respiratory tract viral infections and coinfections identified by anyplex™ ii rv16 detection kit in pediatric patients at a riyadh tertiary care hospital date: 2017-11-21 journal: biomed res int doi: 10.1155/2017/1928795 sha: doc_id: 306411 cord_uid: dutbxfl4 respiratory infections are caused by an array of viruses, and limited information is available about viral coexistence, comparative symptoms, and the burden of illness. this retrospective cohort study aimed to determine the etiological agents responsible for respiratory tract infections by anyplex ii rv16 detection kit (rv16, seegene), involving 2266 pediatric patients with respiratory infections admitted to the department of pediatrics at king abdul-aziz medical city, ministry of national guard, riyadh, from july 2014 to june 2015. the most frequent respiratory infections were recorded in the 1 to 5 year age group (44.7%). rhinovirus (32.5%), adenovirus (16.9%), and respiratory syncytial virus (rsv) b (10.4%) were most common. in single viral infections, rhinovirus (41.2%), metapneumovirus (15.3%), and bocavirus (13.7%) were most frequent. in multiple viral infections, rhinovirus (36.7%), adenovirus (35.2%), bocavirus (11.2), rsv b (7.8%), and rsv a (6.7%) were most frequent. no significant difference was observed in clinical presentations; however, rhinorrhea and hypodynamia were significantly associated with viral respiratory infections. most respiratory viral pathogens peaked during december, january, march, and april. rhinovirus, adenovirus, and bocavirus circulations were detected throughout the year. winter peaks were recorded for rhinovirus, rsv b, adenovirus, and rsv a, whereas the metapneumovirus, and the bocavirus peaked in march and april. these findings enhance understanding of viral etiology and distribution to improve respiratory infection management and treatment. respiratory tract infections lead to mortality and morbidity in children especially during early years. among children, more than 80% of respiratory infections are associated with different viral infectious agents. respiratory virus infections are a major public health problem, due to the ease of spread and considerable morbidity and mortality. the association between respiratory tract infections and different viral pathogens has been reported to vary between 40% and 90% [1] [2] [3] [4] [5] globally. different studies reported the detection of viruses like human respiratory syncytial virus a (rsv a), human respiratory syncytial virus b (rsv b), human adenovirus (adv), human metapneumovirus (hmpv), human coronavirus, and human parainfluenza virus (piv). children under the age of 5 years were detected with human coronavirus 229e (hcov-229e), human coronavirus nl63 (hcov-nl63), human coronavirus oc43 (hcov-oc43), human parainfluenza virus 1 (piv-1), human parainfluenza virus 2 (piv-2), human parainfluenza virus 3 (piv-3), human parainfluenza virus 4 (piv-4), human rhinovirus (hrv), human enterovirus (hev), and human bocavirus (hbov). coinfections with different multiple viruses were reported in 15% to 61% of patients. [6] [7] [8] [9] . molecular techniques such as multiplex polymerase chain reaction (pcr) are widely used for the detection and identification of respiratory viruses [10] [11] [12] and are helpful in the management and treatment 2 biomed research international of respiratory infections [13] . diagnosing respiratory viruses by isolation in cell cultures and serology is time consuming, laborious, expensive, and less sensitive in some cases. molecular techniques provide quick results with high sensitivity and specificity. multiplex pcr has been reported as a fast and sensitive assay for respiratory infection detection. anyplex ii rv16 (seegene, korea) is a multiplex real-time pcr based kit with tagging oligonucleotide cleavage extension (toce) technology. the pitcher and catcher are two novel components used in toce assay for unique signal generation in real time. in toce assay detection point is moved from the target sequence to the catcher so it provides the predictable melting temperature analysis for catcher duplex. this process offers the multiplex real-time pcr capability to anyplex ii rv16 kit. [14] [15] [16] [17] . respiratory infections are mostly reported in children living in developing countries. the spread of respiratory infections varies between populations and countries, depending on differences in geography, climate, and socioeconomic conditions [18] [19] [20] [21] . the central region (riyadh region) of saudi arabia has a dense population of locals and immigrants whose interaction can affect the transmission patterns of different respiratory viruses. previous studies have reported the prevalence of a small number of respiratory viruses within different regions of saudi arabia, and limited information is available on the seasonal distribution of viruses [22] [23] [24] [25] . a better understanding of the local epidemiology and risk factors is critical for the prevention and control of respiratory infections. this study aimed to determine the distribution of 16 different viruses causing respiratory infections in children, by using rv16, and to compare data on demographic characteristics, symptoms, and single infections or coinfections. design. this retrospective cohort study included 2266 patients within an age range of 0 to 14 years from july 2014 to june 2015 with suspected acute respiratory illness and respiratory infection. the patients were examined clinically and initially diagnosed by an admitting physician. nasopharyngeal aspirates, bronchoalveolar lavages, and nasopharyngeal swab specimens were sent for pcr analysis of respiratory viruses to the division of microbiology, pathology and laboratory medicine, king abdul-aziz medical city, riyadh, saudi arabia. when more than one virus was detected simultaneously from single or multiple samples of the same patient, the findings were recorded as multiple infections. patient demographic information was obtained from medical records. demographic and clinical data were recorded in a standardized performa, including age, sex, and clinical presentation. the study was approved by the ethics committee at king abdul-aziz medical city. respiratory virus samples were collected from patients presenting to hospital for the first time with symptoms of respiratory infection or within 7 days of admission. the time frame included for the sampling criteria was based on the incubation periods of these viruses [26, 27] . patient samples collected after 7 days from the date of admission were excluded from the study, as these samples were considered nosocomial infections. nucleic acids were extracted from all samples using microlab nimbus ivd (seegene inc.), and rnas were used for cdna synthesis using cdna synthesis premix (seegene inc.). the samples were tested by using anyplex ii rv16 detection kit (seegene inc.) according to the manufacturer's instructions. the assay was used to detect flu-a, flu-b, rsv a, rsvb, adv, hmpv, hcov-229e, hcov-nl63, hcov-oc43, piv-1, piv-2, piv-3, piv-4, hrv, hev, and hbov. reaction mixtures for virus detection were divided into two panels: a and b. each panel was used to detect 8 viruses with appropriate controls. two types of dna and 14 types of rna viruses were amplified and detected by using cfx 96 real-time pcr thermal cycler (bio-rad). seegene viewer software was used to analyze the amplification results. the study was approved by the research and ethical committee of king abdul-aziz medical city, riyadh. analysis. data analysis was performed by using spss (version 22.0; ibm). differences in the distribution of categorical variables were compared using chi-square or fisher's exact tests. a p value of ≤0.05 was considered significant. from among 2266 hospitalized patients, different respiratory infectious viruses were detected in 2041 (91.6%) samples (1082 male and 959 female participants). among age group of 1 to 5 years 44.7% respiratory infections were recorded and 7.8% infections were detected in age group of 11 to 14 years ( table 1) (table 2) . statistically significant relationships were found between the detection of single and multiple virus infections in the adv and hev groups ( < 0.05). in multiple viral infections coinfections were recorded among hrv (36.7%), adv (35.2%), hbov (11.2), rsv b (7.8%), and rsv (6.7%), respectively (table 3) . patients' clinical presentations with different etiological agents were compared and listed in table 4 . the monthly distribution patterns of different respiratory viruses were shown in figure 1 . the prevalence rate of sixteen respiratory viruses during spring, summer, autumn, in [29] . our findings are in line with previous studies that reported viral detection rates ranging between 47 and 95% [1, 3, 7, 30] . the wide ranging differences in viral pathogen detection rates may be attributed to heterogeneity within the study population, genetic variability, the types and numbers of viral pathogens included for testing, and the methods used for testing [1, 20, 31] . most of the respiratory infections were recorded in the 1to-5-year-old age group, followed by the age group >1 year old (table 1) , whereas a previous study [25] detected a higher ratio of respiratory pathogens among children less than 1 year of age. the higher rate of infections in infants and young children may be related to underdeveloped or weak immune systems, less healthy living conditions, greater pathogen exposure, and poorer hygiene [1, 4, 10, 31] . the detection of disease-causing viral pathogen plays an important role in patient management and treatment. in this study, the most common respiratory viruses detected were hrv (32.5%), adv (16.9%), and rsv b (10.4%). a previous study [25] reported detection rates for rsv (23.9%), hrv (14.7%), and adv (11%) in saudi arabia. in another study in china, influenza viruses (18.50%), rsv (7.86%), and adv (3.47%) were found to be the most common respiratory pathogens [32] . in multiple viral infections, hrv (36.7%), adv (35.2%), hbov (11.2), rsv b (7.8%), and rsv a (6.7%) were found most frequently. in single viral infections, hrv (41.2%), hmpv (15.3%), and hbov (13.7%) were detected most frequently (table 3) . adv and hev were found to be significantly higher in number than other viruses. this finding suggests that the occurrence of these viruses may facilitate other viral pathogens to infect the patient. the mixed viral infection rate was 42.3% for all samples in our study. hrv (36.7%), adv (35.2%), hbov (11.2), rsv b (7.8%), and rsv a (6.7%) were found among most frequent coinfected groups. another study has reported detecting multiple viral infections involving rhinovirus, adv, and hcov-oc43 groups [2] . hrv and adv have also been reported as the leading viral pathogens involved in mixed viral infectious agents among children [33] . these results further suggest that some viral groups facilitate infection or colonization by other viruses in the same patients. viral coinfection or the detection of two or more viral pathogens in a single patient may be attributed to asymptomatic persistence or the shedding of viruses [3] . the great variations in detection rates of multiple viral infections, in different studies, may be related to differences in study populations, locations, study periods, environmental factors, the number of viral pathogens tested, and differences in diagnostic techniques [2, 8, 34] . similarities in clinical presentations of patients infected with different respiratory viruses make it difficult to diagnose the precise etiological agent based on clinical signs. in our study, fever and cough were observed as common symptoms in patients infected with different respiratory viruses, and a strong association of rhinorrhea and hypodynamia was observed alongside other nonsignificant clinical symptoms. in the present study, rsv a, rsv b, hrv, and hev showed peaks of activity during december, whereas hmpv and hbov peaked in march and april. hrv, adv, and hbov circulations were observed throughout the year. winter seasonal peaks were recorded for rsv a and rsv b. an increase in the detection of viral pathogens frequencies occurred during cold and rainy seasons. seasonal variations of different respiratory viruses have been studied [35] and a significantly higher number of viruses have been detected during winter (54.7%) compared to summer (31%), with hrv being the most common pathogen in all seasons. other studies have reported detecting most influenza viruses in november and december, and rsv was detected most frequently between december and february [4, 8, 9, 28] . this study lacked further information on bacterial cultures taken from the samples used for viral pathogen detection. provision of this information would have been helpful for the assessment of concomitant infection by respiratory viruses and bacterial pathogens to help control and treat respiratory infections. this study incorporated data for a period of one year only, but the inclusion of data from the previous and following years could have provided more information on circulatory patterns of respiratory viruses. finally, this study was conducted on a single site in the riyadh region, and virus spread and circulation patterns are likely to differ in other regions of saudi arabia, such as jeddah and dammam. figure 2 : distribution of sixteen respiratory viruses during spring, summer, autumn, and winter. in conclusion, our study provides information regarding the circulatory patterns and seasonal distribution of human respiratory pathogens in the central region of saudi arabia. rhinoviruses, adenoviruses, and rsv were found to be the most common pathogens in pediatric patients. the rv16 based pcr diagnostic approach increased our understanding of viral etiology for better management, control, and treatment of respiratory infections. the authors declare that there are no conflicts of interest regarding the publication of this paper. respiratory pathogens in children with and without respiratory symptoms epidemiology of respiratory viral infection using multiplex rt-pcr in 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associated meteorological factors in southern china human metapneumovirus and human coronavirus infection and pathogenicity in saudi children hospitalized with acute respiratory illness detection of bocavirus in children suffering from acute respiratory tract infections in saudi arabia viral agents causing acute lower respiratory tract infections in hospitalized children at a tertiary care center in saudi arabia viral etiology of respiratory infections in children in southwestern saudi arabia using multiplex reverse-transcriptase polymerase chain reaction nosocomial spread of viral disease incubation periods of acute respiratory viral infections: a systematic review etiology and seasonality of viral respiratory infections in rural honduran children evaluation of viral co-infections in hospitalized and non-hospitalized children with respiratory infections using microarrays viral etiology of acute respiratory infection in gansu province viral etiology of acute febrile respiratory illnesses in hospitalized children younger than 24 months epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china epidemiology and microbiological investigations of community-acquired pneumonia in children admitted at the emergency department of a university hospital prevalence and seasonal distribution of respiratory viruses in patients with acute respiratory tract infections seasonal variations of 15 respiratory agents illustrated by the application of a multiplex polymerase chain reaction assay the authors would like to extend their sincere appreciation to the deanship of scientific research at king saud university through the research grant no. rgp-vpp-253 and national guard hospital for funding of this work. key: cord-352231-awkkper2 authors: bakri, faris ghalib; alqadiri, hamzah m.; adwan, marwan hmoud title: the highest cited papers in brucellosis: identification using two databases and review of the papers' major findings date: 2018-04-11 journal: biomed res int doi: 10.1155/2018/9291326 sha: doc_id: 352231 cord_uid: awkkper2 citation classics represent the highest impact work in a given field. we aim to identify and analyze the most frequently cited papers on brucellosis. we used the databases scopus and web of science to determine the most frequently cited papers. the most cited fifty papers in each database were identified. we then ranked the papers according to the highest citation count recorded from any of the two databases. the most frequently cited paper received 964 citations and was by delvecchio vg et al. reporting the complete genomic sequencing of brucella melitensis. the papers were published in 30 journals led by the “infection and immunity” journal and the “veterinary microbiology” journal (each had 7 papers). citation classics in brucellosis were all in english except one in french and were mostly of basic science type. in addition, we noticed that 12 articles that were identified among the highest fifty articles in one database were missed by the other database and vice versa. therefore, we suggest that searching in more than one database would detect additional citation classics. brucellosis is a zoonotic granulomatous disease that can affect any organ. it is caused by brucella species which are small, gram-negative, and coccobacilli bacteria. clinical presentation varies from an acute, nonspecific febrile illness to chronic, debilitating forms with features of osteoarticular and neuropsychiatric abnormalities [1] . brucellosis was first described in 1887 by david bruce, a british surgeon, who isolated gram-negative coccobacilli from the spleens of five british soldiers who died of fever in malta. in 1905, zammit, a maltese bacteriologist, showed that infected goats transmitted brucellosis and that banning the use of their milk would be effective in eliminating the disease. the observation that apparently healthy goats could be carriers of the disease has been termed one of the greatest advances ever made in the study of epidemiology [2] . the disease has wide geographic distribution and it is one of the most economically important zoonosis. in a review of 76 diseases of animals, brucellosis lies within the top 10 in terms of impact on poor people [3] . in low-income countries, brucellosis is endemic and neglected. it also causes large disease in animals and people and lacks effective control [1, [4] [5] [6] . accurate epidemiological data are not available for many endemic areas, but it has been estimated that more than 500,000 new human cases occur annually [7] . in 1987, garfield listed the "top 100" best cited articles ever published in jama and named them "citation classics" [8] , and these classics represent the highest impact work in a given field [9] . citation analysis in the field of infectious diseases and microbiology was reported for tuberculosis [10] , nontuberculous mycobacteria [11] , anthrax [12] , severe acute respiratory syndrome (sars) [13] , jc virus [14] , herpes simplex virus [15] , ebola virus [16] , schistosomiasis [17] , sepsis [18] , and neglected infectious diseases [19] . here, for the first time to our knowledge, we identify and analyze the citation classics for brucellosis. two electronic databases, scopus and web of science (wos), were searched for the 50 most cited articles using the keyword "brucell * ." for the search in scopus, we selected the "title, abstract, keyword" choice. for the search in wos, we selected the "topic" and "all database" choices. the search in both databases was performed on january 30, 2017, for papers published in all times. textbooks were excluded. the most fifty cited papers were identified in both databases. the articles' abstracts were read by the two study investigators (fgb and mha) to determine whether the articles were specific to brucellosis [20] . we recorded the citation count from the two databases for each selected article. for articles that were among the top fifty articles in one database but not in the other, the citation count in the other database for that article was looked up and recorded. we then ranked the articles according to the highest citation count obtained from any of the two databases. we analyzed the papers according to number of citations, publication year, authors, journal impact factor, country of origin, and article type (basic science, observational study, interventional clinical trial, and review) [21] . basic science articles included genetic studies [22] , in vitro studies, animal studies, or in vivo studies that focused on physiology [23] . observational studies included case-control studies, case series, and cohort studies. to classify the article type, two study investigators (fgb and mha) reviewed all articles independently and in cases of disagreement, they discussed the article until consensus was achieved [21] . the most recent impact factor, year 2015, from journal citation report was used for analysis. in cases where the journal has continued as a new title, the impact factor of the new title was used in the analysis [21] . the list of the most cited articles found in the scopus and wos searches is shown in table 1 . the list included 62 articles. of the total articles, 38 articles appeared in both databases within the highest 50 cited articles. however, among the highest 50 cited articles that were identified by the scopus search, 12 (24%) articles were not among the top 50 articles within the wos search. similarly, among the highest 50 articles that were identified by the wos search, 12 (24%) articles were not among the highest 50 articles within the scopus search. all articles eventually appeared in both databases except for one article (position 21) which appeared only in wos. the mean number of the highest citation count per article from any of the two databases was 284.6 citations (sd = 192.7) and the median number was 197.5 (interquartile range = 169 to 314.5). articles that were identified within the highest 50 articles in wos but not in the highest 50 article in scopus were at positions 7, 8, 11, 21, 22, 29, 41, 49, 51, 55, 56 1950-1959 1960-1969 1970-1979 1980-1989 1990-1999 2000-2009 2010 our results provide a clear picture of the main cited articles in brucellosis research publications history. for example, in the group of genome sequencing, we find at positions 1, 2, 5, 7, and 22 the articles that reported the complete genome sequence for brucella melitensis, brucella suis, brucella abortus strain 9-941, b. abortus strain 2308, and brucella ovis, respectively. at [26, 27] . in the group of articles on molecular diagnostic tests, we find the following: bricker et al. (position 18, in 1994) described a pcr assay that can identify and differentiate most brucella species and biovars found in the united states. prior to this assay, pcr assays did not discriminate among species. baily et al. (position 25, in 1992) developed the first pcr assay around the brucella cell surface protein (bcsp31). this target became one of the most popular targets used in molecular assays [28] . romero et al. (position 61, in 1992) published a brucella 16s rrna based pcr assay, and although similar assay was previously described by herman and de ridder in 1992 [29] , the assay by romero et al. was taken up more widely [28] . in the group of articles on vaccination, we find the following: at position 13, schurig et al. in 1991 produced a live attenuated rb51 strain for vaccination. "r" stands for "rough," "b" for brucella, and "51" for an internal laboratory nomenclature used at the time it was derived. the vaccine has become one of the most commonly used vaccines [30] . first to identify a new member of type iv secretion system family encoded by virb operon in b. suis during a screen for virulence factors. they also showed that the system is essential for the intracellular growth during infection [32] . the system is one of few classical virulence factors identified to date [33] . the type iv secretion system is a pumping system that selectively transports proteins or other macromolecules through membranes [34] . after brucella is taken up by vesicles in macrophage, acidification is thought to induce virb expression. the virb system interacts with components of the endoplasmic reticulum, neutralising the ph and allowing the brucella to undergo regulated cell division [34] . other classics that further the oldest citation classic article was published in 1950 and was at position 29. it was by harris who described the side effects associated with the use of aureomycin and chloramphenicol in treatment of brucellosis. prior to the development of these treatments, chemotherapy of brucellosis yielded unsatisfactory results [37] . the list of classics did not include any article on outbreaks. we suggest the following explanations: (a) papers on brucella outbreaks receive lower citations compared to articles in basic science: in both databases (scopus and wos) the highest cited article on brucellosis outbreaks was "canine brucellosis: outbreaks and compliance, theriogenology, 2006" (78 citations in scopus and 72 citations in wos); (b) outbreaks in brucella have been recognized at very early time; therefore, their findings might have become well known: we found reports of outbreaks as early as 1939 (water-borne outbreak of brucella melitensis infection. am j public health nations health, 1939); (c) identifying brucella outbreaks could be difficult: brucella is difficult to detect and identify [38] ; and (d) brucella species are genetically homogeneous, and thus, the typing of brucella species for epidemiological purposes by conventional molecular typing methods has remained elusive [39] . we also observed the lack of papers on brucellosis in animal health and for this we suggest two explanations: (a) journals in the categories of agriculture and food sciences receive fewer citations than those in basic and clinical sciences as evidenced by the impact factor in these categories. for example, in the wos, in the categories of "agriculture, dairy, and animal sciences" and "food science and technology," the highest impact factor for a journal was 4.7 and 7.3, respectively. while in the category of "medicine, general and internal" and "microbiology," the highest impact factor for a journal was 72 and 23.6, respectively. (b) the possible low productivity of research that is performed on brucella as evidenced by the lower number of articles on brucella in agricultural journals. for example, a combined search for the word "brucell * " and the journals "veterinary research" and "journal of dairy science" yielded 20 and 25 papers, respectively, while the same search in the journals "clinical infectious diseases" and "journal of bacteriology" yielded 52 and 237 papers, respectively. furthermore, we doubt that our search missed important journals from the agricultural fields because the databases scopus and wos include large collection of agricultural journals. scopus has 2608 journals included under the "agricultural and biological sciences" subject area and wos has 58 journals included under the category "agriculture, dairy, and animal sciences" and 130 journals under the category "food science and technology." studies on citation classics that used more than one databases are few and have ranked the articles according to the mean of the citation counts in the databases [40] [41] [42] [43] . here, we ranked the articles according to the highest obtained citation count from any of the two databases. we believe that our method is more accurate because relying on the mean for ranking might lower the rank of a given article. this is because the databases differ in reporting the citation count for a particular article. the variation in citation count between databases results from differences in journal coverage and quality [44] . scopus includes a more expanded spectrum of journals than wos, and its citation analysis is faster and includes more articles than the citation analysis of wos [45] . however, scopus tends to miss older citations which results in omission of studies before 1980 [46, 47] . here, we identified 12 articles that were listed in the highest 50 articles in one database but were not identified within the highest 50 articles in the other database and vice versa. articles that were identified by wos and not by scopus tended to be older and of basic science type, while articles identified by scopus and not by wos were more recent and mostly of observational and review type. we found that many countries had contributed to the classics including american, european, african, and mediterranean countries (table 3 ). this might reflect the epidemiological distribution of brucella. in addition, the finding that the most recent classic article was in 2011 indicates that brucellosis is a dynamic field of study [21, [48] [49] [50] [51] . our study has several limitations that are similar to other studies in citation classics [21] . these limitations include the presence of inherent problems in the citation process itself, for example, incomplete or inappropriate citations, biased citation [44, 45, 52, 53] ; changes in the list of citation classics with time making it a snapshot of the current state of research [54] ; absence of articles with languages other than english which is mostly because authors are more likely to cite articles in their own language, and english articles are more likely to be cited overall [20] ; and finally, missing of important studies because their findings became well known [55] . the latter point is relevant here because brucellosis was discovered in 1887 and it is possible that some important studies were not indexed in current database but their findings are now considered well known. despite these limitations, the study provides a picture for the main cited articles in brucellosis research publications since the discovery of brucella 130 years ago. in conclusion, the citation classics in brucellosis were (a) all in english except one in french, (b) contributed by authors from several countries where brucellosis was or is still endemic, (c) mostly of basic science type, and (d) published in relatively high numbers in recent years indicating a dynamic field of study. in addition, we suggest that performing the search in more than one database would detect additional articles. the authors declare that they have no conflicts of interest. brucellosis in low-income and middle-income countries how themistocles zammit found malta fever (brucellosis) to be transmitted by the milk of goats global burden of human brucellosis: a systematic review of disease frequency neglected tropical diseases of the middle east and north africa: review of their prevalence, distribution, and opportunities for control neglected zoonotic diseases-the long and winding road to advocacy economics of brucellosis impact and control in low-income countries the new global map of human brucellosis 100 citation classics from the journal of the american medical association the most cited works in epilepsy: trends in the "citation classics the 100 top-cited tuberculosis research studies the 100 most-cited articles on non-tuberculous mycobacterial infection from 1995 to 2015 the seminal literature of anthrax research the highly cited sars research literature mapping the history and current situation of research on john cunningham virus -a bibliometric analysis citation classics of herpes simplex virus tracing the scientific outputs in the field of ebola research based on publications in the web of science analysis of highly cited schistosomiasis related papers from the top cited clinical research articles on sepsis: a bibliometric analysis bibliometric assessment of european and sub-saharan african research output on poverty-related and neglected infectious diseases from top-cited articles in digestive system disease from 1950 to 2013 citation classics in chronic granulomatous disease: a bibliometric analysis types of study in medical research: part 3 of a series on evaluation of scientific publications evaluating research in cardiovascular medicine: citation counts are not sufficient brucella microti sp. nov., isolated from the common vole microtus arvalis from the discovery of the malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a reemerging zoonosis brucella abortus 16s rrna and lipid a reveal a phylogenetic relationship with members of the alpha-2 subdivision of the class proteobacteria brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization recent advances in molecular approaches to brucella diagnostics and epidemiology identification of brucella spp. by using the polymerase chain reaction brucellosis vaccines: past, present and future on the biochemical mode of action of levamisole: an update type iv secretion system of brucella spp. and its effectors the brucella virb type iv secretion system human brucellosis the two-component system bvrr/bvrs: a master regulator of brucella virulence clinical manifestations and complications in 1028 cases of brucellosis: a retrospective evaluation and review of the literature present knowledge of brucellosis; a summary dna genotyping suggests that recent brucellosis outbreaks in the greater yellowstone area originated from elk evaluation of a multilocus variable-number tandem-repeat analysis scheme for typing human brucella isolates in a region of brucellosis endemicity analysis of the 100 most-cited articles in periodontology the 100 most-cited original articles in cardiac computed tomography: a bibliometric analysis the top 50 articles on minimally invasive spine surgery an audit of top citations published in pediatric emergency care coverage and quality: a comparison of web of science and scopus databases for reporting faculty nursing publication metrics comparison of pubmed, scopus, web of science, and google scholar: strengths and weaknesses three options for citation tracking: google scholar, scopus and web of science top 100 cited articles in cardiovascular magnetic resonance: a bibliometric analysis top-cited articles in rehabilitation the most cited papers in osteoporosis and related research the top 100 cited articles in clinical orthopedic sports medicine bibliometric analysis of the top 100 cited cardiovascular articles citation classics in anesthetic journals citation classics in critical care medicine the most cited works in essential tremor and dystonia citation classics in obstetrics and gynecology: the 100 most frequently cited journal articles in the last 50 years key: cord-271106-srym2kh4 authors: de rosa, nicoletta; giampaolino, pierluigi; lavitola, giada; morra, ilaria; formisano, carmen; nappi, carmine; bifulco, giuseppe title: effect of immunomodulatory supplements based on echinacea angustifolia and echinacea purpurea on the posttreatment relapse incidence of genital condylomatosis: a prospective randomized study date: 2019-04-11 journal: biomed res int doi: 10.1155/2019/3548396 sha: doc_id: 271106 cord_uid: srym2kh4 introduction. hpv infection is a highly infectious disease; about 65% of partners of individuals with genital warts will develop genital condylomatosis. only in 20-30% it regresses spontaneously and relapse rates range deeply (9-80%). echinacea extracts possess antiviral and immunomodulator activities. the aim of this study was to evaluate the efficacy of the therapy, using a formulation based on hpvadl18® (on dry extracts of 200 mg echinacea purpurea (ep) roots plus e. angustifolia (ea)), on the posttreatment relapse incidence of genital condylomatosis. materials and methods. it is a prospective single-arm study. patients with a satisfactory and positive vulvoscopy, colposcopy, or peniscopy for genital condylomatosis were divided into two random groups and subjected to destructive therapy with co2 laser. group a (n=64) immediately after the laser therapy started a 4-month treatment with oral hpvadl18®; group b (n=61) did not undergo any additional therapy. patients were subjected to a follow-up after 1, 6, and 12 months. differences in relapse incidence between the two groups during follow-up controls were evaluated by χ2-test; the groups were stratified by age, gender, and condylomatosis extension degree. results and discussion. gender, age, and condyloma lesions' extension degree showed no statistically significant differences between the two trial groups. the relapse incidence differs statistically between the two studied groups and progressively decreases during the 12 months after treatment in both groups. statistically significant reduction of relapse rates has been shown in group a in patients over 25 years old. this difference is significant for both men and women. the relapse incidence is superior in case of extended condylomatosis. conclusions. in conclusion, the presence of a latent infection causes condylomatosis relapse; in order to reduce the relapse risk an induction of a protective immune response seems to be essential to allow rapid viral clearance from genital areas surrounding lesion and treatment zones. echinacea promotes this process. ep and ea dry root extracts seem to be a valid adjuvant therapy in reducing relapse incidence of lesions in patients treated for genital condylomatosis. hpv infection is one of the most common sexually transmitted infections in the world. more than 50% of sexually active adults contract the infection during their life. in the two years after a sexual debut the sexual risk of infection varies from 40 to 80% depending on the studied population and the hpv type [1] . there is a similar incidence of genital condylomatosis in males and females (0-2% and 0-7%) [2] [3] [4] . in men, compared to women, infections with multiple genotypes and low-oncogenic risk genotypes are more frequent [5] . only 20-30% of the genital condylomatosis regresses spontaneously. this is a highly infectious disease; about 65% of partners of individuals with genital warts will develop genital condylomatosis. the risk of infection and the risk of progression of hpv-associated lesions are related to several factors including number of sexual partners experienced during the life and early age of the first intercourse; tobacco smoking; and eating habits [6] [7] [8] . it has long since known that the above-ground portion and the roots of echinacea angustifolia (ea) and of e. purpurea (ep) possess anti-inflammatory and immunostimulatory properties. numerous in vitro and in vivo studies have been recently conducted in an effort to validate some of the traditional uses of echinacea extracts [9] . early studies have shown that only a few echinacea extracts possess significant antiviral activity. in particular, above-ground portions and roots of ep show a strong antiviral activity, as they have a virucidal effect against influenza virus, herpes simplex virus, and coronaviruses [10, 11] . the ep appeared much less effective against intracellular viruses [12, 13] , which could be resistant to the ep inhibitory effect; on the contrary, viral particles located in the extracellular fluids appeared to be vulnerable. therefore, ep can act during an initial contact with virus, that is, at the beginning of infection and also during the transmission of the virus from the infected cells. numerous viral and bacterial infections cause an increase of expression of proinflammatory cytokines, in particular, of il-6 and il-8, which are therefore considered as markers of an inflammatory state [14, 15] . any compound or herbal extract that inhibits or inverts the increase of il-6/8 can be considered a potential anti-inflammatory agent. all the portions of the roots, leaves, stems, and flowers of ep show this effect [16] . these studies make it evident that echinacea not exactly acts as an "immunostimulant" or "immune system booster," but more likely has an immunomodulatory action, rather than a generalized immunostimulatory effect [17] [18] [19] [20] . the aim of the present study was to evaluate the efficacy of the therapy, using a formulation based on 200 mg of hpvadl185 (equal to 4 mg polyphenols plus 0.6 mg of echinacosides), on the post-treatment relapse incidence of genital condylomatosis. between july 2014 and july 2017, all patients with a genital condylomatosis diagnosis received in the colposcopy and cervical-vaginal pathology unit of university federico ii, naples, were invited to participate in a prospective randomized trial. patients were properly informed and provided their written consent to participate in the trial and to undergo ambulatory diagnostic examinations; afterwards, colposcopy or peniscopy was conducted and, if appropriate, biopsy examinations. all procedures performed in the study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. the criteria for participation in the trial were as follows: satisfactory and positive colposcopy / peniscopy for genital condylomatosis (cervix, vagina, perianal vulva, or perineum for females and penis, scrotum, or anal region for males) and / or histological examination for koilocytosis or condylomatosis in case of positive cervical biopsy. patients with h-sil cytological diagnosis, cin 1-3 histologic diagnosis, or invasive cervical carcinoma, pregnant women, immunosuppressed patients, and individuals infected with human immunodeficiency virus (hivpositive) were not enrolled in the trial. colposcopy and peniscopy were conducted after an application of 3% acetic acid. visible acetowhite lesions have been classified in accordance with the criteria of the international federation of cervical pathology and colposcopy [21] . in case of genital condylomatosis, to standardize extension of the lesions, genitals were divided into 10 genital areas for women, that is, cervix, left/right vaginal wall, left/right major labia, left/right minor labia, clitoris, pubis, perineum, and perianus and into 5 genitals arear for men, that is, pubis, scrotum, glans, preputial balanus grooves, and penis. patients were classified into 3 lesion degrees, according to the number of genital areas affected by condylomas and the number of the condylomas: (1) from 1 to 5 condylomas on 1-2 genital areas (mild and localized condylomatosis) (2) > 5 condylomas on 2-3 genital areas (mild and diffuse condylomatosis) (3) > 5 condylomas on > 3 genital areas (extended condylomatosis). patients with low grade (ztag1) or high grade (ztag2) cervical lesions were subjected to a targeted biopsy using a biopsy forceps (cfs chimo schumacher pliers) with 5-6 mm jaw in order to obtain 4-5 mm tissue specimens. two serial 4 micron sections of the formalin-fixed and paraffin-embedded sample were stained with hematoxylin and eosin. the specimens were examined by optical microscope and classified as normal, cin 1, cin 2, and cin 3 carcinoma in situ or microinvasive carcinoma according to the criteria of the world health organization. patients with low grade (cin 1) or high grade (cin2-3) preneoplastic lesions were excluded from the trial and carried on all the therapeutic and diagnostic procedures as recommended by national and international guidelines. patients with genital condylomatosis, diagnosed through colposcopy, vulvoscopy, peniscopy, and/or biopsy examinations, were included in the study. all enrolled individuals were divided into two random groups and subjected to destructive therapy with co2 laser. group a immediately after the laser therapy started a 4month treatment with oral immunomodulatory supplements based on hpvadl185; group b did not undergo any additional therapy (control group). the medical device administered to group a was composed of 200 mg of hpvadl185 (equal to 4 mg polyphenols plus 0.6 mg of echinacosides), 40 mg vitamin c, 5 mg of zinc, and 0.5 mg of copper. patients were subjected to a follow-up colposcopy after 1, 6, and 12 months. in case the infection persisted and relapse condyloma lesions occurred, patients were again subjected to destructive therapy until the full lesion elimination. all colposcopy, peniscopy and biopsy examinations and therapies were performed by our team. statistical analysis of the data was executed by spss software 20.0 (spss inc., chicago, il, usa). data with p-values <0.05 were considered statistically significant. demographic and clinical data of the two groups were compared by student's t-test for the data with parametric distribution (age) and by 2-test for ordinal variables (gender and condylomatosis extension degree). differences in relapse incidence between two groups during follow-up controls were evaluated by 2-test; the groups were stratified by age, gender, and condylomatosis extension degree. one hundred and forty women appeared to be suitable for destructive therapy with co2 laser and were divided into group a (n = 70) and group b (n = 70) at random. of these, 6 patients did not undergo a required operation and 9 patients did not undergo a programmed follow-up or interrupted the therapy before the 4-month period expired. one hundred and twenty-five patients, 90 (72%) women and 35 (28%) men, completed the diagnostic-therapeutic procedure as scheduled by the protocol and were therefore included in the analysis. of the studied population, 64 women (51.2%) underwent echinacea therapy after the treatment (group a) and 61 (48.8%) did not undergo any additional therapy (group b, control group). the mean age of female patients in group a is 33.0±8.4 years, in group b 32.1±7.3 years (p = n.s.); the mean age of male patients in group a is 31.4±7.2 years, in group b 34.4±7.1 years (p = ns). table 1 shows epidemiological data and condyloma lesions' extension degree for groups a and b. there were no statistically significant differences in these data in the two trial groups. no severe side effects were recorded in group a. only 5 (7.8%) patients reported some digestive difficulties. the relapse incidence differs statistically between the two studied groups (table 2, figure 1 ) and progressively decreases during the 12 months after treatment in both groups. therapy does not seem to modify the relapse incidence in very young female patients under the age of 25. instead, statistically significant reduction of relapse rates has been shown in patients over 25 years old. this difference is significant for both men and women. the relapse incidence is superior in case of extended condylomatosis (extension degree n.3) (table 2, figure 1 ). clinical trials conducted on patients with genital condylomatosis show quite different relapse rates, depending on the studies and on the treatment and range from 9% to 80% [22] [23] [24] [25] . our data show a global relapse rate of about 30%. therapy with hpvadl18 is effective in reducing relapse incidence of lesions in patients treated for genital condylomatosis. our data prove, indeed, that the relapse incidence of lesion is greater in the control group compared to the treatment group at the first, second, and third follow-up controls. spontaneous remission of genital condylomatosis is possible, but not frequent; the percentage of spontaneously recovered patients varies considerably and ranges from 0% to 50% [24, 25] . most commonly used therapy is cryotherapy or diathermocoagulation (65% and 28%); drug therapy is much less frequent (6%). approximately 50% of patients undergo a single treatment procedure; the number of patients that undergo more than one treatment procedures progressively decreases; 3% of patients undergo 5 or more treatments [26] . this pattern is similar for both sexes and is according to the anatomical site [26] . in compliance with these data, the difference in relapse incidence between the two trial groups is statistically significant even when these are stratified by gender and extension degree of the lesion. on the other hand, age appears to be a determinant factor; in fact, in individuals under the age of 25, the therapy does not seem to influence significantly the relapse incidence of lesion. the small numbers of younger age groups, however, cannot induce us to generalize this data. based on these data, it follows that in very young individuals additional therapy with hpvadl18 could be superfluous. moreover, individuals under the age of 25 show greater relapse incidence at the first follow-up. the relapse incidence decreases progressively in both groups as the time passes and is related to the extension degree; in fact, the extension degree 3 of condylomatous lesions corresponds to a higher relapse incidence than degrees 1 and 2. the presence of a latent infection causes lesion relapse; in order to reduce the relapse risk after the treatment of condyloma lesions, an induction of a protective immune response seems to be essential to allow rapid viral clearance from genital areas surrounding lesion and treatment zones. introduction of an immunostimulatory substance such as echinacea seems to promote this process. the hpv-induced immune response is both humoral and cell mediated. a humoral immune response to hpv capsid protein l1 is weak during natural infection. the humoral immune response to the viral capsid can be detected averagely starting from 6 months after the infection, though 30-50% of patients with persistent infection will never present a seroconversion [27] . the seropositivity to the infectious genotype persists only in 50% of the cases, even when the initial lesion transformed to a cervical cancer [28] . when viral dna has been eliminated, specific antibodies can be detected only in half of cases after 5 years [29] . hpv infection promotes a cellular immune response, especially in the active phase of the clearance of genital condylomatosis infection, when a cell infiltration of macrophages and t cells develops in correspondence to the lesion [30] . in the blood, an immune response of cd4+ t cells against e2, e6, and e7 proteins is associated with hpv 16 and hpv 18 infection and occurs in particular in early disease phases and in case of regressing lesions, less when a persistent disease takes place. in individuals with a deficiency of cell-mediated immune response, hpv infection, genital condylomatosis, or precancerous lesions are destined to persist. therefore, this type of response seems to be essential for the viral clearance. the ep immunomodulatory effect has been widely demonstrated. ep extract was used for the preventive care and for the treatment of various viral infections [31] . in vitro studies have shown that ep acts directly on a number of cell types, including natural killer cells [32] , polymorphonuclear leukocytes [33] , and macrophages [34] . ep induces a proliferation of t cells. this has been conferred to the activation of macrophages that stimulates a production of ifn-and, consequently, a secondary activation of t lymphocytes [35] . ifn-is one of the fundamental mediators for the latency prevention [36] ; it has been proven that this mechanism is responsible for reducing the latency incidence of herpes virus simplex infection and, consequently, reducing the relapse risk of hsv lesions [36] . it is possible that an analogous mechanism induces a cell-mediated response to hpv infection, which allows the reduction of the persistence of infection and, therefore, the lesion relapse. this study has some limitations: this is a single institution study with a small number of participants and it lacks placebo controls. on the other hand, the strengths of this study are as follows: the rigorous inclusions criteria, the evaluation of patients at colposcope (so not only grossly visible genital warts were evaluated and treated but also small lesions), and the treatment modality with laser co2 for all patients. in conclusion, hpvadl185 seems to be a valid adjuvant therapy in reducing relapse incidence of lesions in patients treated for genital condylomatosis. the data used to support the findings of this study are included within the article. the authors state that there are no conflicts of interest. meta-analysis of 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treatment of human papillomavirus-mediated genital lesions an evidence-based review of medical and surgical treatments of genital warts external genital warts: diagnosis, treatment, and prevention management of genital warts a retrospective analysis of the costs and management of genital warts in italy comparison of human papillomavirus types 16, 18, and 6 capsid antibody responses following incident infection human papillomavirus 16 and 18 l1 serology compared across anogenital cancer sites determinants of human papillomavirus 16 serological conversion and persistence in a population-based cohort of 10 000 women in costa rica characterization of human antibody-reactive epitopes encoded by human papillomavirus types 16 and 18 treatment of the common cold with unrefined echinacea: a randomized, double-blind, placebocontrolled trial echinacea purpurea and melatonin augment natural-killer cells in leukemic mice and prolong life span the effect of aerial parts of echinacea on the circulating white cell levels and selected immune functions of the aging male sprague-dawley rat echinacea stimulates macrophage function in the lung and spleen of normal rats the current trend in genital herpes: progress in prevention cd4 t cell control of acute and latent murine gamma herpes virus infection requires ifn key: cord-259823-ia1g5dt4 authors: gowin, ewelina; bartkowska-śniatkowska, alicja; jończyk-potoczna, katarzyna; wysocka-leszczyńska, joanna; bobkowski, waldemar; fichna, piotr; sobkowiak, paulina; mazur-melewska, katarzyna; bręborowicz, anna; wysocki, jacek; januszkiewicz-lewandowska, danuta title: assessment of the usefulness of multiplex real-time pcr tests in the diagnostic and therapeutic process of pneumonia in hospitalized children: a single-center experience date: 2017-01-15 journal: biomed res int doi: 10.1155/2017/8037963 sha: doc_id: 259823 cord_uid: ia1g5dt4 the aim of the study was assessment of the usefulness of multiplex real-time pcr tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. methods. the study group included 97 children hospitalized due to pneumonia at the karol jonscher teaching hospital in poznań, in whom multiplex real-time pcr tests (ftd respiratory pathogens 33; fast-track diagnostics) were used. results. positive test results of the test were achieved in 74 patients (76.3%). the average age in the group was 56 months. viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). the presence of comorbidities was established in 90 children (92.78%). on the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. conclusions. our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. multiplex real-time pcr tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations. acute respiratory tract infections are the most common infectious diseases among children. the incidence of community-acquired pneumonia in europe is 33/100,000 in the age group < 5 years [1] . its clinical manifestation includes symptoms ranging from mild rhinitis to severe pneumonia leading to respiratory failure. the risk group of severe disease course includes children < 5 years of age, especially boys, individuals in immunosuppression, and patients suffering from chronic diseases [2] . establishing the etiological factor is a difficult task. there are no clinical, radiological, or biochemical markers that would enable the differentiation between bacterial and viral infections [3] . this frequently results in the overuse of antibiotics in the therapy of acute respiratory infections. british, american, and polish guidelines state that, in children hospitalized due to pneumonia, microbiological examinations should include blood cultures, the detection of the presence of viruses with the use of pcr (polymerase chain reaction) or immunofluorescence in material collected from the nasopharynx (smear or upper respiratory aspirate), the assessment of antibodies against mycoplasma and chlamydophila in classes igm and igg, and the comparison of antibody levels in the acute phase of the disease and during convalescence [4] [5] [6] . in children it is difficult to collect reliable material for microbiological analysis in a low or noninvasive manner and retain its representativeness of the lower respiratory tract flora. nasal smear cultures are not useful in establishing the etiology of pneumonia. the pathogens grown in a such manner include both physiological flora as well as flora that could potentially cause pneumonia. on the other hand, microbiological cultures need to be secured before antibiotic therapy is commenced, which is impossible in many cases. the solution to this problem is the detection of microbial genetic material in, for example, the material acquired from nasal smear cultures. it is, however, important to remember that pcr results may be positive in the case of persistent infections; in some pathogens prolonged shedding is observed even up to 7 months since the beginning of an infection [7] . another problem is that traditional bacterial cultures are insufficient to establish the etiology, mainly due to the significant participation of viruses in the etiopathogenesis of these infections. this especially concerns children in their first year of life, in whom viral infections may be responsible for even 67% of pneumonia cases [8] . the currently used molecular examinations enable quick identification of numerous pathogens. detecting the influenza virus with pcr is a widely accepted and utilized method of confirming infection [9] . this is also true for the respiratory syncytial virus (rsv). the main drawback of tests detecting just a single pathogen is the necessity of requesting every examination separately, collecting samples for analysis multiple times, and making decisions concerning the selection of pathogens for analysis. test panels used for establishing the presence of the most important bacterial and viral pathogens enable simultaneous detection of the most significant etiological factors. a separate problem is that, apart from the accepted viral etiology, pneumonia may also be caused by new viruses, such as the human metapneumovirus (hmpv), human coronavirus, or bocavirus [10] . molecular techniques are more sensitive and capable of diagnosing more viruses [11] . prompt and accurate diagnosis is important for infection control and surveillance, patient cohorting, treatment choices, and avoiding antibiotics. previous experience with the use of the multiplex realtime pcr tests in the population of children is limited [11] [12] [13] [14] . aim of the study is the assessment of the usefulness of multiplex real-time pcr tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. the study group was composed of children hospitalized due to pneumonia in the period between 01.2014 and 02.2015 at the karol jonscher teaching hospital in poznan, in whom multiplex real-time pcr tests (ftd respiratory pathogens 33; fast-track diagnostics) were used in the diagnostic process. ftd respiratory pathogens 33 is an in vitro test with eight multiplex real-time pcr reactions for the qualitative detection of the following viruses, bacteria, and fungi causing respiratory infections: influenza a, b, and c; parainfluenza viruses 1 the analysis included the following factors: age, sex, comorbidities, immunosuppression, low body mass, airways obstruction, and respiratory failure requiring admission to icu (intensive care unit). respiratory samples (throat or nasal swabs) were collected from all patients. the next step was extraction of pathogens' genetic material either dna or rna, followed by amplification of specific regions by real-time polymerase chain reaction. the presence of specific pathogen sequence in the reaction is reported as a cycle threshold value. apart from the multiplex real-time pcr method, also traditional microbiological culture tests were performed in the studied children: blood cultures, nasopharynx smear cultures, and respiratory aspirate cultures. positive microbiological cultures were defined a ≥105 cfu/ml. all the described tests were done as diagnostic tests during hospitalization. informed consent was obtained from parents/legal guardians on admission to hospital. analysis. data were presented as percentages or medians and range means and standard deviations. interval data were analyzed by mann-whitney test since data did not follow normal distribution (kolmogorov-smirnov test). nominal data were analyzed by chi-square test of independence or in case of zero observed frequencies an exact fisher-freeman-halton test was used. data were analyzed with the use of statistical packages statistica 10 (statsoft, inc.) and statxact 8.0 (cytel); all tests were considered significant at < 0.05. the study group included 97 patients, 52 boys and 45 girls; the average age in the group was 56 months. the presence of comorbidities was established in 90 children (92.78%), including respiratory system diseases in 25 cases, cardiac diseases in 21 cases, neurologic diseases in 13 cases, and neoplastic diseases in 9 cases. immunosuppression was identified in 11 patients (11.34%). the details are provided in table 1 . positive test results were achieved in 74 patients (76.3%). the presence of a viral factor was established in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). the presence of the genetic material of a single pathogen was found in 34 children (45% of all positive results). the details of the analysis are presented in table 2 . on the basis of the obtained results, 5 groups of patients were established: when comparing these groups, no differences were found in terms of age, day of material collection in relation to hospitalization time, or the necessity of icu stay. radiological image analysis was not sufficient to unambiguously diagnose the etiology of pneumonia or respiratory infection. the presence of airflow obstruction was significantly higher in patients with a viral etiology ( = 0.0011). on admission 54% of children with negative real-time pcr results had elevated procalcitonin levels. this proportion is significantly higher when compared to patient with established etiology ( = 0.0186). such difference was not observed while comparing proportion of patients with markedly elevated crp levels (>50 mg/dl). the prevalence of increased procalcitonin levels (>2 ng/ml) was higher in patients without established etiology. the difference is of statistical significance. such differences were not observed when comparing number of patients with increased crp levels. airflow obstruction was found in 44 patients (45%); 75% of them had viral etiology of pneumonia. the difference was of statistical significance ( = 0.0011). the most frequently identified pathogen was rsv in 23 children (23.7%), and rhinovirus (rh) was found in 19 patients (19.6%), adenovirus (adv) in 11 (11.3%), cytomegalovirus (cmv) in 10 (10.3%), and pneumocystis jiroveci (pnp) in 9 (9.3%). purely bacterial etiology was established in 8 patients (8.2%). the details are presented in figure 1 . relations were found between age and the presence of individual microorganisms: infections of rsv etiology were more frequent in younger children (median age 10.5 months versus 42 months; = 0.0133); airflow obstruction symptoms were also more frequent in this group ( = 0.0008). in children under the age of one, the dominant pathogen was rsv, which was found in 32% of patients in this age group. 56% of all positive results for rsv occurred in children in their first year of life. blood cultures were performed in 75 children, in 62 cases they were sterile (82,67%), bacterial flora growth occurred in 13 cases, and in 3 (4%) cases it was pathological (s. aureus, serratia sp., and e. coli). respiratory aspirate cultures were performed in 64 children, they were sterile in 15, physiological respiratory tract flora growth was achieved in 28 cases, and in 23 it was pathological; in 8 of these cases the results were confirmed with multiplex real-time pcr tests. nasal smear cultures were performed in 58 children, they were sterile in 15, and the presence of pathological flora was found in 17 cases (29.31%); in 5 of these cases the results were confirmed with multiplex real-time pcr tests. detailed results of microbiological analyses of patients with at least one positive microbiology results are presented in table 3 . in the group of children with negative results of multiplex pcr tests, blood cultures were positive in 3 cases (e. coli, serratia, and s. aureus), tracheal aspirate cultures were positive in 7 (in 4 of them pathological flora was grown), and nasal smear cultures indicated the presence of pathogenic flora in 5 cases (see details in table 3 ). test results enabled the implementation of targeted therapy in 13 patients; details are presented in table 4 . the acquired results confirm the usefulness of multiplex real-time pcr tests in establishing the etiology of severe pneumonia in hospitalized children. probable etiology of pneumonia was diagnosed in 76% of children with the use of this method. after the exclusion of children with neoplastic diseases from the group, this percentage increased to 79.54%. similar percentages of positive results are often encountered in the literature [8, 12, 14] . bierbaum et al. achieved positive results of multiplex real-time pcr tests detecting only viral factors in 76% of cases in a group of children below the age of six with symptoms of respiratory tract infection and the dominant pathogen was rsv [12] . in a study conducted by mengelle et al. on a group of 914 children with symptoms of respiratory infection, 90% of the collected samples were positive, with rh, rsv, and if (influenza virus) as the most frequent pathogens [15] . in our analysis, we found a more significant participation of rsv; however, only patients with pneumonia underwent the analysis. the general lower participation of the viral factor in comparison to the [16] . in an italian study within a group aged 5-14 years, etiology was established in 77% of cases with the use of molecular analysis. the presence of viruses was revealed in 65% of samples and bacteria in 40% of samples, while mixed etiology was present in 28% of children [17] . mengelle et al. demonstrated that infections caused by more than one virus are common and occurred in 30% of the children included in his study. the sensitivity of virus detection of the multiplex realtime pcr method is high and reaches almost 90% [11] . when genetic material of several pathogens is revealed with the use of the pcr method, it may result from a simultaneous infection caused by two pathogens or from an infection in a patient who is already a carrier of another pathogen. it may also result from establishing the presence of viral genetic material after a previous infection, through the so-called virus shedding. it is estimated that the etiology of pneumonia is mixed in about 30% of children [18] . some works point out that there is a relation between the type of viral factor and the risk of coinfection. martic et al. found coinfections with different pathogens in 50% of infections caused by adenovirus [19] . the high percentage of coinfections in our study may be explained by the specificity of the study group. the majority of the children were burdened with the presence of chronic diseases, and some of these patients were in immunosuppression. the presence of bacterial coinfection is frequent in viral etiology of pneumonia; however, bacteria are not always responsible for active infections. in children with pneumonia, establishing the presence of viruses in the upper respiratory tract is considered as a probable etiological factor. positive results may also be achieved in individuals in temporary asymptomatic carrier state during convalescence after a previous infection [13] . it is, therefore, necessary to remain very cautious when interpreting examination results. the clinical significance of human rv rna detection in respiratory samples remains unclear [20] . the pcr method does not enable unambiguous differentiation between infection and carriage, even with the use of the quantitative method. it needs to be stated, however, that comparative studies of carriage significantly more often indicated the presence of s. pneumoniae in children with pneumonia compared to the healthy population [21] . the available literature contains reports stating that higher numbers of viruses were usually associated with milder disease course [20] . the exception was the coinfections with rsv and hmpv [22] . infections of this type are characterized by intensified obstruction symptoms [22] . in our group, the children with infections caused by mixed pathogens did not differ from the children with infections caused by single pathogens, in terms of both the severity of disease course and intensity of inflammatory reaction. the high percentage of positive results of examinations detecting viral genetic material points to the significance of viruses in the occurrence of pneumonia in children. the literature indicates that viruses may be responsible for the majority of pneumonia cases in children [8, 16] . in our group, in the case of hospitalized children, some patients received outpatient antibiotic therapy, which was then continued during inpatient care in all cases. therefore, all samples were collected during antibiotic therapy, on the fifth day on average. hence, it is not possible to completely exclude the presence of bacterial factors, which were not identified in the upper respiratory tract later on due to the ongoing antibiotic therapy. this may explain the relatively low percentage of bacterial genetic material detected in the group we analyzed. the current guidelines recommend the use of antibiotic therapy in children hospitalized due to pneumonia, but the lack of clinical improvement despite the application of broadspectrum antibiotic therapy may suggest a viral cause of infection [4] [5] [6] . the studied group was unique due to the high participation of patients with comorbidities, some of them in immunosuppression. such patients are at a higher risk of severe course of infection and prolonged hospitalization. the diagnosis of a viral etiology of infection enables moderate use of antibiotics and allows for more emphasis on symptomatic treatment, which is the most beneficial for such patients. because viruses are easily transmitted from patient to patient, advanced infection control methods are critical in controlling the spread of viruses [20] . rsv turned out to be the most frequently detected pathogen, especially in the group of children under the age of one. moreover, it was frequently the only identified pathogen. in children from risk groups, rsv causes infections with severe course, and it is characterized by high infectiousness. diagnosing rsv as the etiological factor enables the isolation of or cohorting the patients in order to reduce the spread of the infection and the implementation of the recommended management for severe obstruction [4, 5] . research is currently being conducted on the implementation of antiviral drugs which are more effective than the previously used ribavirine [23] [24] [25] . in the case of viral infections such as influenza, the establishment of the etiological factor enables the implementation of specific treatment. in the studied group, a single case of influenza was found, but the year in which the study was conducted was not an epidemic year. molecular tests for detecting the influenza virus only are widely applied. they are very useful during epidemic seasons, but it is important to remember that the disease may occur also outside such periods. in certain situations, it may be difficult to select a diagnostic test only on the basis of symptoms. moreover, in the case of influenza, the time of treatment implementation is crucial. therefore, including influenza in the standard diagnostic set prevents the omission of this important infection factor. the conducted analysis showed limited usefulness of traditional microbiological examinations in the diagnostic process of pneumonia. taking blood cultures is also considered standard in severe pneumonia, but its usefulness is not significant. in a group of patients with radiologic confirmed pneumonia, esposito revealed the presence of s. pneumoniae in 14.3% of cases; 91.8% of the diagnoses were based on positive results of real-time pcr tests conducted on material collected from the respiratory tract, while positive results of blood cultures were present only in 8.14% of cases [22] . nasopharyngeal swabs cultures often show flora growths that may constitute colonization. in our analysis, traditional microbiological examinations revealed the presence of potentially pathogenic bacteria in material collected from 31 children; in 11 of these cases there was agreement with the results of multiplex real-time pcr tests. in 8 patients it was not possible to assess the agreement of research methods due to the fact that in the ftd respiratory pathogens 33 test microorganisms such as pseudomonas aeruginosa, escherichia coli, or serratia sp. are not detected. the majority of previous works assessed the results of multiplex real-time pcr tests conducted in admission rooms on all patients reporting with respiratory symptoms [15] [16] [17] . because of the costs associated with these tests, it is difficult to use them in such a manner in daily practice. the mentioned analyses seem to be more epidemiological in nature. in our work we made an attempt to assess the usefulness of tests which were conducted during hospitalization in children with severe pneumonia not responding to standard therapy. in this group of patients, the benefits of the applied treatment were satisfactory in relation to the diagnostic cost incurred. the confirmation of viral etiology was very important, especially in the group of children at a risk of severe course of infection. in such a unique group of patients, constituted by children burdened with comorbidities, under immunosuppression, and after marrow stem cell transplants, the implementation of targeted treatment should be taken into consideration with the use of modern antiviral drugs, as well as such steps as isolation and the reduction of immunosuppression. in the guidelines of the neutropenia management it is strongly advised to isolate patients with documented respiratory viruses until symptoms resolve [20] . there are some limitations to our study. we have collected a heterogenic group of patients (different indication for testing, different comorbidities, and chronic diseases). the number of patients and the number of specimens were too small to perform elaborate statistical analysis. sample collection for microbiological cultures was incomplete. the exact timing of sample collection relative to antibiotic administration was not accurately documented. similar problems were described in other clinical studies in this subject topic. our analysis demonstrated that coinfection with viruses is common in severe lung infections in children with comorbidities. multiplex real-time pcr tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations widely used in the applicable diagnostic process to date. informed consent was obtained from parents/legal guardians on admission to hospital. this article does not contain any studies with human participants performed by any of the authors. this study is done based on analysis of patients' medical records; all tests were done as diagnostic tests during hospitalization. estimates of world-wide distribution of child deaths from acute respiratory infections acute respiratory infections in children procalcitonin, creactive protein and leukocyte count in children with lower respiratory tract infection british thoracic society guidelines for the management of community acquired pneumonia in children: update the management of community-acquired pneumonia in infants and children older than 3 months of age: clinical practice guidelines by the pediatric infectious diseases society and the infectious diseases society of america rekomendacje postępowania w pozaszpitalnych zakażeniach układu oddechowego viral pneumonia impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses clinical and socioeconomic impact of different types and subtypes of seasonal influenza viruses in children during influenza seasons development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay multiplex pcr and emerging technologies for the detection of respiratory pathogens performance of a novel microarray multiplex pcr for the detection of 23 respiratory pathogens (symp-ari study) clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial epidemiological investigation of nine respiratory pathogens in hospitalized children in germany using multiplex reversetranscriptase polymerase chain reaction the use of a multiplex real-time pcr assay for diagnosing acute respiratory viral infections in children attending an emergency unit etiology of community-acquired pneumonia in hospitalized children based on who clinical guidelines etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections epidemiology and virology of acute respiratory infections during the first year of life: a birth cohort study in vietnam multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children viral infections in immunocompromised patients frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction antibiotic therapy for pediatric community-acquired pneumonia: do we know when, what and for how long to treat? new options in the treatment of respiratory syncytial virus disease successful treatment of parainfluenza virus respiratory tract infection with das181 in 4 immunocompromised children chemotherapy of respiratory syncytial virus infections: the final breakthrough all authors declare that they have no conflict of interests. key: cord-280908-o1z4ka3r authors: vieira, sandra e.; thomazelli, luciano m.; de paulis, milena; ferronato, angela e.; oliveira, daniele b.; martinez, marina baquerizo; durigon, edison l. title: infections caused by hrsv a on1 are predominant among hospitalized infants with bronchiolitis in são paulo city date: 2017-05-24 journal: biomed res int doi: 10.1155/2017/3459785 sha: doc_id: 280908 cord_uid: o1z4ka3r human respiratory syncytial virus is the main cause of respiratory infections in infants. several hrsv genotypes have been described. goals. to describe the main genotypes that caused infections in são paulo (2013–2015) and to analyze their clinical/epidemiological features. methods. 94 infants (0–6 months) with bronchiolitis were studied. clinical/epidemiological information was collected; a search for 16 viruses in nasopharyngeal secretion (pcr-real-time and conventional, sequencing, and phylogenetic analyses) was performed. results. the mean age was 2.4 m; 48% were male. the mean length of hospital stay was 4.4 d (14% in the intensive care unit). the positive rate of respiratory virus was 98.9%; 73 cases (77.6%) were hrsv (76,7% hrsva). hrsva formed three clusters: on1 (n = 34), na1 (n = 1), and na2 (n = 4). all hrsvb were found to cluster in the ba genotype (ba9-n = 10; ba10-n = 3). clinical analyses showed no significant differences between the genotype aon1 and other genotypes. conclusion. this study showed a high rate of hrsv detection in bronchiolitis. hrsva on1, which has recently been described in other countries and has not been identified in previous studies in the southeast region of brazil, was predominant. the clinical characteristics of the infants that were infected with aon1 were similar to infants with infections by other genotypes. hrsv infections are frequent worldwide, and the most severe cases mainly affect children during the first year of life, elderly and immunocompromised individuals. hrsv is the most common causal agent of respiratory infections in infants, which occur at predictable annual seasons [1, 2] . hrsv is an enveloped virus (genus orthopneumovirus, family pneumoviridae) [3] . its genome consists of a nonsegmented single-stranded rna that encodes 11 proteins. the surface proteins f and g are important antigenic targets of neutralizing antibodies. variations in the g protein and in the gene regions that encode this protein allowed for the classification of hrsv into two subgroups (a and b) and into many genotypes. different hrsv genotypes of the two subgroups generally cocirculate during a season in the same region, and the predominant genotypes are replaced by others in subsequent years [4, 5] . such antigenic and genetic variability allows the virus to escape immunity acquired by the population of patients that have been subjected to previous infections. several genotypes have been described in subgroups a (ga1 to ga7, saa1 and na1, na2) and b (uru1, uru2, and gb1 to gb4, sab1 to sab3, and ba1 to ba13). the recently described genotype hrsv a on1 is characterized by the duplication of 72 nucleotides in gene g. after being reported in canada, hrsva on1 has also been identified in europe, india, africa, south america, and asia [6] [7] [8] [9] [10] [11] . knowledge on the molecular epidemiology of hrsv infections is important to assess the clinical implications of infections by different genotypes. the clinical presentation, severity, response to treatment, and prophylaxis are 2 biomed research international among these implications. the occurrence of genotypes that have not been previously identified leads to concerns about the severity of new cases or an increased number of infected individuals when considering the possible absence of immunological memory in the affected population. in this study, the authors describe the main genotypes of hrsv a and b, which caused infections in infants hospitalized in the university hospital of universidade de são paulo in são paulo city, from 2013 to 2015. the genotype characteristics and clinical and epidemiological features of hrsv are analyzed, and the infections caused by the new genotype hrsv aon1 are compared to other genotypes that were circulating during the study period. to our knowledge, this is the first report to perform an analysis of the association between clinical features and genotypes in infections caused by hrsv a on1 in the southeast region of brazil. this study included 124 infants aged between zero and six months with diagnoses of bronchiolitis that were admitted to the pediatric clinic division of the university hospital of the university of são paulo between 2013 and 2015. the infants were enrolled in the study by one of the authors (mp) who collected nasopharyngeal secretions after written consent was obtained from the child's parents. on examination, the clinical and socioeconomic backgrounds, clinical signs/symptoms, and diagnosis at admission were recorded on a standard form. diagnosis of bronchiolitis was defined as the first wheezing crisis, beginning no more than 3 days before hospital admission. for clinical analysis, infants diagnosed with or suspected of having bacterial or fungal infections and those who received antibiotics, macrolides, or antifungals prior to or during hospitalization were excluded. infants with codetection of respiratory viruses were also excluded from the clinical analysis. the project was approved by the research ethics committee of the university hospital of the university of são paulo (1011/10) and was funded by fundação de amparo a pesquisa do estado de são paulo (12/22854-9) . nasal wash fluid was obtained after washing the nostrils with 3 ml of a saline solution and collecting the suctioned specimen in a cup within a maximum of 24 hours after admission. hrsv-positive samples were amplified by traditional pcr in two steps for dna sequencing: cdna was synthesized using the super script iii kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. the second hypervariable region of the g protein gene pcr was carried out with the primers gr5_fwd (5 -ctggcaatgataatctcaacttc-3 ) and fv_rev (5 -gttatgacactggtataccaacc-3 ) in a 10 l mixture that contained 5 l of 10x pcr buffer, 25 mm of each dntp, 25 pmol of each primer, and 1.5 u of platinum taq dna polymerase (invitrogen, carlsbad, ca, usa) for a final volume of 50 l. the amplification was performed in a geneamp pcr system 9700 thermocycler (applied biosystems, foster city, ca, usa). a second step with seminested pcr was carried out using the f1ab_rev (5 -caactccattgttatttgcc-3 ) primer corresponding to bases 3-22 of the f gene. the traditional pcr assays were performed with the following program: 95 ∘ c for 5 minutes, followed by 35 cycles, each composed of 30 sec at 95 ∘ c, 30 sec at 55 ∘ c, and 45 sec at 72 ∘ c, and finally 7 minutes of extension at 72 ∘ c. the amplified products were analyzed by agarose gel electrophoresis and visualized under uv light after staining with ethidium bromide. the amplified products of gene g were ≈490 bp, were purified by exosap-it (affymetrix, inc., usa), and were submitted to a cycle sequencing reaction (sanger) using the bigdye terminator kit (applied biosystems, foster city, ca, usa) and gr5, fv or f1ab primers in a 3100 dna sequencer (applied biosystems, foster city, ca, usa). both strands of each amplicon were sequenced at least twice. sequence editing, alignments, and phylogenetic analyses were performed with megalign 5.03 v software (dnastar, inc., madison, wisconsin, usa). standard published sequences from subgroups a (accession numbers from ky828387 to ky828428) and b (accession numbers from ky828374 to ky828386) were downloaded from genbank as references of different lineages and genotypes. the results of the analyses of the clinical and demographic characteristics as well as the categorical variables are presented as absolute numbers and percentages, and the continuous variables are presented as the means and standard deviations. for studies of the associations between the categorical variables, the chi-square test, and fisher's exact test were used. for associations between the continuous variables, student's -test was used. the null hypothesis was rejected when the probability was less than 5% ( < 0.05). we used ibm spss statistic software, version 23. of the 124 infants selected, 94 were studied after the exclusion of 30 cases (infants with clinical diagnoses or suspicion of infection by bacteria or other agents as well as those who received antibiotics). the mean age of the infants was 2.4 months (sd = 1.6) and 48% were male. the mean length of hospital stay was 4.4 days (sd = 3.6) and 14% were admitted to the intensive care unit. figures 3 and 4 . the hrsv a isolates formed three clusters (na1, na2, and on1 genotypes); most of the hrsva isolates were found to cluster in the on1 genotype. the on1 cluster included 40 isolates, the na1 cluster included 2 isolates, and the na2 cluster included 4 isolates. they showed a 0.004-0.113 sequence p-distance at the nucleotide level, but there was a 0.000-0.175 p-distance at the amino acid level compared to the on1 prototype strain (jn257694). the sequenced hrsvb isolates were found to cluster in the ba genotype. the hrsv b isolates formed two subclusters, identified as the ba-9 and ba-10 genotypes. the ba-9 cluster included 10 isolates, and the ba-10 cluster included 3 isolates. they showed a 0.026-0.052 sequence p-distance at the nucleotide level, but there was a 0.045-0.084 p-distance at the amino acid level compared to the ba prototype strain (ay333362). the comparative clinical analyses included 32 infants with a hrsv single infection (22 aon1 and 10 other genotypes) and showed no significant differences between these subgroups (table 1 ). the present study showed the strong predominance of hrsv infections in infants hospitalized with bronchiolitis, predominance of the hrsva on1 genotype, and occurrence of the na1 and na2 genotypes, previously unidentified in southeast region of brazil. the high occurrence of hrsv among infants was expected due to the inclusion criteria that selected children the use of molecular methods for conducting viral research in the selected cases showed low occurrences of 6 other respiratory viruses and a high prevalence of respiratory virus codetections, most of which involved hrsv, which was the main viral agent in both single infections and in codetection. cocirculation of hrsv a and b was observed, with a predominance of hrsva (76.7%), as reported in most studies conducted in other countries [5, 7, 12] . previous studies performed by this group of researchers, in the same service, showed the cocirculation of different genotypes of hrsv a and b during the same viral season. an analysis from 1995 to 2006 showed the predominance of group b only in 1999 in the state of são paulo [13] . after genotype hrsv a on1, ba9 was the second most frequent genotype identified in the present study. the genotypes that predominated in previous seasons were not detected, such as gb1, gb3, ga2, and ga5. in a previous study, the authors included hrsv samples that were collected from hospitalized children in the state of são paulo until seven years before this study. they showed important nucleotide substitutions in the ga2 genotype that are genetically close to the na1 and na2 genotypes identified here between 2013 and 2015 [14] . since the first year of the study (2013), the genotype hrsva on1 was predominant, but the genotypes na1 and na2 and genotypes ba9 and ba10 also circulated. in subsequent years (2014 and 2015) , the genotype on1 remained predominant and a unique representative of hrsv a, but there was cocirculation with the ba9 and ba10 genotypes, with predominance of the ba9 genotype among hrsv b. figure 3 : genotype tree: the topology of the hrsv a tree shows the study samples identified (peg) compared to the specimens from genebank, identified by their access number. clades in red show samples that belong to genotype on1. clades in blue show samples that belong to genotype na2, and clades in green show samples that belong to genotype na1. in molecular analysis, most of the sequenced hrsva isolates were found to cluster in the on1 genotype with prototype reference strain jn257694, which was first reported in ontario, canada [6] . the sequenced isolates had the signature 72-bp duplication in the g protein when aligned with representative sequences from all of the a subgroup (ga1 to ga7, na1, na2, and on1) from genbank. the brazilian hrsv a isolates were most closely related to isolates from the united states, kenya, and new zealand. all of the sequenced hrsvb isolates were found to cluster in the ba genotype, with prototype ba reference strain ay333362, which was first reported in buenos aires, argentina [15] . the sequenced isolates had the signature 60bp duplication in the g protein when aligned with representative sequences from all of the b subgroup (gb1 to gb4, sab1 to sab4, uru1 and uru2, and ba-1 to ba-13) from genbank. the brazilian hrsv b isolates were most closely related to isolates from the united states, new zealand, and vietnam. some authors suggest the occurrence of a greater number of cases during seasons in which new genotypes predominate. this could be a result of the absence of immunity acquired by the population against the new genotypes [4, 15] . genotype a on1 shows a duplication of 72 nucleotides in the cterminal third of the g gene. the region of the g protein encoded by this gene sequence is targeted by specific genotype neutralizing antibodies, which may contribute to the escape of the virus from the population immunity induced by previous contact with other genotypes. however, according to the national registry of hospitalization cases, there was no increase in the frequency of hospitalizations of infants diagnosed with bronchiolitis between 2013 and 2015 in brazil and the state of são paulo compared to the previous five years [16] . it is possible that, acting as a controller, the immunity of the population only contributes to the selection of genotypes that replace each other without necessarily increasing the number of cases when genotypes without recent incidences appear. the demographic and clinical characteristics of the infants that were infected with genotype aon1 were similar to the characteristics of the other genotypes. exclusion of infants with viral codetection and those who received antimicrobials during hospitalization made it possible to avoid confounding factors in the comparative analysis of the severity between the genotypes. a mean age at 2 months showed the precocity of the infection, which was independent of the infecting genotype. clinical characteristics that could differentiate the initial presentation, such as the presence of cough, fever, dyspnea, and apnea crises, also showed similar prevalence among the genotypes. some prognostic factors that are relevant in respiratory infections in infants were also similar, such as exposure to tobacco smoke and breastfeeding. severity, as analyzed by the hospitalization time, need and duration of oxygen therapy and mechanical ventilation, and hospitalization in the intensive care unit, was also similar regardless of the infecting genotype. despite the limited number of cases, the results suggest that the hrsv a on1 genotype was not associated with specific clinical characteristics and was therefore clinically indistinguishable from the other genotypes. these results need to be confirmed by more extensive analyses but are consistent with a previous german study that found no clinical differences between infections by other hrsv genotypes [17] . although nucleotide variation in this region is important for viral antigenicity, other regions may be more relevant determinants of infection severity. in addition, other factors inherent to the host and the environment must be considered [18] . on the other hand, an epidemiological study carried out in vietnam compared community-acquired infections and nosocomially acquired infections and showed a greater severity of the respiratory condition in children infected with hrsv on1 compared to those infected with na1 with consideration of the clinical severity and occurrence of pneumonia. in that study, all children used antibiotics, which may have created a bias since nonexcluded bacterial coinfections could impact clinical evolution. additionally, the age group was a differential factor since children up to 5 years of age were included, including infants with bronchiolitis and also cases of posterior hrsv infections [19] . other authors found fewer signs of severity in children infected by hrsv a on1 compared to other hrsv genotypes, such as na1 [7] , ga2, and ba [20] . another important aspect is the need for clinical followup studies that can assess the possible impacts of infections by different genotypes on the development of recurrent wheezing and asthma in the years following infections. the present study showed a high rate of hrsv detection in infants hospitalized with bronchiolitis. five genotypes were found, with a predominance of genotype a on1, which was recently described in other countries and not identified in previous studies in the southeast region of brazil. the clinical and epidemiological characteristics of infants that were infected with hrsv a on1 were similar to infants with infections by other genotypes identified in the study. respiratory syncytial virus-associated hospitalizations among children less than 24 months of age respiratory syncytial virus infection in elderly and high-risk adults circulation patterns of genetically distinct group a and b strains of human respiratory syncytial virus in a community lower respiratory tract infection caused by respiratory syncytial virus biomed research international 7 in infants: the role played by specific antibodies genetic variability of human respiratory syncytial virus a strains circulating in ontario: a novel genotype with a 72 nucleotide g gene duplication characteristics and their clinical relevance of respiratory syncytial virus types and genotypes circulating in northern italy in five consecutive winter seasons genetic variability of human respiratory syncytial virus in pune, western india epidemiology and molecular characterization of human respiratory syncytial virus in senegal after four consecutive years of surveillance sixteen years of evolution of human respiratory syncytial virus subgroup a in alternate circulation and genetic variation of human respiratory syncytial virus genotypes in influence of respiratory syncytial virus strain differences on pathogenesis and immunity phylodynamics and dispersal of hrsv entails its permanence in the general population in between yearly outbreaks in children positive selection results in frequent reversible amino acid replacements in the g protein gene of human respiratory syncytial virus major changes in the g protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides novel respiratory syncytial virus (rsv) genotype on1 predominates in germany during winter season 2012-2013 immunity to rsv in early-life association of rsv-a on1 genotype with increased pediatric acute lower respiratory tract infection in vietnam epidemiology of respiratory syncytial virus in children in cyprus during three consecutive winter seasons (2010-2013): age distribution, seasonality and association between prevalent genotypes and disease severity the authors declare no conflicts of interest. key: cord-296033-5zyoddl7 authors: hu, xiaoliang; tian, jin; kang, hongtao; guo, dongchun; liu, jiasen; liu, dafei; jiang, qian; li, zhijie; qu, juanjuan; qu, liandong title: transmissible gastroenteritis virus papain-like protease 1 antagonizes production of interferon-β through its deubiquitinase activity date: 2017-10-23 journal: biomed res int doi: 10.1155/2017/7089091 sha: doc_id: 296033 cord_uid: 5zyoddl7 coronaviruses (covs), such as human coronavirus nl63 (hcov-nl63), severe acute respiratory syndrome cov (sars-cov), murine hepatitis virus (mhv), porcine epidemic diarrhea virus (pedv), and middle east respiratory syndrome coronavirus (mers-cov), encode papain-like (pl) proteases that inhibit sendai virus(sev-) induced interferon (ifn-β) production. recently, the crystal structure of transmissible gastroenteritis virus (tgev) pl1 has been solved, which was similar to that of sars-cov pl2(pro), which may antagonize host innate immunity. however, very little is known about whether tgev pl1 can antagonize host innate immune response. here, we presented evidence that tgev pl1 encoded by the replicase gene could suppress the ifn-β expression and inhibit the nuclear translocation of interferon regulatory factor 3 (irf3). the ability to antagonize ifn-β production was dependent on the intact catalytic activity of pl1. furthermore, tgev pl1 exerted deubiquitinase (dub) activity which strongly inhibited the retinoic acid-induced gene i(rig-1-) and stimulator of interferon gene(sting-) dependent ifn expression. our data collectively suggest that tgev pl1 can inhibit the ifn-β expression and interfere with rig-1and sting-mediated signaling through a viral dub activity. our study has yielded strong evidence for the tgev pl1 mechanisms that counteract the host innate immunity. the innate immune system is the first line of defense that protects the host against viral infection, and the induction of ifn-/ is a crucial antiviral mechanism of the innate immune system [1] . the initiation of ifn expression is triggered by pathogen-associated molecular patterns (pamps) through host pattern recognition receptors (prrs) [2] . after viral rnas are sensed by prrs, signals are transmitted to different downstream adaptor molecules (such as ifnpromoter stimulator 1 (ips-1)); and then i b kinase-(ikk-) related kinases are recruited. next, interferon regulatory factor 3 (irf3), nuclear factor b (nf-b), and atf-2/c-jun are activated by the kinase complexes and translocate to the nucleus and directly induce the expression of type i ifns [3] . tgev is an enveloped virus belonging to the coronaviridae (cov) family and the nidovirales order. covs are positive-strand rna viruses that replicate in the cytoplasm of infected cells [4] . covs encode two types of cysteine proteases, m pro , and papain-like proteases, pl1 and pl2, which contained nonstructural protein 5 (nsp5) and nsp3, respectively. pl pro is served mainly as in processing of the replicase pp1a and pp1ab polypeptides [5] . other than their role in replicase polyprotein processing, pl2 domains possess an additional but related enzymatic activity, in hcov-nl63 [6] , mhv [7] , sars-cov [8, 9] , and mers-cov [10] , through their deubiquitination (dub) enzymes, which play a key role in antagonizing ifn induction. however, tgev pl1 processes the nsp2/nsp3 site and is capable of hydrolyzing isopeptide bonds in both lys48-and 2 biomed research international lys63-linked polyubiquitin chains [11] . whether tgev pl1 could antagonize the production of ifns was unknown. in the present study, we found that tgev pl1 encoded by the replicase gene could suppress the ifn-expression and inhibit the nuclear translocation of interferon regulatory factor 3 (irf3) and exerted deubiquitinase (dub) activity which strongly inhibited the retinoic acid-induced gene i-(rig-1-) and stimulator of interferon gene-(sting-) dependent ifn expression. cells and viruses. hek293t cells and pk-15 cells were cultured in dulbecco's modified eagle's medium (hyclone, logan, usa) containing 10% (v/v) fetal calf serum supplemented with penicillin (100 u ml −1 ) and streptomycin (100 g ml −1 ). sendai virus (sev) was obtained from the centre of virus resource and information (wuhan institute of virology, chinese academy of sciences). plasmids and agents. ifn--luc, 4x prdiii/i-luc (referred to as irf3-luc), 4x ap-1-luc, and 4x nf-b-luc luciferase reporter plasmids were constructed according to an earlier protocol [12] . accession numbers of sting, irf3, and mavs were kc860780, kc860781, and kc860782, respectively. expression plasmids for rig-1 (p-flag-rig-1) and tbk-1 (p-flag-tbk-1) were generated with the following primers: rig-1 forward, 5 -tttggatccatgacagca-gagcagcggcggaat-3 , rig-1 reverse 5 -tttaag-cttcactcaaggttcgggattccctg-3 ; tbk-1 forward, 5 -tttgaattcatgcagagcacttctaatcat-cttt-3 , tbk-1 reverse 5 -tttagatcttaaagacag-tcaacattgcgaa-3 . to construct the dna expression vector, pmyc-pl1, pflag-pl1, and ppl1-myc, encoding tgev pl1, standard reverse transcription-(rt-) pcr was applied to amplify cdna of the total rna extracted from pk-15 cells infected with the tgev strain hx, using the following primers: pl1-forward, 5 -gtacaagaa-gctgaacaatttaa-3 (3498-3520 bp), pl1 reverse, 5 -atcgtttttaggactttgaattt-3 (4249-4271 bp). all constructs were validated via dna sequencing. pdsred2-mito was purchased from clontech (tokyo, japan). transfection agent was performed with x-tremegene hp (roche, switzerland) per the manufacturer's instructions. luciferase reporter gene assay. hek-293t cells grown in 24-well plates were cotransfected with 0.2 g/well reporter plasmid, 0.02 g/well prl-tk plasmid (promega, madison, usa) as an internal control for normalization of transfection efficiency, and the indicated expression or empty control vector plasmid. where indicated, cells were also mockinfected or infected with sev (100 hemagglutinating activity units/well) at 10 h after cotransfection. cells were subsequently lysed, and firefly and renilla luciferase activities were determined with the dual-luciferase reporter assay system (promega, madison, usa), according to the manufacturer's protocol. data are presented as mean relative luciferase units ± standard deviation from triplicate samples. for statistical analysis, data were compared between empty vector-and tgev pl1-transfected groups with the unpaired, two-tailed student's t-test using spss 11.0 software. values < 0.05 were considered statistically significant [13] . elisa. cell supernatants of transfected pk-15 cells were centrifuged at 3,000 for 5 min to remove cell debris and stored at −80 ∘ c until use. secreted ifn-in the cell supernatants was determined using commercial porcine ifn-(interferon beta) elisa kit (elabscience, china) according to the manufacturer's instructions. immunoblot analysis. hek293t cells were cultured in 6well plates and 60 mm dishes were transfected with the appropriate plasmids. after 36 h, cells were harvested by the addition of lysis buffer and protein concentrations in whole cell extracts measured. equal amounts of samples were subjected to sds-page and analyzed for tgev pl1, sting, tbk-1, and irf3 proteins via immunoblotting using ha, flag, or gfp-tagged antibodies (sigma, st louis, usa). expression of p-irf3, irf3, and gapdh was detected with the rabbit-anti p-irf3 (ab76493), irf3 (ab68481) (abcam, cambridge, uk), and a mouse anti-gapdh monoclonal antibody (sigma, st louis, usa). assay of deubiquitinase activity in cultured cells. hek293t cells were cotransfected with pcdna3.1-ha-ub plus the indicated amounts of tgev pl1, p-flag-rig-1, and p-flag-sting constructs. the effect of tgev pl1 on ubiquitinated proteins in cultured cells was assessed by immunoblot analysis. coimmunoprecipitation analysis. coimmunoprecipitation experiments were performed on hek293t cells transfected with the indicated expression plasmids as described in an earlier report [14] . immunofluorescence assay. hek293t cells were plated on fibronectin-treated glass coverslips in 24-well plates. to evaluate the localization of tgev pl1, cells were cotransfected with plasmid dna expressing flag-pl (500 ng per well) and pdsred-mito (500 ng per well) using x-treme gene hp, according to the manufacturer's protocol. hek293t cells were cotransfected with irf3-gfp (500 ng per well) and empty vector (500 ng per well) or irf3-gfp (500 ng per well) and flag-pl1 (500 ng per well). 24 h after transfection, cells were infected with sev (100 hemagglutinating activity units/well) for 16 h. next, cells were fixed with 4% paraformaldehyde for 30 min and permeated with 0.1% triton x-100 for 15 min at room temperature. after three washes with pbs, cells were blocked with pbs containing 5% bovine serum albumin for 2 h, followed by incubation with a mouse monoclonal antibody against flag (1 : 100) for 1 h at room temperature. cells were treated with fluorescein isothiocyanate-labeled goat anti-mouse (sigma, st louis, usa) for 1 h, and subsequently with 4 ,6-diamidino-2-phenylindole (dapi) for 15 min at room temperature. samples were washed with pbs, and fluorescent images were acquired under a confocal laser scanning microscope (tcs sp5; leica, solms, germany). to assess the formation of sting dimers, hek293t cells were transfected with flag-sting (500 ng per well) and the lysates were subjected to western blot, as described earlier [15] , with the indicated antibodies. is an ifn antagonist. the crystal structure of tgev pl1 has been determined [11] . the structure of tgev pl1 is similar to that of sars-cov pl2 pro . in order to determine whether tgev pl1 is capable of blocking ifn-production, we assessed ifn-promoter activity in the presence of pl1 (figure 1(a) ). hek 293t cells were cotransfected with tgev pl1 and ifn-luciferase or renilla luciferase reporter plasmids for 24 h and subsequently infected with sev to activate the rig-1-dependent ifn-expression pathway. we observed the inhibition of sev-induced ifn-promoter activation in the presence of pl1, similar to the antagonistic function of nl63 plp2 and porcine epidemic diarrhea virus (pedv) plp2, clearly indicating that tgev pl1 could act as an interferon antagonist. to establish the mechanisms by which pl1 inhibits ifnexpression, transcriptional activities of nf-b, irf3, and ap-1 were analyzed using the luciferase assay to identify the precise transcription factor involved. notably, the luciferase activities of all three transcription factors were significantly inhibited by tgev pl1 in a dose-dependent manner ( figures 1(b) , 1(c), and 1(d)). furthermore, flag-pl1 also significantly inhibited ifn-production in pk-15 cells at protein level ( figure 1(e) ), which was further confirmed with the result that tgev pl1 could block the production of interferon. to further establish whether tgev pl1 affects irf3 phosphorylation or migration from the cytoplasm to nucleus, hek293t cells were transfected with tgev pl1 and/or irf3-egfp. then the result was analyzed using western blot and confocal microscopy. in figure 1(f) , the level of p-irf3 was decreased significantly by tgev pl1 compared with that of sev-induced. furthermore, irf3-egfp was located in the cytoplasm compared with mock-infected hek293t cells but translocated to the nucleus when the cells were inoculated with sev. in contrast, after being inoculated with sev, it was found that irf3-egfp was translocated from cytoplasm to nuclear in mock infected hek293t cell, which was not observed in cells transfected with tgev pl1 (figure 1(g) ). our results collectively suggested that tgev pl1 suppressed ifn-transcription by interfering with nf-b-, irf3-, and ap-1 signaling-mediated ifn expression. to determine whether tgev pl1 is capable of blocking stingmediated activation of the ifn-promoter, we assessed promoter activity in the presence of sting along with increasing amounts of tgev pl1. stimulation of hek293t cells with sting alone resulted in a robust increase in ifnpromoter activity. coexpression of sting and tgev pl1 induced a dose-dependent decrease in ifn-activity, clearly indicating antagonistic activity of tgev pl1 on stingmediated activation of the ifn-promoter (figure 2(a) ). sting dimerization is reduced in the presence of hcov-nl63. sting dimmers are visualized as an 80 kd band on sds-page. to further determine whether tgev pl1 inhibits sting-mediated signaling through disrupting the stability of sting dimers, hek293t cells were cotransfected with plasmid dna expressing sting in the presence or absence of tgev pl1 and sev, and cell lysates were evaluated for dimmers via immunoblotting (figure 2(b) ). interestingly, the results indicated that sting dimerization was not affected by tgev pl1. inhibiting ifn-expression. to determine whether catalytic activity is required for tgev pl1-mediated inhibition of ifn-expression, hek293t cells were cotransfected with alanine mutants of three conserved catalytic residues of tgev pl1 (c32a, h183a, and d196a) with or without rig-1, mavs, sting, or tbk-1, and ifn--luc and prl-tk plasmids, followed by infection with sev to activate ifnpromoter activity. tgev pl1 mutation at two of the catalytic sites (c32a and h183a) led to almost complete loss of ifn antagonistic activity, relative to wild-type tgev pl1, but the d196a mutant showed a little inhibition for ifn-promoter activity (figures 3(a), 3(b) , 3(c), 3(d), and 3(e)). based on the results, we conclude that the intact catalytic triad of tgev pl1 is required to inhibit activation of the ifn-promoter driven by sting and tbk-1. recent studies have revealed that sting acts as a scaffold protein for tbk-1 and irf3 and links them to the mavs complex in mitochondria upon viral infection [16] . moreover, activation of sting is critical for stimulation of irf-3 activity. here, we observed that tgev pl1 protein inhibits sting-and tbk-1-induced activation of ifn-. additional localization experiments showed that pl1 existed in mitochondria (figure 3(f) ). modification of signaling molecules by ubiquitin (ub) plays a critical role in activation of the ifn response. tgev pl1 has been shown to possess dub activity. here, we investigated the dub activities of tgev pl1 and its catalytic mutants. hek293t cells were cotransfected with pcdna ha-ub and tgev pl1, and the level of ubiquitinated proteins was assessed via western blot. the level of ub-conjugated proteins was reduced dramatically in cells transfected with wild-type tgev pl1, while the ubiquitinated ub-ha level was not reduced in the presence of the c32a, h183a, and d196a mutants (figure 4(a) ). next, we investigated whether tgev pl1 recognizes and deubiquitinates the key regulators, rig-i and sting, in the ifn signaling pathway. tgev is known to induce robust expression of ifn-at the late step of the replication and is distinct from covs [17, 18] . moreover, tgev infection activates transcription factors nf-b, irf3, and ap-1 in porcine kidney cells and a delayed activation of the ifn response in intestinal epithelial cells [19, 20] . however, the mechanism of its evasion of the innate immune system has never been reported. the current study firstly showed antagonistic function of the tgev pl1 protein against the irf3 signaling pathway to inhibit ifninduction through its dub activity. to combat the host antiviral effects, coronaviruses likely take advantage of pl activity to escape from the host innate antiviral response. hcov-nl63 (pl2-tm) and sars-cov (plpro-tm) inhibit sting-mediated activation of irf-3 nuclear translocation and induction of irf-3-dependent promoters [6, 8] . pl2 of mhv strongly inhibits cardif-, tbk1-, and irf3-mediated ifn-reporter activities and prevented nuclear translocation of irf3 [7] . pedv plp2 negatively regulated rig-i and sting-mediated ifn-expression [14] . moreover, tgev pl1 displays a similar structure to sars-cov pl2 [11] and gives rise to the speculation that tgev pl1 may similarly act as an ifn antagonist. in the present study, we first found that overexpressed tgev pl1 inhibited sting-and tbk-1-mediated ifntranscription and antagonized the type i ifn response stimulated by sev in pk-15 cells. the catalytic activity of tgev pl1 is essential for inhibiting ifn-transcription. furthermore, sting dimerization is reduced in the presence of hcov-nl63 pl2-tm, which was not affected by tgev pl1. these results suggested that tgev pl1 acted as an ifn antagonist to negatively regulate host antiviral innate immunity. ubiquitination and deubiquitination are critically involved in regulation of virus-induced type i ifn signaling pathways [21, 22] . recently, dubs have been reported in a variety of viruses, such as foot-and-mouth disease virus, lpro [23] , human cytomegalovirus, ul48 [24] , herpes simplex virus type 1, ul36 [25] , and porcine reproductive and respiratory syndrome virus, nsp2 [26, 27] . interestingly, all covs have evolved to encode dub enzymes, which may contribute to modulation of the innate immune response. plp of hcov-nl63, sars-cov, mhv, pedv, and mers-cov dramatically reduced the levels of ubiquitinated sting, rig-i, tbk1, and irf-3 to negatively regulate host antiviral innate immunity. here, we showed that tgev pl1 interferes with and significantly inhibits ubiquitination of rig-1 and sting, which are essential activators of type i ifn signaling. then, the levels of phosphorylated irf-3 were reduced, which blocked nuclear translocation of irf3 to activate the transcript of ifns. three catalytically inactive mutants of tgev pl1 (c32a, h183a, and d196a) found to be defective in dub activity failed to inhibit virus-induced inf-expression, indicating that the dub function of tgev pl1 is directly involved in inhibition of type i ifn induction. however, the membrane protein m and envelope protein e of tgev were translated at the late step of the replication as the major inducing component of ifns. further studies are required to establish the precise functions of pl1 protease/dub activity in coronavirus interactions with the host innate immune response. our results are the first report identifying tgev pl1 that is responsible for inhibiting the induction of ifn-. we found that tgev pl1 displayed ifn antagonist activity dependent on the intact catalytic triad (c32, h183, and d196) and interfered with rig-1-and sting-mediated signaling through a viral dub activity. these characteristics of tgev pl1 served as a multifunctional protein with a critical regulatory role in tgev interactions with the host antiviral innate immune response. moreover, these findings contribute to our understanding of the molecular mechanisms of innate immunity evasion strategies utilized by tgev. antiviral signaling through pattern recognition receptors rna recognition and signal transduction by rig-i-like receptors rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity development of protection against coronavirus induced diseases: a review virus-encoded proteinases and proteolytic processing in the nidovirales deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity mers-cov papain-like protease has deis-gylating and deubiquitinating activities papainlike protease 1 from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates molecular cloning and functional characterization of porcine ifn-promoter stimulator 1 (ips-1) replicates and repeatswhat is the difference and is it significant? a brief discussion of statistics and experimental design the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase eris, an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation interferon-response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes transmissible gastroenteritis virus infection induces nf-b activation through rlr-mediated signaling transmissible gastroenteritis virus does not suppress ifn-induction but is sensitive to ifn in ipec-j2 cells ubiquitylation in innate and adaptive immunity ubiquitination, ubiquitinlike modifiers, and deubiquitination in viral infection the leader proteinase of foot-andmouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase cleavage specificity of the ul48 deubiquitinating protease activity of human cytomegalovirus and the growth of an activesite mutant virus in cultured cells a deubiquitinating enzyme encoded by hsv-1 belongs to a family of cysteine proteases that is conserved across the family herpesviridae 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 the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions this work was supported by the national key technology support program (2013bak11b01-31), the state of international science and technology cooperation projects (2010dfb33620), and the national natural science foundation of china (3140220). interferon regulatory factor 3 dub:deubiquitinase rig-1:retinoic acid-induced gene i sting:stimulator of interferon gene prr:pattern recognition receptors pamp:pathogen-associated molecular patterns mda5:melanoma differentiation gene 5 ips-1:ifn-promoter stimulator 1 ikk: i b kinase tbk1:tank binding kinase 1 nf-b:nuclear factor b nsp5:nonstructural protein 5. the authors declare that they have no conflicts of interest regarding the publication of this paper. key: cord-274056-9t3kneoo authors: abd elwahaab, marwa a.; abo-elkhier, mervat m.; abo el maaty, moheb i. title: a statistical similarity/dissimilarity analysis of protein sequences based on a novel group representative vector date: 2019-05-08 journal: biomed res int doi: 10.1155/2019/8702968 sha: doc_id: 274056 cord_uid: 9t3kneoo similarity/dissimilarity analysis is a key way of understanding the biology of an organism by knowing the origin of the new genes/sequences. sequence data are grouped in terms of biological relationships. the number of sequences related to any group is susceptible to be increased every day. all the present alignment-free methods approve the utility of their approaches by producing a similarity/dissimilarity matrix. although this matrix is clear, it measures the degree of similarity among sequences individually. in our work, a representative of each of three groups of protein sequences is introduced. a similarity/dissimilarity vector is evaluated instead of the ordinary similarity/dissimilarity matrix based on the group representative. the approach is applied on three selected groups of protein sequences: beta globin, nadh dehydrogenase subunit 5 (nd5), and spike protein sequences. a cross-grouping comparison is produced to ensure the singularity of each group. a qualitative comparison between our approach, previous articles, and the phylogenetic tree of these protein sequences proved the utility of our approach. sequence comparison is used to study structural and functional conservation and evolutionary relations among the sequences. the importance of similarity/dissimilarity of biological sequences returns to its relationship with the structures and functions. proteins with similar sequences usually have similar structures. the rate of addition of new sequences to the databases is increasing exponentially [1] . comparing these new sequences to those with known functions is a key way of understanding the biology of an organism. thus, sequence analysis can be used to assign function to genes and proteins by the study of the similarities between the compared sequences. there are many tools and techniques that provide the sequence comparisons. sequence comparison can be classified into alignmentbased methods and alignment-free methods [2, 3] . alignment-based methods assign scores to different possible alignments, picking the alignment with the highest score. some algorithms do global alignment or local alignment [4] [5] [6] . blast [7] and fasta [8] are the most widely used applications. alignment-based methods are computationally difficult with multiple sequence alignments at the same time. a wide range of scoring systems has been proposed such as amino acid substitution scoring matrices pam and blosum for protein alignment [9] . alignment-free approaches overcome the limitations of alignment-based methods. graphical representation approaches are one of them. graphical representations are usually accompanied by numerical characterization and then a descriptor to describe each protein sequence. a similarity/dissimilarity analysis is then done using these descriptors by evaluating euclidean distance or correlation angle among them. the smallest euclidean distance or correlation angle is the more similar. many graphical representations of dna and protein primary sequences have been proposed. some other approaches characterize numerically protein sequences without previous graphical representation and nongraphical representation methods [10, 11] . in this article, an alignment-free method is introduced. it is considered a nongraphical representation method. three groups of protein sequences are selected to illustrate our approach. they are beta globin, nadh dehydrogenase subunit 5 (nd5), and spike protein sequences. they are selected as each group has sequences of similar range of lengths. the 1 human aaa16334 147 2 chimpanzee caa26204 125 3 gorilla caa43421 121 4 mouse caa24101 147 5 rat caa29887 147 6 gallus caa23700 147 7 opossum aaa30976 147 opossum np 007105 602 most common sequences of each group are selected. the selected sample is assumed to be unbiased and the population distribution of each group is normal. therefore, the selected sample represents the group. statistics can be used to estimate the population's parameters. the adjacency vector is introduced as a novel descriptor for protein sequences. it is computed for each sequence in the selected sample of three groups. a reference vector is then computed for each group. this vector acts as a representative of the group. each sequence's degree of similarity in each group is measured according to its group's representative vector. so, a similarity/dissimilarity vector is constructed instead of ordinary similarity/dissimilarity matrix. our approach is independent of the protein sequence length. it does not require any previous graphical representation. it is a mathematically simple approach. the protein sequences used in this article are listed in tables 1, 2 , and 3. the sequences are downloaded from the national center for biotechnology information (ncbi) "https://www.ncbi.nlm.nih.gov/" as fasta files. these fasta files are imported into wolfram mathematica 8 where all the results and figures are produced. the phylogenetic tree of these protein sequences is also created by the basic local alignment search tool (blast) "https://blast.ncbi.nlm.nih .gov/blast.cgi". table 1 shows the 1 st sample set that consists of seven species of beta globin protein sequences. their range of lengths is from 121 to 147. this sample set is applied before in [12] . table 2 shows the 2 nd sample set which consists of nine nd5 protein sequences. their range of lengths is from 602 to 610. this sample set is applied before in [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . table 3 shows the 3 rd sample set which consists of 29 spike protein sequences. their range of lengths is from 1162 to 1447. these viruses are coronavirus. they are classified into four classes: class i that includes the porcine epidemic diarrhea virus (pedv) and the transmissible gastroenteritis virus (tgev). class ii includes the bovine coronavirus (bcov), human coronavirus oc43 (hcov-oc43), and the murine hepatitis virus (mhv). class iii contains the infectious bronchitis virus (ibv). the others are severe acute respiratory syndrome coronaviruses (sars-cov). this sample set is applied before in [26] . in this approach, a new vector is suggested to be a descriptor of a protein sequence. this vector is called the adjacency vector ( ); x refers to the species' protein sequence and y refers to its related group. it counts the occurrence of all possible pairwise adjacencies obtained by reading the protein primary sequence from left to right. the protein sequence table 4 aa ar an ad ac aq ae ag ah ai al ak am af ap as at aw ay av 1 0 1 0 0 0 0 1 4 0 3 0 0 1 0 0 1 0 1 2 table 5 va table 6 aa ar table 7 va is composed of 20 common different amino acids which are "a," "r," "n," "d," "c," "q," "e," "g," "h," "i," "l," "k," "m," "f," "p," "s," "t," "w," "y," and "v" as ordered alphabetically according to 1 st letter code. therefore, the adjacency vector (a xy ) consists of 400 elements. every 20 elements are related to each amino acid. the first 20 elements are related to "a" amino acid. the second 20 elements are related to "r" amino acid. the third 20 elements are related to "n" amino acid and so on by the same order which is illustrated previously according to 1 st letter code. we borrow our idea from the 20 ×20 adjacency matrix [27] . the adjacency vector counts the possibilities of each pair. in other words, it counts the number of times that each pair is repeated along the sequence length. if the pair does not exist, its value in the adjacency vector is zero. for example, to evaluate the adjacency vector of the two short segments of "yeast saccharomyces cerevisiae" protein [16, 19, [22] [23] [24] 28] protein i: "wtfesrndpakdpvilwlnggpgcssltgl" protein ii: "wffesrndpandpiilwlnggpgcssftgl" the two protein sequences are composed of 30 amino acids. protein i is converted to 29 adjacent pairs that are wt, tf, fe, es, sr, rn, nd, dp, pa, ak, kd, dp, pv, vi, il, lw, wl, ln, ng, gg, gp, pg, gc, cs, ss, sl, lt, tg, gl as reading sequence from left to right. protein ii is converted to 29 adjacent pairs that are wf, ff, fe, es, sr, rn, nd, dp, pa, an, nd, dp, pi, ii, il, lw, wl, ln, ng, gg, gp, pg, gc, cs, ss, sf, ft, tg, gl as reading sequence from left to right. for example, "nd" pair has a count one in protein i and two in protein ii. "dp" pair has a count two in both protein i and protein ii. "sl" and "lt" pairs have a count one in protein i and zero in protein ii. our approach is applied on three selected groups of protein sequences. the groups are beta globin, nd5, and spike protein sequences as illustrated in tables 1, 2 , and 3, respectively. the most common protein sequences are selected in each group. the selected sample is assumed to be unbiased and the population distribution of each group is normal. therefore, the selected three samples can represent the three groups. the samples consist of seven beta globin, nine nd5, and 29 spike protein sequences. seven adjacency vectors for beta globin proteins, nine adjacency vectors for nd5 protein sequences, and 29 adjacency vectors for spike proteins are evaluated. for example: (1) human (beta globin) protein sequence's first 20 elements of its adjacency vector (a human beta globin ) are as shown in table 4 . (2) gorilla (nd5) protein sequence's last 20 elements of its adjacency vector (a gorilla nd5 ) are as shown in table 5 . the adjacency vector is used to describe each protein sequence individually in its corresponding group. this article provides a descriptor to the group itself. the median vector is selected to play the role of the group representative (gr y ); y refers to its group. it acts as a reference vector for each group. the median is a better measure of central tendency. it separates the higher half from the lower half of the sample's data. it is not sensitive to extreme values like average. the suggested group representative vector (gr y ) is a vector which is composed of also 400 elements. each element of 400 is the median of the corresponding elements in all adjacency vectors related to its sample that represents the group. beta globin, nd5, and spike protein sequences' representative vectors are computed. for example: (1) beta globin representative vector's (gr beta globin ) 1 st 20 elements are as shown in table 6 . (2) nd5 representative vector's (gr nd5 ) last 20 elements are as shown in table 7 . (3) spike proteins representative vector's (gr spike proteins ) 1 st 20 elements are as shown in table 8 . biomed research international table 8 aa ar an ad ac aq ae ag ah ai al ak am af ap as at aw ay av 9 1 3 5 2 4 3 6 0 7 6 2 1 3 6 4 7 1 5 3 a similarity/dissimilarity vector is introduced instead of the regular similarity/dissimilarity matrix [10, 11] . the similarity/dissimilarity matrix is a square symmetric matrix with zeros in its main diagonal. in order to evaluate this matrix, it is required to measure the degree of similarity between each protein sequence and others in the same group. if the 1 st row represents human and the 2 nd row represents gorilla, the similarity of all species according to human in 1 st row is measured. then the similarity is measured again of all species in 2 nd row according to gorilla and so on. the calculations' number of this matrix equals ∑ 1 = (k − 1)/2 where n is the number of compared species. the similarity/dissimilarity vector is suggested to save time and number of calculations. it is a vector that has a number of elements equal to the number of protein sequences in the selected sample of each group. it measures the degree of similarity between each protein sequence's adjacency vector and the group representative vector. in other words, it measures the degree of similarity between each protein's descriptor and the "group representative." it is simpler than previous matrix. it is calculated only one time for each sequence. the calculations' number of this vector equals n where n is the number of compared species. to measure the degree of similarity, we suggest two methods: (ii) e nd method. compute the angle between each sequence's adjacency vector (a xy ) and the group representative vector (gr y ) in radians by for beta globin protein sequences, seven species are selected in our sample set: human, chimpanzee, gorilla, mouse, rat, gallus, and opossum, as illustrated in table 1 . there are seven adjacency vectors corresponding to them. the group representative gr beta globin is evaluated based on these seven adjacency vectors. therefore, the similarity/dissimilarity vector has seven elements. the 1 st element corresponds to human, 2 nd element corresponds to chimpanzee, and so on, by the same order as in table 1 . in the tables 2 and 3 , respectively. the similarity/dissimilarity vectors that are corresponding to beta globin, nd5, and spike protein sequences are illustrated in tables 9, 10, and 11, respectively, based on the two methods discussed before. the results in table 9 show that the magnitude ( , where x: species) cannot measure the similarity/dissimilarity degree well among all beta globin sequences. the human, chimpanzee, and gorilla have the same value that is equal to 0.5568, while the similarity is well measured between mouse and rat. also, the dissimilarity between opossum and human is very clear. the angle ( ) is successfully measured similarity/dissimilarity among all the species as shown in figure 1 . the closest values of both and mean more similarity. the results in table 10 show that both the magnitude ( 5 ) and the angle ( 5 ) can measure similarity/dissimilarity degree well among nd5 protein sequences as shown in figure 2 . it is obvious that pigmy chimpanzee, common chimpanzee, human, and gorilla are very similar. also it shows the similarity of the blue whale, fin whale, and the mouse and rat as pairs and the dissimilarity between human and opossum. these results are satisfied with [13, 14, 16, 18, 19, [21] [22] [23] [24] [25] . the results in table 11 show that both and classified the 3 classes of viruses and sars covs well each as a single coherent class except only the "mhvjhm" virus. this virus belongs to class ii but our approach cannot classify it well. the classification of 29 spike proteins into classes by our approach is illustrated in figure 3 . the mhvjhm virus is the only wrong classified sequence. it is colored red. despite the wrong classification of mhvjhm virus, our approach corrects the broken classification of class i in [26] . according to the results in tables 9, 10 , and 11, the angle is preferred to be used as shown in figures 1, 2 , and 3. the group representative vector ( ) carries the information of its group. a cross-group comparison is done to prove the singularity of each group. tables 9, 10, and 11 are evaluated based on the group's sample set of protein sequences related to their corresponding group representative vector. tables 12, 13, 14, and 15 are evaluated based on each group sample set of protein sequences with another group representative vector. the similarity/dissimilarity analysis among the seven beta globin sequences measured according to ( 5 ) is illustrated in table 12 and shown in figure 4 . the similarity/dissimilarity analysis among the nd5 sequences measured according to ( ) is illustrated in table 13 and shown in figure 5 . the similarity/dissimilarity analysis among the beta globin sequences measured according to (gr spike ) is illustrated in table 14 and shown in figure 6 . the similarity/dissimilarity analysis among the nd5 sequences table 15 and shown in figure 7 . the results show a big distortion that ensures the individuality of each group. the phylogenetic tree is a branching diagram showing the evolutionary relationships among various biological species based upon similarities and differences in their sequences. a qualitative comparison between our results and the phylogenetic tree of protein sequences is used to prove the utility of our approach. the matching between the results and phylogenetic trees means matching with the naïve measure of sequence similarity (sequence homology). the basic local alignment tool (blast) is used to draw the phylogenetic trees. the phylogenetic trees of beta globin's seven species, nd5 nine species, and 29 spike protein sequences are illustrated in figures 8, 9 , and 10, respectively. the qualitative comparison of the results of tables 9, 10, and 11 and figures 8, 9 , and 10 shows the utility of our work especially the angle results. the proposed method is an alignment-independent method. an adjacency vector is suggested as a descriptor of any protein sequence. it does not require any graphical representation. a group representative vector is introduced to represent each group of protein sequences. a similarity/dissimilarity vector is produced instead of the regular similarity/dissimilarity matrix. the similarity/dissimilarity analysis is done by two methods. our approach is applied on three sample sets of three groups of protein sequences. each sample has a different range of lengths than the others. our approach does not depend on protein sequence length. it successfully measured similarity/dissimilarity among different lengths. it is very mathematically simple. a cross-grouping comparison is introduced to prove the singularity of each group. the results approved the utility of our approach compared with previous articles and phylogenetic tree obtained by blast program. we hope to make the method available to include ambiguous amino acid residues and nonstandard amino acids. we hope also to include the analyses of partial or gapped sequences. all data is mentioned clearly in the manuscript in section 2 under the title "dataset." in this section, we illustrate the data in three tables: tables 1, 2, and 3. we also mention in the 1st paragraph of dataset that data are downloaded from "gene bank." all data files are with extension ". fasta". the authors declare that they have no conflicts of interest. dna sequence comparison by a novel probabilistic method linear regression model of short kword: a similarity distance suitable for biological sequences with various lengths sequence comparison via polar coordinates representation and curve tree a general method applicable to the search for similarities in the amino acid sequence of two proteins identification of common molecular subsequences an improved algorithm for matching biological sequences basic local alignment search tool rapid and sensitive protein similarity searches amino acid substitution matrices from protein blocks graphical representation of proteins similarity/dissimilarity calculation methods of dna sequences: a survey 3-d maps and coupling numbers for protein sequences a novel descriptor for protein similarity analysis similarity analysis of protein sequences based on 2d and 3d amino acid adjacency matrices a new method to analyze protein sequence similarity using dynamic time warping a 2d graphical representation of protein sequence and its numerical characterization graphical representation and similarity analysis of protein sequences based on fractal interpolation adld: a novel graphical representation of protein sequences and its application comparative analysis of protein primary sequences with graph energy uc-curve: a highly compact 2d graphical representation of protein sequences the graphical representation of protein sequences based on the physicochemical properties and its applications f-curve, a graphical representation of protein sequences for similarity analysis based on physicochemical properties of amino acids a novel method of 2d graphical representation for proteins and its application 3d graphical representation of protein sequences and their statistical characterization novel numerical characterization of protein sequences based on individual amino acid and its application similarities/dissimilarities analysis of protein sequences based on pca-fft on novel representation of proteins based on amino acid adjacency matrix a sequence-segmented method applied to the similarity analysis of long protein sequence it is a figure which summarizes our approach. it is submitted under the name of graphical abstract. (supplementary materials) key: cord-344061-gsl84nv6 authors: pariani, elena; martinelli, marianna; canuti, marta; jazaeri farsani, seyed mohammad; oude munnink, bas b.; deijs, martin; tanzi, elisabetta; zanetti, alessandro; van der hoek, lia; amendola, antonella title: influenza and other respiratory viruses involved in severe acute respiratory disease in northern italy during the pandemic and postpandemic period (2009–2011) date: 2014-06-12 journal: biomed res int doi: 10.1155/2014/241298 sha: doc_id: 344061 cord_uid: gsl84nv6 since 2009 pandemic, international health authorities recommended monitoring severe and complicated cases of respiratory disease, that is, severe acute respiratory infection (sari) and acute respiratory distress syndrome (ards). we evaluated the proportion of sari/ards cases and deaths due to influenza a(h1n1)pdm09 infection and the impact of other respiratory viruses during pandemic and postpandemic period (2009–2011) in northern italy; additionally we searched for unknown viruses in those cases for which diagnosis remained negative. 206 respiratory samples were collected from sari/ards cases and analyzed by real-time rt-pcr/pcr to investigate influenza viruses and other common respiratory pathogens; also, a virus discovery technique (vidisca-454) was applied on those samples tested negative to all pathogens. influenza a(h1n1)pdm09 virus was detected in 58.3% of specimens, with a case fatality rate of 11.3%. the impact of other respiratory viruses was 19.4%, and the most commonly detected viruses were human rhinovirus/enterovirus and influenza a(h3n2). vidisca-454 enabled the identification of one previously undiagnosed measles infection. nearly 22% of sari/ards cases did not obtain a definite diagnosis. in clinical practice, great efforts should be dedicated to improving the diagnosis of severe respiratory disease; the introduction of innovative molecular technologies, as vidisca-454, will certainly help in reducing such “diagnostic gap.” most cases of influenza a(h1n1)pdm09 infection have a mild outcome; however some present as severe acute respiratory infection (sari) and require admission to intensive care unit (icu) [1, 2] . the main reason for admission to icu is a pulmonary inflammatory syndrome characterized by diffuse alveolar damage (acute respiratory distress syndrome: ards), which can be fatal. since the beginning of 2009 pandemic, international health authorities recommended monitoring severe and complicated cases of influenza infection [3, 4] . considering the serious outcome of these respiratory diseases, the contribution of other respiratory pathogens besides a(h1n1)pdm09 should be envisaged [5] . additionally, in clinical practice, a specific causative agent which explains the respiratory symptoms is often unidentified, owing to the lack of sensitive tests or the presence of an asyet unknown pathogen. the recently developed vidisca-454 (virus discovery using cdna amplified fragment-length polymorphism combined with roche-454 high-throughput sequencing) is a sensitive sequence-independent virus discovery technique which can be used to reveal as-yet unknown viruses [5, 6] . this study aimed at evaluating the proportion of sari/ards cases and deaths due to a(h1n1)pdm09 infection and assessing the impact of other respiratory pathogens during pandemic and postpandemic period (2009) (2010) (2011) in northern italy as well as searching for unknown viruses in those cases for which diagnosis remained negative. to this end, common respiratory pathogens were investigated and vidisca-454 methodology was applied on samples which remained negative for all tested pathogens. in the capacity of reference laboratory operating within influnet network [7] , our laboratory is in charge of carrying out the virological surveillance of severe forms of influenza infection in lombardy (nearly 10 million inhabitants). from october 1, 2009, to april 30, 2011, 206 respiratory samples were collected from patients hospitalized due to severe respiratory illness. of these, 61.2% were males with a median age of 44.3 years (iqr: 49.7 years; range: 1 month-89 years); 17.5% were children ≤ 5 years and 23.3% were ≥65 years. data on comorbidities presence were available for nearly 70% of study patients: 64.3% reported medical conditions [3, 4] ; in detail, 25.6% had weakened immune system (due to cancer, hiv/aids, or long-term steroid treatment), 19.7% heart disease, 11.6% asthma/chronic lung disease, and 10.4% neurological/neurodevelopmental conditions. out of 206 patients, 91 (59.3% males; 18.7% aged ≤ 5 years, 58.2% aged 6-64 years) were sari cases who required admission to icu and extracorporeal membrane oxygenation (ecmo) therapy, and 115 (62.6% males; 16.5% aged ≤ 5 years, 60% aged 6-64 years) were ards cases, as defined by the european consensus conference [8] . nine ards patients (median age: 35.6 years, iqr: 21.4 years) died during hospitalization: case fatality rate (cfr) in our ards series was 7.8% (9/115). no sari case was fatal. respiratory specimens (paired nasal/oral swab and bronchoalveolar lavage) were collected from each sari/ards case. nucleic acids were purified by nuclisens easymag (biomérieux, france) and analyzed by real-time rt-pcr assay to identify influenza virus. in detail, a one-step realtime rt-pcr assay was performed to simultaneously detect influenza viruses type a and b [9] . the subtyping of influenza a positive samples was carried out by a one-step realtime rt-pcr assay using specific primer/probe sets for the hemagglutinin gene [10] . the clinical specimens that resulted negative to influenza virus detection were then screened by real-time rt-pcr/pcr for a panel of respiratory pathogens (respiratory mws r-gene real-time pcr, biomérieux, france) to detect respiratory syncytial virus (rsv) a and b; human metapneumovirus (hmpv) a and b; human rhinovirus (hrv) and enterovirus (hev); adenovirus (adv); human bocavirus (hbov) 1-4; human coronavirus (hcov) 229e, nl63, oc43, hku1; human parainfluenza virus (hpiv) 1-4; chlamydophila pneumoniae; mycoplasma pneumoniae. cases which resulted negative to all diagnostic assays were further investigated by vidisca-454 technique. this is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors, and subsequent amplification by pcr combined with high-throughput sequencing 454 flx/titanium system (roche, usa) [5] . influenza a(h1n1)pdm09 virus was detected in 58.3% (120/206) of sari/ards cases (61.7% males; 13.3% aged ≤ 5 years, 67.5% aged 6-64 years). moreover, the presence of another condition possibly increasing the risk for developing influenza-related complications [3, 4] forty-six (46/206: 22.3%) sari/ards cases (including two fatalities) resulted negative to all diagnostic assays (58.2% males; 18.2% aged ≤ 5 years, 45.4% aged 6-64 years) and were further investigated by vidisca-454 [5, 11] . vidisca-454 revealed no sequence reads that could belong to a novel virus or viral variant in any of the 46 specimens; however it enabled the identification of one case of undiagnosed measles, thus increasing the proportion of cases with a diagnosis to 78.2% (161/206). hence, the overall proportion of cases with unknown diagnosis was 21.8% (45/206); most (34/45: 75.6%) cases that could not be diagnosed were ards and two (2/45: 4.4%) were fatal. figure 1 summarizes study results. during pandemic and postpandemic period, several pathogens cocirculated and were associated to severe respiratory infections; however, influenza a(h1n1)pdm09 virus had the greatest impact (58.3%) in our sari/ards series. more than half (51.7%) of a(h1n1)pdm09 infection resulted in ards. it is interesting to note that most (67.5%) severe respiratory diseases due to a(h1n1)pdm09 infection were identified among 6-64-year-old individuals. the a(h1n1)pdm09 case fatality rate in our ards series was 11.3% fatal cases in young adults, and 42.8% did not belong to any at-risk category [3, 4] . this data is in agreement with other studies; van kerkhove et al. have reported a median age of 46 years among fatal laboratory-confirmed a(h1n1)pdm09 cases [12] . mccallum has described that during the 2009 pandemic only 1% of laboratory-confirmed cases and 13% of laboratory-confirmed deaths were among persons 65 years of age or older [13] . the global pandemic mortality (glamor) project has evaluated that although the pandemic mortality estimate was similar in magnitude to that of seasonal influenza, a marked shift toward mortality among persons 65 years of age occurred, so that many more life-years were lost [14] . such an age shift has been documented as well by several studies on a(h1n1)pdm09 mortality [15] [16] [17] . the proportion of sari/ards cases associated with respiratory viruses other than a(h1n1)pdm09 was significantly lower (19.4% versus 58.3%, value <0.0000001). severe respiratory diseases associated with respiratory viruses other than a(h1n1)pdm09 were detected more frequently among children ≤ 5 years (13.3% versus 30%, value = 0.02). this piece of evidence is in accordance with the results of other studies reporting a notable burden of respiratory viruses in children under 5 years [18] [19] [20] . studies published to date have suggested that influenza viruses and rhinoviruses are the leading causes of severe respiratory disease leading to hospitalization [21, 22] , similarly to what was observed in our sari/ards series, where hrv/hev were the most common identified viruses along with influenza viruses. also influenza a(h3n2) virus played a significant role in our sari cases and caused ards in one patient with a weakened immune system due to hiv/aids. overall, the proportion of sari/ards correlated to an influenza a virus infection was 62.1% (128/206), thus emphasizing the central role of influenza a virus in severe respiratory infection [23, 24] . the use of molecular assay has notably contributed to identifying pathogens possibly involved in severe respiratory disease, thus allowing getting to a diagnosis of viral infection in nearly 80% of study sari/ards cases. other studies that have not used nucleic acid amplification assays have typically reported that 5-20% of cases of acute respiratory infection have a viral etiology [25] . in addition, it is noteworthy that vidisca-454 enabled the identification of one measles infection that escaped clinical diagnosis in one sari case. hence, measles infection should be considered in complicated pulmonary disease, as also suggested by others [26] , since measles virus is not generally included in respiratory diagnostic panels. during pandemic and postpandemic period, several pathogens cocirculated and were associated to severe respiratory infections, with influenza a(h1n1)pdm09 virus having the greatest impact. nearly 22% of sari/ards cases did not obtain a definite diagnosis, and among these cases two were fatal. in clinical practice, great efforts should be devoted to improving diagnosis of severe respiratory infec-tions and to reducing such "diagnostic gap. " the advantage from relying upon more accurate diagnosis could benefit the patient, in terms of receiving the more appropriate antiviral drugs, and could provide more detailed information on viruses circulating in the community, thus making public health authorities aware so as to adjust their policies accordingly. vidisca-454 proved to be a sensitive and specific methodology that can be successfully applied to surveillance of viral respiratory infections that represent an ever-changing field due to the continuous emergence of new viruses (i.e., influenza a(h5n1) and a(h7n9) viruses, mers-cov). pandemic novel 2009 h1n1 influenza: what have we learned 2009 h1n1 influenza pandemic: field and epidemiologic investigations in the united states at the start of the first pandemic of the 21st surveillance of severe disease due to influenza in europe surveillance of severe forms of influenza a(h1n1)pdm09 infection a sensitive assay for virus discovery in respiratory clinical samples identification of a new human coronavirus the american-european consensus conference on ards: definitions, mechanisms, relevant outcomes, and clinical trial coordination world health organization (who) global influenza surveillance network (gisn), "manual for the laboratory diagnosis and virological surveillance of influenza 2009 protocol cdc protocol of real-time rt pcr for swine influenza a(h1n1) performance of vidisca-454 in feces-suspensions and serum risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis epidemiological characteristics of the influenza a(h1n1)2009 pandemic in the western pacific region global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study mortality burden of the a/h1n1 pandemic in mexico: a comparison of deaths and years of life lost to seasonal influenza mortality burden of the 2009 a/h1n1 influenza pandemic in france: comparison to seasonal influenza and the a/h3n2 pandemic all-cause mortality during first wave of pandemic (h1n1) epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries global burden of childhood pneumonia and diarrhoea surveillance for hospitalized acute respiratory infection in guatemala incidence and characteristics of viral community-acquired pneumonia in adults the role of viruses in the aetiology of community-acquired pneumonia in adults incidence of respiratory viruses in patients with community-acquired pneumonia admitted to the intensive care unit: results from the severe influenza pneumonia surveillance (sips) project viral infection in patients with severe pneumonia requiring intensive care unit admission community-acquired pneumonia viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord-283092-t3yqsac3 authors: shah, kamal; abdeljawad, thabet; mahariq, ibrahim; jarad, fahd title: qualitative analysis of a mathematical model in the time of covid-19 date: 2020-05-25 journal: biomed res int doi: 10.1155/2020/5098598 sha: doc_id: 283092 cord_uid: t3yqsac3 in this article, a qualitative analysis of the mathematical model of novel corona virus named covid-19 under nonsingular derivative of fractional order is considered. the concerned model is composed of two compartments, namely, healthy and infected. under the new nonsingular derivative, we, first of all, establish some sufficient conditions for existence and uniqueness of solution to the model under consideration. because of the dynamics of the phenomenon when described by a mathematical model, its existence must be guaranteed. therefore, via using the classical fixed point theory, we establish the required results. also, we present the results of stability of ulam's type by using the tools of nonlinear analysis. for the semianalytical results, we extend the usual laplace transform coupled with adomian decomposition method to obtain the approximate solutions for the corresponding compartments of the considered model. finally, in order to support our study, graphical interpretations are provided to illustrate the results by using some numerical values for the corresponding parameters of the model. mathematical models are powerful tools to study various physical phenomena of real world problems. the respective idea was initiated by bernouli in 1776. after that, the first mathematical model of infectious disease was formulated in 1927 by mckendrick and karmark. following that, this area got considerable attention and lots of models, which describe numerous physical or biological processes, were formed; the reader may refer to [1] [2] [3] [4] [5] for more information about some models. by using mathematical models for the description of infectious diseases, we can get information about the transmission of a disease in a community, its mortality rates, and how to control it. therefore, this area has been established in the last few decades very well, (see [6] [7] [8] [9] ). several outbreaks in the form of pandemics have been come out like in 1920 and 1967 in which more than 100 million people died. during the start of this century, there also occurred some outbreaks in saudi arabia, china, and mexico. in these outbreaks, thousands of people lost their lives. but due to the rapid advancement in medical science, vaccines were prepared and made those diseases curable. in the end of 2019, a serious outbreak has occurred in hubei province of china due to a virus known as corona which has been named the novel covid-19. this outbreak is in progress and more than three million people have been infected in almost every country of the globe. nearly 0.22 million people have died due this disease. three months have gone but, up to date, neither proper cure nor some suitable vaccine have been prepared yet [10] . the world health organization (who) reported the presence of a novel coronavirus (2019-ncov) in wuhan city, hubei province of china, on 31 december 2019. the virus which caused this infection belongs to the previous family of sars. investigating the present literature, there are many theories behind the origin of the virus. some researchers have investigated that it originated from bats to human due unlawful full transmissions of animals in a market of seafood in wuhan. the concerned virus has been identified in pangolin and also in dogs. therefore, it has been considered that many infected cases claimed that they had been working in a local fish and wild animal market in wuhan from where they caught infection of coronavirus-19. after that, researchers confirmed that the widespread of the disease is due to person-to-person contact. also, the mentioned city of the republic of china is a great trade center from where the infection was transferred to many countries of the world through immigration; for details, we refer to [11, 12] . keeping in mind what we have mentioned above, numerous researchers started to model the disease to figure out the properties in different ways. recently, some researchers in [13] have constructed the following mathematical models under ordinary derivative, as a modification to some previously studied prey-predator models [14] [15] [16] , as follows: in the above model, h stands for healthy individual, i for infected individuals, and β for the infection rate, where the rate of immigration of healthy individuals from one place to another place is denoted by α. further, the rate at which infection take immigration is γ, while death rate is denoted by δ and cure rate by ρ. since immigration of people is also a big cause of spreading of this disease, it is evident to check the impact of the immigration of individuals on the transmission dynamics of the current disease. on the other hand, such a study may help in forming some precautionary measures to protect more people from this infection. the study of the mathematical models under fractional derivatives instead of usual ordinary derivatives produces more significant results which are more helpful in understanding. in fact, numerous fractional order derivatives have been introduced and used in literature including "caputo and riemann-liouville" derivatives which are the most popular differential operators. there are large number of applications in real world problems due to fractional calculus; see [17] [18] [19] [20] [21] [22] [23] . recently, some authors replaced the singular kernel in classical nonlocal fractional derivatives by a nonsingular kernel of "mittag-leffler" type; for details, see [24, 25] and the references therein. it is remarkable that fractional derivatives in fact are defined by means of convolutions which contain ordinary derivatives as a special case. further, the geometry of fractional derivatives tells us about the accumulation of the whole function. actually, fractional operators, either of singular or without singular kernels, are nonlocal with memory effect unlike ordinary differential operators which are local in nature. fractional order operators involving "mittag-leffler" kernels have been proved more practical and efficient like the classical nonlocal fractional operators; see [25] [26] [27] [28] [29] . further investigating dynamics problems under fractional derivatives instead of integer order derivative produces global dynamics of the concerned problems which include the integer order derivative as a special case [30] [31] [32] [33] [34] [35] [36] . inspired from the aforesaid discussion, in this paper, we investigate the covid-19 model (1) under the new type derivatives as where, in the above model, abc d θ 0 stands for atangana-baleanu-caputo (abc) derivative of order 1 > θ > 0. we shall analyze the above model from various aspects including existence theory and series type solution. we shall also investigate some stability results of ulam's type for the considered model. for the existence theory, we use the classical fixed point theorems of krasnoselskii's and banach. in addition to the series type solutions, we shall use the integral transforms given by laplace and decomposition technique of adomian. numerical interpretations are given via graphs to demonstrate the obtained results. also, it is necessary that the right hand side of the above covid-19 abc-model must vanish at 0 (see theorem 3.1 in [30] ). 1.1. organization of the paper. section 1 is devoted to introduction of the paper. in section 2, some fundamental results are given. also, in section 3, we establish the existence results while in section 4, the required analytical results are constructed. section 5 is related to the graphical presentations of the results and their discussion. in section 6, we provide a brief conclusion and some future directions. here, we provide some necessary results that may be found in [29] and the references therein such as [24, 25] . definition 2.1. if φ ∈ h1ð0, tþ and θ ∈ ð0, 1, then the abc derivative is defined by we remark that if we replace e θ ½ð−θ/1 − θþðt − yþ θ by e 1 = exp ½ð−θ/1 − θþðt − yþ, then we get the so-called caputo-fabrizo differential operator. further, it is to be noted that here, kðθþ is known as the normalization function which is defined as kð0þ = kð1þ = 1. also, e θ stands for famous special function called mittag-leffler which is a generalization to the exponential function [17] [18] [19] . 2 biomed research international definition 2.2. let φ ∈ l½0, t, then the corresponding integral in abc sense is given by lemma 2.3. (see proposition 3 in [28] ). the solution of the given problem for 1 > θ > 0, is provided by definition 2.4. the laplace transform of abc derivative of a function φðtþ is defined by note: for the qualitative analysis, we define banach space as z = x × x, where x = c½0, t under the norm kðh, iþk = max t∈½0,t ½jhðtþ+|iðtþj. the following fixed point theorem will be used to proceed to our main results. theorem 2.5. [36] . let b be a convex subset of z and assume that f and g are two operators with (1) fðh, iþ + gðh, iþ ∈ b for every h, i ∈ b (2) f is contraction (3) g is continuous and compact then, the operator equation fðh, iþ + gðh, iþ = ðh, iþ has at least one solution. (2) here, we are going to discuss existence and uniqueness of solution for our main model. let us write model (2) as where if we apply the fractional integral ab i θ 0 of order θ on both sides of (9) and make use of lemma 2.3 together with the use of the initial conditions, we get to derive the existence and uniqueness, we imposed some growth conditions on the nonlinear functions f , g : under the continuity of f , g together with assumption (a2), system (5) has at least one solution if proof. by the help of krasnoselskii's fixed point theorem, we shall prove the existence result. we define the operators f = ðf 1 , f 2 þ, g = ðg 1 , g 2 þ by using (6) as follows: which implies that and similarly, one has from (8) and (9), one has which implies that f is a contraction. let us define a closed subset b of z as for g to be compact and continuous, let any ðh, iþ ∈ b, we have from (10) and (10), we have hence, f is bounded. next, we show that f is equicontinuous. let t 1 < t 2 ∈ ½0, t, then consider similarly, now, from (12) and (14), we see as t 1 ⟶ t 2 , then the right sides tend to zero. hence, we see that consequently, we claim that hence, g is a equicontinuous operator. by using arzelà-ascoli theorem, the operator g is a completely continuous operator and also uniformly bounded proved already. hence, g is relatively compact. by krasnoselskii's fixed point theorem, the given system has at least one solution. next, we establish results about uniqueness of solution as follows: with max fl f , l g g = l. proof. define the operator p = ðp 1 , p 2 þ: z ⟶ z using (6) as and in same fashion, one has from (16) and (17), we have hence, p is a contraction. by banach contraction theorem, the considered system has unique solution. next, we give a results about ulam-hyers stability. given by ja + bj < 1, where proof. let ðh, iþ ∈ z be any solution of the model (2) and ð h, iþ ∈ z is unique solution of the same model; then, we have where a and b are given as in (19) . hence, the solution of the given system is ulam-hyers stable. since the eigenvalues of square matrix are λ 1 = 0, λ 2 = a + b and spectral radius of the matrix is given by max fjλ1j: i = 1, 2g = ja + bj < 1: in this section, we apply the proposed novel analytical method to find the series type solution of the suggested model (2) . to this end, we take the laplace transform of both sides of (2) and use the initial conditions to get after rearranging the terms in (34), one has now, we are interested to find the required solution in the form of infinite series, therefore taking the unknown solutions further, the nonlinear term hðtþiðtþ in the system (2) may be decomposed in terms of adomian polynomials as where we compute few terms for n = 0, 1, 2, ⋯, as and so on. plugging the above series type representation in (21) , one has comparing terms on both sides in (40), we get taking inverse transform of laplace on both sides and after computation with using biomed research international we get few terms of the series solution as and so on. in this way, the remaining terms will be generated. now, we take some various values for parameters taken in [16] as α = 0:0, β = 0:03, γ = 0:05, δ = 0:05, and ρ = 0:05 and take a random community where the total population is divided in such a way that 70 percent of the population is healthy and 30 percent is infected, that h0 = 0:7, i = 0:3: clearly, using these values in model (2), we have l f = 0:03, l g = 0:03, kðθþ = 1: from which we have l = 0:03. hence, the condition of existence of at least one solution holds by using theorem 3.1. also, the condition of theorem 3.3 is valid under suitable value of t. in the current situation, the solution is going to become stable. further, taking kðθþ = 1, we compute few terms from (43) of series solution up to four terms as follows: and so on. we plot the solutions (43) for different fractional order by using matlab in figures 1-4 . from figure 1 , we see that at when the rate of healthy immigrants is zero, it means that protection rate is increasing and hence the population of infected class is decreasing while the population of healthy class is increasing at different rates due to fractional order derivative by evaluating the solution up to twenty terms via using matab. as the order is increasing, the growth rate of healthy class is increasing and thus becomes stable first as compared to the small fractional order. on the other hand, the decaying process of infected class is fastest on the small fractional order as compared to the large order. thus, in this case, the stability is achieved first at the smallest fractional order derivative rather than at the greater order. further at the given values of the parameters, the infection to vanish in a locality will take days between 220 and 250. in figure 2 , in the presence of immigration and less protection rate, we plot the solution corresponding to different fractional orders. we see that infection is increasing while the population of healthy class is decreasing at various rates due to fractional order. from figure 3 , when we involve immigration of infected class and cease the immigration for the healthy class, we see that the population density of infected class is going up with different rates due to fractional order derivative during in first 250 days. on the other hand, the healthy population is going on instability in the first 50 days, that is; it increases and then decreases suddenly. to achieve stable position, it requires nearly 110 days. this means that immigration of infected population from one place to another will cause instability in the healthy population of a community. from figure 4 , we see that the straight increase in both populations is due to immigration of healthy population but using strong protection rate at different fractional orders. in this article, we have examined a population model of the novel covid-19 under abc fractional order derivatives. we have proved sufficient results about the existence and uniqueness of solution for the considered model and proved that it has at least one solution. hence, the fixed point theory always works as an effective tool that can be used to check the existence and uniqueness of various physical problems. a stability result has also been established. through a novel method, we have derived approximate solutions for the corresponding compartments of the model under investigation. further, some numerical results have been presented for different fractional orders through matlab by taking various values of the immigration rates. we observed that as the immigration of infected class is increasing, it will cause the decrease in healthy population and hence the population of infected people increases. therefore, an important factor which increases the infection of the current outbreak is free biomed research international immigration. when people do not avoid the unnecessary traveling from one place to another, there is greater chance to infect. if this term decreases, then infection may be sufficiently decreased in a population. if in society, the immigration of infected people is strictly controlled, then we may protect our society from further hazard. in the future, the concerned model may be further extended by involving exposed class, recovered class, and asymptotically infected class to form five compartment models. this new model will further produce more significant information basis on which better controlling policies and procedure may be made to save our society from this infection. no data were used to support this study. there exist no competing interest regarding this research work. semianalytical study of pine wilt disease model with convex rate under caputo-febrizio fractional order derivative dynamic behavior of leptospirosis disease with saturated incidence rate global stability analysis and control of leptospirosis complex mathematical models of biology at the nanoscale mathematical 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transform method on a new modified fractional analysis of nagumo equation a comparative study on solving fractional cubic isothermal auto-catalytic chemical system via new efficient technique numerical treatment for studying the blood ethanol concentration systems with different forms of fractional derivatives a fixed-point theorem of krasnoselskii all authors have read and approved the final revised version of this paper. the second and third authors will financially support this article from their own sources. h 0 t ð þ = h 0 , i 0 t ð þ = i 0 ,h t ð þ = 0:7 + 0: all authors have equal contribution in this work. the first and second authors were involved in the analysis while the last two authors have done the verification and checking of the formal analysis of this paper. key: cord-279528-41atidai authors: abo-elkhier, mervat m.; abd elwahaab, marwa a.; abo el maaty, moheb i. title: measuring similarity among protein sequences using a new descriptor date: 2019-11-22 journal: biomed res int doi: 10.1155/2019/2796971 sha: doc_id: 279528 cord_uid: 41atidai the comparison of protein sequences according to similarity is a fundamental aspect of today's biomedical research. with the developments of sequencing technologies, a large number of protein sequences increase exponentially in the public databases. famous sequences' comparison methods are alignment based. they generally give excellent results when the sequences under study are closely related and they are time consuming. herein, a new alignment-free method is introduced. our technique depends on a new graphical representation and descriptor. the graphical representation of protein sequence is a simple way to visualize protein sequences. the descriptor compresses the primary sequence into a single vector composed of only two values. our approach gives good results with both short and long sequences within a little computation time. it is applied on nine beta globin, nine nd5 (nadh dehydrogenase subunit 5), and 24 spike protein sequences. correlation and significance analyses are also introduced to compare our similarity/dissimilarity results with others' approaches, results, and sequence homology. information encoded in the genome of any organism plays a central role in defining the life of that organism. e nucleotide sequence that forms any gene is translated into its corresponding amino acid sequence. is sequence of amino acids becomes functional only when it adopts its tertiary structure. experimental methods such as x-ray diffraction and nuclear magnetic resonance are considered authoritative ways for obtaining proteins' structure and function. ese experimental methods are very expensive and time consuming. erefore, computational methods for predicting protein structure have become very useful. proteins with similar sequences are usually homologous, typically displaying similar 3d structure and function. sequence alignment is the first step of 3d structure prediction for protein sequences. alignment approaches are classified into alignment-based and alignment-free methods. blast (basic local alignment search tool) and clustalw are the most widely used computer programs for alignmentbased approaches [1] [2] [3] . results of these programs provide an approximate solution to the protein alignment problem. on the other hand, many alignment-free approaches are proposed for sequence comparison. most biological sequence analysis methods still have weaknesses, including having low precision and being time consuming [4, 5] . similarity/dissimilarity analysis of biological sequences is used to extract information stored in the protein sequence. many mathematical schemes have been proposed to this end. graphical representations of biological sequences identify the information content of any sequence to help biologists choose another complex theoretical or experimental method. graphical representation provides not only visual qualitative inspection of gene data but also mathematical characterizations through objects such as matrices. some 2d and 3d graphical representations are created by selecting a geometrical object that is used to describe nucleic acid bases or residues [6] [7] [8] [9] [10] . others are based on assigning vectors of two or three components to nucleic acid bases or amino acids [11] [12] [13] [14] [15] [16] [17] . adjacency matrices are also introduced in some articles [18] [19] [20] [21] , where an exact solution is obtained to the protein alignment problem. additional methods use discrete fourier transform (dft) in which dna sequences are mapped into four binary indicator sequences, followed by the application of dft on these indicator sequences to transform them into a frequency domain [22, 23] . dynamic representation is used to remove degeneracies in the previously mentioned approaches [24] [25] [26] [27] [28] [29] [30] [31] . another method is based on the simplified pulsecoupled neural network (s-pcnn) and huffman coding where the triplet code was used as a code bit to transform dna sequence into numerical sequence [32] . in this study, we introduce a new alignment-free method for protein sequences. each amino acid in the protein sequence is represented by a number, and a new 2d graphical representation is suggested. a new descriptor is introduced, comprising a vector composed of the mean and standard deviation of the total numbers of each protein sequence (a t , sa t ). our graphical representation eliminates degeneracy and has no loss of information. it is suitable for both short and long sequences. as a proof of concept, our approach is applied on nine beta globin protein sequences and nine nd5 (nadh dehydrogenase subunit 5) protein sequences. it can be applied on any sequence length with the same efficiency. correlation and significance analyses are introduced among our results, along with pid% [15] and clustalw [33] to demonstrate the utility of our approach. all the protein sequences used in this study were downloaded from e national center for biotechnology a new 2d graphical representation is introduced. each amino acid in any protein sequence is represented by the suggested intensity y x (i) and intensity level a x (i). e intensity (y x (i)) of each amino in the sequence depends on its abundance and location in the different sequences. it is calculated using where f x is the frequency of amino acid x in the sequence, number of times of x/n. n is the protein sequence length, number of residues in protein sequence. i is the position of each amino acid x in a sequence. en, the intensity level a x (i) of each amino acid (x) in the sequence is calculated by using the natural logarithm function as in erefore, each amino acid has its own intensity level which is a vector of n elements according to equation (2) . finally, the combined intensity level of the protein sequence a t (i) is obtained by the summation of the 20 intensity levels' vectors a x (i) of the protein sequence by using equation (3) . e combined intensity level a t (i) is also a vector of n elements: each amino acid has its own graph. now, twenty graphs are obtained for each sequence of the 20 different amino acids. e combined graph is obtained by combining these 20 graphs within a single graph. is combined intensity level is our new 2d graphical representation. our approach is first applied on two short segments of protein from "yeast saccharomyces cerevisiae": protein i: "wtfesrndpakdpvilwlnggpgcs-sltgl" protein ii: "wffesrndpandpiilwlnggpgcs-sftgl" ese two short proteins consist of 30 amino acids each. e two sequences are different in amino acids at positions 2, 11, 14, and 27. e values y x (i) and a x (i) for each amino acid in the two sequences are calculated. for protein i, the g amino acid is repeated four times in the protein sequence. ese four repeats occur in positions 20, 21, 23, and 29. e frequency, f g , equals (4/30). by substituting in equations (1) and (2), the results of y g (i) and a g (i) are presented in table 4 . by summing the values of a x (i) for all amino acids in protein i, the total value of a t (i) is obtained, as shown in figure 1 (a). e position i of each amino acid is located on the x-axis, and the total intensity level a t (i) is located on the y-axis. we next apply our approach on nine beta globin and nine nd5 (nadh dehydrogenase subunit 5) protein sequences, which are illustrated in tables 1 and 2 . e 2d graphical representation for human, chimpanzee, and opossum beta globin protein sequences is illustrated in representations for fin whale and rat nd5 protein sequences are illustrated in figures 3(a) and 3(b), respectively. we finally apply our approach on 24 coronaviruses protein sequences which are illustrated in table 3 . e 2d graphical representation of tgevg from class i and gd03t0013 from sars_cov protein sequences is illustrated in figures 4(a) and 4(b) respectively. mathematical descriptors help in recognizing major differences among similar protein sequences quantitatively. a new descriptor for protein sequences is suggested, which is a vector composed of the arithmetic mean a t and standard deviation sa t of the combined intensity level value a t (i) of the protein sequence. ey are evaluated according to the following equations: is descriptor compresses the information from primary protein sequences into a single vector composed of only two values. e beta globin, nd5, and coronaviruses protein sequence descriptors are illustrated in tables 5-7 , respectively. table 7 shows that the mean of all 24 coronaviruses is around 38.7 and with a range from 38.601 to 38.838 while their standard deviation varies according to their class. ey are divided into four classes. e first four viruses belong to class i. e fifth to the ninth coronaviruses belong to class ii. class iii contains the tenth and eleventh viruses. e rest viruses from the 12th to the 24th belong to sars-cov. according to our approach, the standard deviation of class i ranges from 10.94 to 11.17. class ii's standard deviation ranges from 10.68 to 10.77. class iii's standard deviation has values from 10.6271 to 10.6458. sars-cov's standard deviation almost equals 10.58. e resulting standard deviation values of the 24 coronaviruses classify them correctly to the four classes. e coronaviruses classes' ranges according to our approach are shown in figure 5 . to compare the species' protein sequences, the euclidean distance among species' descriptors is evaluated. for example, the human beta globin protein sequence's descriptor is (37.145, 11 .505) and the chimpanzee beta globin protein sequence's descriptor is (36.912, 11.586) . to measure the degree of similarity between human and chimpanzee, the euclidean distance between these vectors is evaluated. e similarity/dissimilarity matrices of beta globin and nd5 protein sequences are illustrated in tables 8 and 9 , respectively. table 8 results show that human and chimpanzee sequences are similar. ere is also striking similarity between mouse and rat sequences, while human and opossum sequences are obviously dissimilar. species id length 1 gorilla caa43421 121 2 chimp caa26204 125 3 human aaa16334 147 4 rat caa29887 147 5 mouse caa24101 147 6 gutta ach46399 147 7 duck caa33756 147 8 gallus caa23700 147 9 opossum aaa30976 147 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 pigmy chimpanzee, common chimpanzee, human, and gorilla nd5 protein sequences are similar, while the blue whale is similar to the fin whale, and mouse is similar to rat. similar to the other sequence, human and opossum are still dissimilar. however, our algorithm cannot measure the degree of similarity very well for pigmy chimpanzee. e distance between human and pigmy chimpanzee is 0.1826, while the distance between human and gorilla is 0.0575, as shown in table 9 . e results of both tables 8 and 9 are approximately comparable to previous reports [13, 15, 21, 33-39]. we got the phylogenetic trees of beta globin and nd5 protein sequences by applying the upgma (unweighted pair group method with arithmetic mean). e phylogenetic tree based on tables 8 and 9 of our method is presented in figures 6 and 7 , respectively. figure 6 proves the utility of our similarity/dissimilarity analysis for beta globin protein sequences. figure 7 shows our analysis of similarity/dissimilarity of nd5. it is mentioned that our algorithm cannot measure the degree of similarity very well for pigmy chimpanzee with human. is appears of course in figure 7 . e p. chimp branch should be close to c. chimp. despite this error, the tree shows that human, common chimpanzee, pigmy chimpanzee, and gorilla belong to the same cluster. to check the effect of this error on our algorithm, the results of our algorithm are compared to sequence homology. a correlation and significance analysis is also provided. e results of our algorithm are compared to the sequence homology by two methods. first, we use the smith waterman algorithm to calculate the number of identical residues in each pair of protein sequences [15] . e results of the pid% of nine beta globin sequences are illustrated as a similarity/dissimilarity matrix in table 10 . e larger pid% represents the more similar protein sequences. a correlation and significance analysis is provided to compare our approach in table 8 with pid% in table 10 . e correlation of the two sets of data is sufficiently strong when the correlation coefficient (r) is greater than 0.7. e negative sign of (r) indicates that when the first data set increases, the second data set decreases. we then assess statistical significance for correlation coefficient values greater than 0.7 to ensure that they likely do not occur by chance. our sample set is composed of nine protein sequences. erefore, we use 7 degrees of freedom. a t-value of 2.385 or greater indicates that a less than 0.05 chance of the results occurred by coincidence. e results for correlation coefficients and t-values for our approach are illustrated in table 11 . second, clustalw is a widely used system for aligning any number of homologous nucleotides or protein sequences [33] . e clustalw program's distance matrix of nine nd5 protein sequences is illustrated in table 12 . correlation and significance analyses are also provided to compare our approach in table 9 with clustalw results in table 12 . e results of the correlation and significance analyses of our approach and other approaches [15, 33] are illustrated in table 13 . our sample set of nd5 is also composed of nine protein sequences. erefore, we use 7 degrees of freedom and a t-value of 2.385 or greater. despite the unusual result for pigmy chimpanzee that appeared in table 11 : e correlation and significance analysis between our similarity analysis results of beta globin protein sequences in table 8 and pid% similarity matrix in table 10 . tables 9 and 7 in [33] and table 3 in [15] and clustalw similarity matrix in table 12 . correlation coeff. (r) of our approach t-value of our approach correlation coeff. (r) of [33] t-value of [33] correlation coeff. (r) of [15] (table 3) t-value of [15] ( table 9 , the correlation coefficient of pigmy chimpanzee in our similarity matrix and clustalw matrix is 0.8811. is value likely does not occur by chance, as the t-value equals 4.928, as illustrated in table 13 . e comparison between our results and both pid% and clustalw and other approaches' results indicate the utility of our approach. a new graphical representation of protein sequences is introduced. it is the combined intensity level of the 20 amino acids composing any protein sequence. each amino acid in a given protein sequence has its own intensity and intensity level. ey are vectors of n elements as n is the protein sequence length. e combined intensity level is then computed and graphed to represent any protein sequence graphically. our 2d graphical representation effectively displays differences between protein sequences without degeneracies. e graph does not overlap or intersect with itself. our new descriptor suggested a vector of two elements, which are the mean and standard deviation of the combined intensity level (a t and sa t ). a similarity/dissimilarity analysis is evaluated by computing euclidean distance between each two species' descriptors. examination of similarity/dissimilarity among nine beta globin, nine nd5, and 24 coronaviruses protein sequences provided good results compared to previous approaches. e suggested approach is effective for both short and long sequences, and the computations are very simple. furthermore, loss of sequence information is avoided. correlation and significance analyses with pid% and clustalw are also introduced to show the utility of our approach. basic local alignment search tool gapped blast and psi-blast: a new generation of protein database search programs clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice graphical representation of proteins similarity/dissimilarity calculation methods of dna sequences: a survey highly compact 2d graphical representation of dna sequences, sar and qsar unique graphical representation of protein sequences based on nucleotide triplet codons novel 2-d graphical representation of proteins representation of protein sequences on latitude-like circles and longitude-like semi-circles on a geometry-based approach to protein sequence alignment dna sequence comparison by a novel probabilistic method 2-d graphical representation of proteins based on physico-chemical properties of amino acids a 2d graphical representation of protein sequence and its numerical characterization 3-d maps and coupling numbers for protein sequences 3d graphical representation of protein sequences and their statistical characterization dna sequence representation without degeneracy protein map: an alignment-free sequence comparison method based on various properties of amino acids on novel representation of proteins based on amino acid adjacency matrix protein alignment: exact versus approximate. an illustration very efficient search for protein alignment-vespa similarity analysis of protein sequences based on 2d and 3d amino acid adjacency matrices a measure of dna sequence similarity by fourier transform with applications on hierarchical clustering a new method to cluster dna sequences using fourier power spectrum descriptors of 2d-dynamic graphs as a classification tool of dna sequences distribution moments of 2d-graphs as descriptors of dna sequences similarity studies of dna sequences using genetic methods 2d-dynamic representation of dna sequences 3d-dynamic representation of dna sequences spectral-dynamic representation of dna sequences four-component spectral representation of dna sequences 20d-dynamic representation of protein sequences a novel dna sequence similarity calculation based on simplified pulse-coupled neural network and huffman coding f-curve, a graphical representation of protein sequences for similarity analysis based on physicochemical properties of amino acids a novel descriptor for protein similarity analysis adld: a novel graphical representation of protein sequences and its application comparative analysis of protein primary sequences with graph energy e graphical representation of protein sequences based on the physicochemical properties and its applications a novel method of 2d graphical representation for proteins and its application novel numerical characterization of protein sequences based on individual amino acid and its application all data are mentioned clearly in the manuscript in section 2 under the title "dataset, technology, and tools." in this section, we illustrate the data in three tables: tables 1, 2, and 3. we also mention that data are downloaded from "gene bank." all data files are with extension", fasta". e authors declare that they have no conflicts of interest. key: cord-293503-e7be12qb authors: xiang, chao; lu, ji; zhou, jun; guan, li; yang, cheng; chai, changzhu title: ct findings in a novel coronavirus disease (covid-19) pneumonia at initial presentation date: 2020-08-15 journal: biomed res int doi: 10.1155/2020/5436025 sha: doc_id: 293503 cord_uid: e7be12qb background: covid-19 first broke out in china and spread rapidly over the world. objectives: to describe the ct features of covid-19 pneumonia and to share our experience at initial diagnoses. patients and methods. data from 53 patients (31 men, 22 women; mean age, 53 years; age range, 16-83 years) with confirmed covid-19 pneumonia were collected. their complete clinical data was reviewed, and their ct features were recorded and analyzed. results: the average time between onset of illness and the initial ct scan was six days (range, 1-42 days). a total of 399 segments were involved and distributed bilaterally (left lung: 186 segments [46.6%], right lung: 213 segments [53.4%]) and peripherally (38 [71.7%] patients). multiple lobes (45 [84.9%]) and bilateral lower lobes (left lower lobe: 104 [26.1%], right lower lobe: 107 [26.8%], and total: 211 [52.9%]) were the most commonly involved. ground-glass opacity with consolidation (24 [45.3%]) and pure ground-glass opacity (28 [52.8%]) were the main findings. the other findings were crazy-paving (14 [26.4%]), bronchiectasis (12 [22.6%]), atelectasis (7 [13.2%]), parenchymal bands (6 [11.3%]), air bronchogram (6 [11.3%]), interlobular thickening (5 [9.4%]), reticular pattern (1 [1.9%]), and pleural effusion (1 [1.9%]). conclusions: most covid-19 pneumonia patients had abnormalities on chest ct images at initial presentation. imaging features combined with patient's exposure history and onset symptoms could facilitate the identification of the suspected patient for further examinations. an epidemic novel coronavirus infection broke out in wuhan (the capital city of hubei province in central china) in december 2019. the infected people were initially considered highly associated with exposure to the huanan seafood wholesale market. from december 31, 2019, to january 3, 2020, a total of 44 patients with pneumonia of unknown etiology were reported to who [1] . as this infection broke out during the spring-festival travel rush, it spread rapidly to all provinces of china, and cases were subsequently found in 26 countries worldwide [2] . as of march 25 th 2020, 372,757 confirmed cases and 16,231 deaths were reported globally [3] . on january 7, 2020, a new type of coronavirus (sars-cov-2) was isolated by the wuhan institute of virology at the china academy of sciences. sars-cov-2 was identified as the causative virus by chinese authorities on january 7, 2020. this novel coronavirus had not been previously identified in humans and was tentatively named the 2019 novel coronavirus (2019-ncov) by who on january 13, 2020, and was officially named covid-19 on february 11, 2020 [4] . covid-19 leads to respiratory infections similar to those of sars and mers, causing pneumonia, severe acute respiratory syndrome, kidney failure, and even death. data have suggested that the sars-cov-2 is generally less pathogenic than sars-cov and is much less pathogenic than mers-cov [5] . due to previous reported cases which are biased to more severe cases at the early stages of the epidemic, the death rate was relatively high [5] . but the true mortality risk might be much lower, and it was reported that the death rate caused by pneumonia-related disease is about 3% [5] . the pathological findings of covid-19 have indicated that it is greatly similar to those seen in sars and mers coronavirus infections [2] . ct is an important tool for identifying the infected patients, and it is helpful for the follow-up evaluation of the treatment. although the ct features of covid-19 pneumonia have been reported in the literature, none of them have excluded patients with basic lung disease (such as copd, pulmonary tuberculosis, and interstitial lung disease) in their studies. these diseases may have the same features and patterns that are similar to those caused by covid-19 pneumonia. here, we aim to analyze and discuss the ct features of covid-19 pneumonia in patients without severe basic pulmonary disease and share our experience in ct diagnoses. the patients' clinical and image data used in this study were approved by the review board of our institution, and the patients' privacy was well protected. informed consent was waived by the review board for this retrospective study. between december 2019 and february 20, 2020, 185 covid-19-infected patients were confirmed at the first college of clinical medical science, china three gorges university, and the yichang central people's hospital. some patients (n = 102) were excluded for the following reasons: (a) respiratory motion artifacts on ct images, (b) incomplete clinical and/or imaging data, (c) severe basic pulmonary disease (including severe pulmonary interstitial fibrosis, severe chronic obstructive pulmonary disease, pneumoconiosis, and pulmonary tuberculosis), and (d) primary or secondary pulmonary tumor. after the selection, 53 eligible patients (31 males, mean age: 53 ± 16 years, range: 16-83 years) were included in our study ( figure 1 ). all imaging data were transferred to picture archiving and communication systems (pacs, united imaging, shanghai, china) and reviewed independently by two radiologists (c.x. and j.l.) with more than 10 years of experience in thoracic ct image interpretation. the location, distribution, morphology, ct features, and patterns of the lesions were recorded and analyzed. disagreements were resolved by consensus. the ct image characteristics were recorded as follows: (a) lesion's location (segment), (b) morphology (patchy, nodular, and linear), (c) distribution (single or multiple, peripheral or/and central), (d) type (ground-glass opacity, consolidation, and linear opacity), (e) pattern (reticulation, parenchymal bands, crazy-paving, and interlobular thickening), (f) atelectasis, (g) cavitation, (h) pleural effusion, (i) hilar or mediastinal lymphadenopathy, (j) bronchiectasis, and (k) air bronchogram. ground-glass opacity was defined as hazy increased opacity in the lung that did not obscure the bronchial and vascular architecture. consolidation was defined as homogeneously increased opacity in the lung without clear margins of vessels and bronchus. peripheral distribution was defined as the lesion located at the outer one-third of the lung; otherwise, it was defined as central. other abnormalities, if any, were noted. we used the diagnosis and treatment protocols for covid-19 pneumonia (6 th edition) promulgated by the national health commission of the people's republic of china [6] . the suspected cases comprised any one of the criteria in epidemiology and any two of the criteria in clinical manifestations or any three of clinical manifestations with uncertain epidemiological history. the epidemiology and clinical manifestations were as follows: (1) history of travel to or resident in wuhan or exposure to patients with respiratory symptoms or/and fever from wuhan within 14 days before the onset of illness the average time between onset and the initial ct scan was six days, range from 1 to 42 days. all the patients were confirmed to have covid-19 infection via rt-pcr. of the 53 patients, three patients (5.7%) showed normal findings on the first thoracic ct scan, but their second ct scan manifested abnormal changes ( figure 2 ). after reviewing initial ct image data, a total of 399 involved segments were found, including 186 segments (46.6%) in left lobes and 213 segments (53.4%) in the right lobes. in the right upper lobe, 22 lesions (5.5%) were located in the anterior segment, 30 (7.5%) in the posterior segment, and 18 (4.5%) in the apical segment. in the right middle lobe, 14 lesions (3.5%) were seen in the medial segment and 22 (5.5%) in the lateral segment. in the right lower lobe, 8 lesions (2%) were seen in the medial basal segment, 18 (4.5%) in the anterior basal segment, 30 (7.5%) in the lateral basal segment, 16 (4%) in the superior segment, and 35 (8.8%) in the posterior basal segment. in the left upper lobe, there were 22 lesions (5.5%) in the apicoposterior segment, 24 (6.0%) in the anterior segment, and 36 (9%) in lingual segment (including the superior and inferior segments). in the left lower lobe, there were 11 lesions ct is an important tool for identifying sars-cov-2-infected patients. ground-glass opacity with or without consolidation combined with bilateral, multilobe, and peripheral involvement of the lung was the main feature and distribution of covid-19 pneumonia. previous researches showed that which host is susceptible to sars-cov infection is mostly decided by the affinity between host angiotensin-converting enzyme 2 (ace2) and the viral receptor-binding domain (rbd) [7] . the recent studies show that the genome sequence of sars-cov-2 is quite similar to that of sars-cov but distant from mers-cov [8] [9] [10] . these findings indicate the following: (1) ace2 is used as a receptor by sars-cov-2, which is similar to sars-cov; (2) sars-cov-2 can infect the human cell; and (3) sars-cov-2 can spread from person to person [7] . ace2 is expressed in human airway epithelia, lung parenchyma, and the gastrointestinal tract, which are the major sites of replication of the virus [11, 12] . the lower respiratory tract symptoms (including fever, cough, and shortness of breath) are the major onset symptoms and are similar to those caused by sars-cov [7, 13] . diarrhea was also reported in the literature [14] , and there was one patient (1.9%) in our group. pan et al. reported that infected individuals can be infectious prior to the onset of symptoms [15] . in our group, 5 patients (9.4%) were asymptomatic at initial presentation but confirmed by viral nucleic acid test. this suggested that clinical symptoms are not essential components for identification or diagnosis. there was a slight predilection for males (male: 31 [58.5%]) and old age (>60 years in 30 [56.6%] patients) in our cohort, and this is similar to previous findings [16] . however, other studies showed no obvious sex predilection [17, 18] . this could be explained by different demographic features and the small size of our group. chest radiographs are less sensitive than a ct scan in detecting small lesions; thus, the ct scan is the first choice for initial identification [19] . most types of covid-19 pneumonia have abnormal radiographic changes at initial presentation, and ct features and patterns are similar to those of viral pneumonia. symptomatic patients without radiographic changes have been reported in the literature [18, 20] , and these patients are also found in our group (3 [5. 7%]), suggesting that there is an incubation period (1-14 days) prior to positive findings of the ct scan. negative findings of the ct scan could not exclude the infected patients. [20] . shi et al. reported that 79% of patients had bilateral lung involvement and 54% of patients showed peripheral distribution [17] . in our cohort, the involvement of 399 segments (left lobe: 186 [46.6%] seg, right lobe: 213 [53.4%] seg) at initial ct scan distributed bilaterally. the lower lobes (especially the anterior basal segment, lateral basal segment, and posterior basal segment) are the most commonly affected sites. this may be because of the anatomical structure of the trachea and bronchi-the bronchus of lower lobes is relatively straight, and the virus arrives more easily in the lower lobes [17] . here, 46 (86.8%) of our cases show multiple lobe involvement with lower lobe predominance. only 8 patients (13.2%) had infected single lobes, and of the 8 patients, 3 patients involved a single segment. our study shows that the distribution of lesions preferred to affect the peripheral zone, which is similar to those in a radiological study with sars and mers [21, 22] . this might be due to the viral capability of reaching the terminal bronchioles and alveoli. the extensive bilateral lung involvement, like sars-cov, might be consistent with high initial viral loads [15, 23] . ground-glass opacity with or without consolidation are main features of the disease. these features are highly suggestive of acute interstitial pneumonia of the disease and are consistent with its histopathological findings that covid-19 pneumonia involves both parenchyma and interstitial lung tissue [2] . these features could also be found in sars and mers. pure consolidation opacity at an initial ct scan is rare. other findings include interlobular and intralobular septum thickening, crazy-paving pattern, reticular pattern, air bronchogram, atelectasis, and bronchiectasis. when an inflammatory response occurred, lung macrophages reside in the lung interstitium, and alveoli play critical roles in initiating and maintaining inflammation [23] . a histopathological report of covid-19 pneumonia showed that lung tissue displayed pulmonary edema with hyaline membrane formation and lymphocytes predominated the infiltration of interstitial mononuclear inflammatory and alveolar damage with cellular fibromyxoid exudates [2] . these features are similar to those of sars and mers [24, 25] . these pathological findings are consistent with the features of ground-glass appearances, consolidation, interlobular and intralobular septum 7 biomed research international thickening, and crazy-paving pattern. the crazy-paving pattern was originally reported in patients with alveolar proteinosis. this pattern also could be found in other pulmonary diseases (such as usual interstitial pneumonia, pulmonary edema, and adult respiratory distress syndrome) that affect both the interstitial and airspace compartments [26, 27] . the reticular pattern is associated with intralobular lines or interlobular septum thickening indicating interstitial changes (interstitial inflammation or fibrosis). parenchymal bands were found in 6 (11.3%) patients. this reflects fibrosis and distortion of the lung architecture [26] . fibrosis may demonstrate in the late stage of sars or can be due to steroid therapy [28] . parenchymal bands manifested at the early ct images suggested the preexisting obsolete lesions. if the alveolar collapsed, then segmental atelectasis could be seen on ct images with a thick parenchymal band pointing to the hilus. an air bronchogram can be seen within ground-glass lesions and consolidation, suggesting unobstructed proximal airways. bronchiectasis is associated with dilatation of bronchioles suggesting fibrotic changes within the lesion. pleural effusion and lymphadenopathy were reported in the literature [17] , but they are rare features of sars and mers; only 1 patient showed pleural effusion in our cohort. it was hard to say if these changes were caused by sars-cov-2 infection because comorbid patients were not excluded in other studies, and iatrogenic reasons cannot be excluded either. no evidence of cavitation was seen, and the result is similar to recent literature and sars and mers pneumonia [20, 27, 29] . it is noticeable that, of the 5 asymptomatic infected patients, 3 patients had a clear exposure history. thus, an epidemiological survey of a patient is very important in the initial identification of a suspected patient. if a clear exposure history is obtained, then the patient should be quarantined for further examination. unlike patients in wuhan city, most our patients do not have a clear exposure history. thus, during the outbreak, a patient with symptoms of low respiratory infection should be isolated for chest ct scan and nucleic acid testing. repeat ct scan and nucleic acid testing might be done, because the negative result of both the ct scan and nucleic acid testing can be obtained at the initial time. in our experience, exposure history is the most important clue for identifying high-risk individuals. clinical diagnosis should combine with the patient's symptom, ct changes, and exposure history. none of these components could be used for diagnosing covid-19 alone. although a patient with exposure history may be asymptomatic and obtained negative results of ct findings and viral nucleic acid test at initial presentation, the potential infection cannot be totally excluded, and performing repeating ct scan and coronavirus rna test is needed. our study had several limitations. first, we had a small cohort, performed a single-center study, and excluded the comorbid patients. this may lead to selection bias. second, some of our patients had a ct scan with a thickness of 5 mm. this might overlook subtle changes in the lesion. in conclusion, covid-19 infection appears clinically milder than sars or mers in terms of severity and fatality, but stronger in terms of transmissibility. an exposure history is extremely important for identifying high-risk groups for quarantine and further examination, even if they are asymptomatic. chest ct scans are helpful in identifying the suspected patient, even though negative results may be obtained during the incubation period. if the features are consistent with viral pneumonia, then a viral nucleic acid test should be done, and these patients should be admitted to a hospital for isolated observation and further examination. ground-glass opacity and consolidation with multiple, bilateral, and lower lobe distribution are the main features of covid-19 pneumonia at initial ct scan. other ct findings are crazy-paving, bronchiectasis, air bronchogram, and atelectasis, which are similar to features found in sars and mers, and these features and patterns are nonspecific for diagnosing covid-19 pneumonia. viral nucleic acid test is the golden standard for confirmation, but it has high specificity for identifying covid-19 infection and relatively low sensitivity. thus, negative results will not exclude the highrisk group; repeated nucleic acid testing should be done. severe adult respiratory syndrome sars-cov: severe adult respiratory syndrome coronavirus mers: mideast respiratory syndrome mers-cov: mideast respiratory syndrome coronavirus pacs: picture archiving and communication system real-time fluorescence polymerase chain reaction aec2: angiotensin-converting enzyme 2 rbd: receptor-binding domain rna: ribonucleic acid. the data may be available upon email request. institutional review board approval was obtained. novel coronavirus (2019-ncov) situation report-1world health organizationjanuary pathological findings of covid-19 associated with acute respiratory distress syndrome covid-19) situation report-64world health organizationhttps novel coronavirus(2019-ncov) situation report-22world health pathogenicity and transmissibility of 2019-ncov-a quick overview and comparison with other emerging viruses national health commission of the people's republic of receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus genome composition and divergence of the novel coronavirus (2019-ncov) originating in china a pneumonia outbreak associated with a new coronavirus of probable bat origin diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus animal origins of the severe acute respiratory syndrome coronavirus: insight from ace2-s-protein interactions ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia clinical characteristics of coronavirus disease 2019 in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study viral load of sars-cov-2 in clinical samples clinical features of patients infected with 2019 novel coronavirus in wuhan, china radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study chest ct findings in coronavirus disease-19 (covid-19): relationship to duration of infection imaging profile of the covid-19 infection radiologic findings and literature review ct imaging features of 2019 novel coronavirus (2019-ncov) ct correlation with outcomes in 15 patients with acute middle east respiratory syndrome coronavirus a major outbreak of severe acute respiratory syndrome in hong kong anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection pulmonary pathological features in coronavirus associated severe acute respiratory 9 biomed research international syndrome (sars) histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study fleischner society: glossary of terms for thoracic imaging thin-section ct of severe acute respiratory syndrome: evaluation of 73 patients exposed to or with the disease late-stage adult respiratory distress syndrome caused by severe acute respiratory syndrome: abnormal findings at thin-section ct middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. jun zhou is the guarantor of integrity of the entire study; chao xiang and ji lu conceptualized and designed this study; chao xiang and changzhu chai performed the literature research; li guan collected covid-19 pneumonia cases and recorded the patient's clinical and radiographic data; cheng yang performed the 3d reconstruction; chao xiang and ji lu reviewed the conventional ct and 3dvr images and drafted the manuscripts; and jun zhou edited the manuscript. chao xiang and ji lu contributed equally to this work. key: cord-261633-r4qlbnc5 authors: xie, guo-hao; chen, qi-xing; cheng, bao-li; fang, xiang-ming title: defensins and sepsis date: 2014-08-19 journal: biomed res int doi: 10.1155/2014/180109 sha: doc_id: 261633 cord_uid: r4qlbnc5 sepsis is a leading cause of mortality and morbidity in the critical illness. multiple immune inflammatory processes take part in the pathogenesis of sepsis. defensins are endogenous antimicrobial peptides with three disulphide bonds created by six cysteine residues. besides the intrinsic microbicidal properties, defensins are active players which modulate both innate and adaptive immunity against various infections. defensins can recruit neutrophils, enhance phagocytosis, chemoattract t cells and dendritic cells, promote complement activation, and induce il-1β production and pyrotosis. previous publications have documented that defensins play important roles in a series of immune inflammatory diseases including sepsis. this review aims to briefly summarize in vitro, in vivo, and genetic studies on defensins' effects as well as corresponding mechanisms within sepsis and highlights their promising findings which may be potential targets in future therapies of sepsis. sepsis, severe sepsis, and septic shock represent a continuum of clinical syndromes which are common complications observed in patients with infection, trauma, and major surgeries [1] [2] [3] . these syndromes start with infection induced systemic inflammatory response syndrome (sirs) and evolve to sepsis induced acute organ dysfunction and cardiovascular collapse. epidemiology studies demonstrated that severe sepsis has a population prevalence of 300/100 000 in the united states and counts for 10-30% of the intensive care unit (icu) patients [4] [5] [6] . and severe sepsis has already been acknowledged as the first cause of death in noncoronary icus with a high mortality rate of approximately 25-50% [7] . in the past one or two decades, steady progresses in treatment of sepsis have been made due to the advanced supportive care in icu and the implementation of bundle therapies [7] . however, searching for specific remedies and reliable predictors within the pathophysiological mechanisms of sepsis is still the emphasis of today's studies [8, 9] . defensins are classified as a subfamily of cationic antimicrobial peptides, which are major components of the human innate immunity. they are small endogenous peptides with three disulphide bonds created by six cysteine residues. defensins are categorized into three subtypes, -, -, and -defensin, based on the spatial structure and the locations of three disulphide bonds within the peptide. in the past decade, cumulative evidences have suggested that defensins play an important role and may be a potential intervention target in sepsis. this review hereby will summarize in vitro, in vivo, and genetic studies on defensins' effects as well as corresponding mechanisms within sepsis and its sequential syndromes. defensins have broad spectrum antimicrobial activities against most pathogens in sepsis. [10] [11] [12] . they can inhibit a large variety of gram-positive bacteria, gram-negative bacteria, and some species of fungi and viruses [11] . the -defensins are mainly distributed in the epithelial cells of the respiratory system, digestive system, and genitourinary system [10] [11] [12] . they can effectively kill a number of gram-negative bacteria, such as e. coli and p. aeruginosa, gram-positive bacteria, such as s. aureus and streptococcus pyogenes, and candida albicans. -defensin-3 even has bactericidal effect towards multiresistant s. aureus and vancomycin-resistant enterococcus [11, 13] . the -defensins, which have a unique macrocyclic structure, are isolated from leukocytes from some species of monkey and have not been detected in humans [14] . they are also reported to have antimicrobial activity against a spectrum of pathogens including e. coli, s. aureus, and c. albicans [15] . also, they are found to have protective effect in a mouse model from a lethal pulmonary infection by a mouse adapted strain of sars-coronavirus [16] . the classic mechanism of defensins' bactericidal effect is the "pore formation" theory. these positively charged antimicrobial peptides target negatively charged bacterial membrane components, such as lipopolysaccharides, teichoic acids, or phospholipids. then they form transmembrane pores, disrupt cell integrity, and lead to bacteria lysis [10, 11] . recently, another mechanism has been reported that defensins kill bacteria by inhibiting the synthesis of bacterial cell wall through interaction with certain precursors such as lipid ii [17] . defensins' bactericidal effect can be limited by high salt concentration of local environment where they encounter with the pathogens [18, 19] . also, the antimicrobial action appears to be regulated by the redox response, as -defensin-1 become more potent after reduction of disulfide bridges by thioredoxin or a reducing environment [20, 21] . defensins are also reported to have modulating effects on both innate and adaptive immune response. it is well known that hnp1-3 participate in the host immune defense via multiple mechanisms, including enhancing macrophage phagocytosis, facilitating neutrophil recruitment, modulating complement activation, and chemoattracting immature t cells and dendritic cells [12, 22] . in vitro studies showed that -defensins have potent chemotactic effects, leading to the recruitment and maturation of naive dendritic cells and memory t cells in the inflammatory sites and the triggering of specific immune response in the host [23] . as the endogenous ligand of tlr-4, -defensins interact with tlr-4 of the immune cells and regulate the expression of inflammatory mediators via the nf-b pathway [18] . in vivo researches have revealed that the abnormal expression of -defensins is associated with sepsis and various infectious diseases, as levels ofdefensins in both plasma and bronchoalveolar lavage fluid in patients with pulmonary infections are elevated [24] [25] [26] , transcription of -defensin-2 in leukocytes of severe septic patients is suppressed [27] , expression of -defensins in burn wound is reduced [28] , and impaired expression of -defensins is associated with inflammatory bowel diseases [29, 30] . in a mouse model of acute lung injury, shu et al. expressed recombinant -defensin-2 in lung tissue via recombinant adenovirus to study its protective effect against p. aeruginosa infection. compared with control mice, they found considerably less p. aeruginosa in the transinfected lung tissue, as well as alleviated alveolar impairment, interstitial edema, and neutrophil infiltration [31, 32] . in subsequent studies, mice transinfected by adenovirus with or withoutdefensin-2 genes received cecal ligation and puncture (clp) twice to generate sepsis models. the impact of -defensin-2 on the inflammatory response (e.g., the level of icam-1 expression), the severity of lung injury, and the sepsis outcome (7-day survival rate) were observed and evaluated. it was found that recombinant -defensin-2 could downregulate the expression of icam-1 in lung tissue 24 h, 36 h, and 72 h after clp and significantly raised the 7-day survival rate in sepsis mice [31, 33] . in the clinical setting, olbrich et al. found preterm neonates had lower level of -defensin-2 in cord blood when compared to term neonates [34] . and among these preterm neonates, lower -defensin-2 level was associated with late-onset sepsis. these studies indicate that -defensin-2 may play an important role in the immune inflammatory response in sepsis and might influence the outcome of sepsis. among the -defensins, rhesus macaque -defensin (rtd), which has six subtypes, has been extensively studied. though not expressed in humans, rtds were reported to significantly reduce levels of tnf-, il-1 , il-6, il-8, mip1, and so on, in human peripheral blood leukocytes that are preincubated with various toll-like receptor agonists [35] . furthermore, in vivo study showed that subcutaneously administration of 5 mg/kg rtd-1 could improve the survival rate and suppress the levels of a number of inflammatory cytokines and chemokines in two sepsis mouse models (received either intraperitoneal injection of e. coli or clp). although detailed mechanisms of the protective effect of rtd-1 have not been illuminated, the authors suggested that the interaction between rtd-1 and leukocyte is the critical determinant of tnf-blockade [35] . the latter is a major proinflammatory cytokine and influences the consequent inflammatory cascades. these results indicate that -defensins may be a potential immune adjuvant in the treatment of sepsis, though they are not expressed in human. in sepsis and other inflammatory disorders, defensins are among a group of rapidly-released host endogenous molecules, which are capable of both recruiting and activating apcs and are also termed the alarmins. recently, in vitro studies have shown that alarmin hnp1-3 have the ability to boost host inflammatory response by promoting macrophage il-1 production and pyroptosis via purinergic p2x7 receptor [36] . however, this effect is a double-edged sword in sepsis since it can promote pathogen elimination as well as mediate organ dysfunction such as acute lung injury [22, 37] . in molecular genetics and molecular biology, knock-out animal model is one of the most convincing means to determine the role of a specific molecule in the physiopathology of a certain disease. however, as members of the defensin family have overlapped biological functions, the function of the knock-out gene in animal models may probably be compensated by other defensins. since the gene cluster coding for the entire defensin family cannot be fully knocked out using the present techniques of molecular biology and genetics as well as human defensins lack of absolute animal analogues, genetic association analysis is a good alternative that can effectively explore the relationship between genetic polymorphism and sepsis. in normal peripheral blood cells, mrna levels of both -defensin-1 and -defensin-2 raise remarkably when stimulated by lps or p. aeruginosa [23] . however, the upregulation of -defensin-1 and -defensin-2 varies among individuals, resulting in interindividual differences in host defense capacity and hence influencing the clinical progression of sepsis. previous studies showed that single nucleotide polymorphism (snp) of -defensin-1 gene (defb1) correlates with chronic obstructive pulmonary disease, asthma, genetic allergy, hiv infection, and pseudomonas species infection in oral mucosa [38] [39] [40] [41] [42] . since sepsis is a multifactorial disease caused by both environmental factors (pathogenic microbes) and host factors (comorbidities and genetic background), its occurrence and outcome are influenced with individual genetic background [43] . chen et al. selected 5 snps in the promote region of defb-1 (-1816a/g, -390a/t, -52a/g, -44c/g, and -20a/g) and one in its extron (1654g/a) as candidate loci and studied 211 patients with severe sepsis and 157 healthy controls [44] . distribution of alleles, gene types, and haplotypes associating with these loci were studied and compared between septic patients and controls, as well as between survivals and victims of severe sepsis. association analysis, logistic regression, and linkage disequilibrium study showed that -44g allele was closely related with susceptibility to severe sepsis and poor outcome. and severe septic patients with haplotype -20g/-44g/-52g had even poorer outcome, while individuals with haplotype -20a/-44c/-52g were less susceptible to severe sepsis. the reason why -44c/g is correlated with the occurrence and outcome of severe sepsis may attribute to the following points. it located in the 5 untranslated region of defb1 and its polymorphism may result in changes in the space conformation of mrna, which would alter the stability of mrna and the efficiency of translation. and its impact on the protein function is more significant than nonsynonymous snp in coding region [45] , as the quantity of protein would change dramatically. however, as any other genetic association analysis, defb1 −44c/g may be only a surface marker of some unknown real genetic marker of sepsis in linkage disequilibrium. although these hypothesis need to be proved by further researches, the above-mentioned study indicated that -defensin-1 might be an influential factor in the process of immune defense and inflammation regulation in sepsis, and the locus of −44c/g may be an important genetic warning indicator of susceptibility to severe sepsis and its outcomes. copy number variation (cnv) is a kind of genetic polymorphism that accounts for approximately 12% of human genomic dna. it refers to a large-scale duplication or deletion of certain dna sections, which causes a variation in the number of copies of one or more genes. previous publications reported that cnv is present in -defensin-2 gene (defb4), -defensin-3 gene (defb103), -defensin-4 gene (defb104), -defensin-1 gene (defa1), and -defensin-3 gene (defa3) [18, [46] [47] [48] . and copy number of defb4 has a positive correlation with its mrna level [35, 45] . recently, chen et al. screened 179 severe sepsis and 233 healthy controls for defa1 and defa3 [49] . an average defa1/defa3 copy number of 7 per genome was observed in the studied population, with a range of 2 to 15. the authors found that patients with high copy number of defa1/defa3 were predisposed to severe sepsis and tended to have lower level of plasma hnp1-3 as well as cytokines such as tnf-, il-6, and il-10. they further validated their findings in an independent cohort. these results indicated that cnvs in the defensin gene may be potential genetic markers for identifying high risk patients or providing individual treatment in sepsis. defensins are emerging therapeutic molecules against pathogens in sepsis because of their broad spectrum antimicrobial properties. in the past decade or two, a number of potent and salt insensitive defensins and their analogs have been screened, structurally modified, and synthesized. however, most of these studies are performed in vitro and not much is known about the in vivo roles of these molecules. in fact, chemoattracting and immunomodulating effects make defensins a double-edged sword in the pathogenesis of sepsis, which leads to facilitation of pathogen clearance as well as exacerbation of inflammation and injury of self-tissues. recently, several investigations showed that the chemoattractant and antimicrobial activities of defensins could be separated, which shed light on the design of defensin-derived pharmaceuticals [50] . in addition, genetic studies help identify high risk patients with susceptibility to sepsis or its adverse outcome, which provides foundation for future individualized sepsis treatments that are targeting defensins. the pathophysiology 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human lung inducibility of the endogenous antibiotic peptide -defensin 2 is impaired in patients with severe sepsis reduced antimicrobial peptide expression in human burn wounds a chromosome 8 gene-cluster polymorphism with low human geta-defensin 2 gene copy number predisposes to crohn disease of the colon hammarström, " -defensin-3 and -4 in intestinal epithelial cells display increased mrna expression in ulcerative colitis protection against pseudomonas aeruginosa pneumonia and sepsis-induced lung injury by overexpression of -defensin-2 in rats the protective effect of beta-defensin 2 in acute lung injury induced by respiratory pseudomonas aeruginosa infection in rats the effect of pretreatment of recombinant beta-defensin 2 on the icam-1 expression in lung of rats with acute lung injury association of human beta-defensin-2 serum levels and sepsis in preterm neonates rhesus macaque theta defensins suppress inflammatory cytokines and enhance survival in mouse models of bacteremic sepsis alarmin hnp-1 promotes pyroptosis and il-1 release through different roles of nlrp3 inflammasome via p2x7 in lps-primed macrophages neutrophil alpha-defensins cause lung injury by disrupting the capillaryepithelial barrier genetic variants of human -defensin-1 and chronic obstructive pulmonary disease association of defensin beta-1 gene polymorphism with asthma a single-nucleotide polymorphism in the human beta-defensin 1 gene as associated with hiv-1 infection in italian children single-nucleotide polymorphisms (snps) in humandefensin 1: high-throughput snp assays and association with candida carriage in type i diabetics and nondiabetic controls contribution of alpha-and beta-defensins to lung function decline and infection in smokers: an association study genomic polymorphisms in sepsis genomic variations within defb1 are associated with the susceptibility to and the fatal outcome of severe sepsis in chinese han population human catechol-o-methyltransferase haplotypes modulate protein expression by altering mrna secondary structure extensive normal copy number variation of a -defensin antimicrobialgene cluster copy number polymorphism and expression level variation of the humandefensin genes defa1 and defa3 screening of copy number polymorphisms in human -defensin genes using modified real-time quantitative pcr increased genomic copy number of defa1/defa3 is associated with susceptibility to severe sepsis in chinese han population analysis and separation of residues important for the chemoattractant and antimicrobial activities of beta-defensin 3 the authors declare that there is no conflict of interests regarding the publication of this paper. this work was supported by the national natural science foundation of china (no. 81201495) and the research project of the department of education of zhejiang province (no. y200909678). key: cord-352190-1987sfyz authors: xia, hongyue; li, xibao; zhao, wenliang; jia, shuran; zhang, xiaoqing; irwin, david m.; zhang, shuyi title: adaptive evolution of feline coronavirus genes based on selection analysis date: 2020-08-13 journal: biomed res int doi: 10.1155/2020/9089768 sha: doc_id: 352190 cord_uid: 1987sfyz purpose: we investigated sequences of the feline coronaviruses (fcov), which include feline enteric coronavirus (fecv) and feline infectious peritonitis virus (fipv), from china and other countries to gain insight into the adaptive evolution of this virus. methods: ascites samples from 31 cats with suspected fip and feces samples from 8 healthy cats were screened for the presence of fcov. partial viral genome sequences, including parts of the nsp12-nsp14, s, n, and 7b genes, were obtained and aligned with additional sequences obtained from the genbank database. bayesian phylogenetic analysis was conducted, and the possibility of recombination within these sequences was assessed. analysis of the levels of selection pressure experienced by these sequences was assessed using methods on both the paml and datamonkey platforms. results: of the 31 cats investigated, two suspected fip cats and one healthy cat tested positive for fcov. phylogenetic analysis showed that all of the sequences from mainland china cluster together with a few sequences from the netherlands as a distinct clade when analyzed with fcov sequences from other countries. fewer than 3 recombination breakpoints were detected in the nsp12-nsp14, s, n, and 7b genes, suggesting that analyses for positive selection could be conducted. a total of 4, 12, 4, and 4 positively selected sites were detected in the nsp12-nsp14, s, n, and 7b genes, respectively, with the previously described site 245 of the s gene, which distinguishes fipv from fecv, being a positive selection site. conversely, 106, 168, 25, and 17 negative selection sites in the nsp12-14, s, n, and 7b genes, respectively, were identified. conclusion: our study provides evidence that the fcov genes encoding replicative, entry, and virulence proteins potentially experienced adaptive evolution. a greater number of sites in each gene experienced negative rather than positive selection, which suggests that most of the protein sequence must be conservatively maintained for virus survival. a few of the sites showing evidence of positive selection might be associated with the more severe pathology of fipv or help these viruses survive other harmful conditions. feline coronaviruses (fcov) belong to the genus alphacoronavirus within the subfamily coronavirinae of the family coronaviridae in the order nidovirales [1] . fcov include the feline enteric coronavirus (fecv) and the feline infectious peritonitis virus (fipv) [2] . similar to other coronaviruses, such as the sars and mers viruses, fipv infections are distributed worldwide and can cause a fatal pathogenic disease fip in their hosts, thus, seriously endangering the life and health of cats [3] . however, the more common variant, fecv, causes an asymptomatic or mild enteric infection [3] . fipv and fecv are antigenically divided into two types (i and ii) based on a difference in the nucleotide sequence of the s gene, which encodes the spike protein [4] . most natural cases of feline coronavirus infection are type i fcov; however, these viruses poorly propagate in cell culture, whereas type ii fcov viruses can grow in several different cell lines [5] . the pathogenesis of fip is not yet completely understood. it has been suggested that large viral quasispecies of fcov, which are due to copying errors of its rna genome, may destroy a weak immune system found in some individuals leading to fip [6, 7] . the coronavirus spike (s) glycoprotein is a typical class 1 viral fusion protein and plays a central role in receptor binding and viral entry [8] . in addition to the s gene, there are several other genes in the fcov genome. nonstructural protein nsp12 encodes the rna-dependent rna polymerase protein, nsp13 encodes the helicase protein, and nsp 14 encodes the exoribonuclease protein, which are all essential for genome replication. the n gene encodes the nucleocapsid, which is commonly used in phylogenetic analysis [9] . the 7b gene is a small orf that is located downstream of the n gene and is important for virulence [10] . compared to the more predominant type i fcov, type ii fcov viruses are only found in 2-30% of infections [11] . at present, a high incidence of type i fcov occurs in europe, japan, australia, korea, and the usa.however, fipv cases in japan and taiwan are more frequently associated with type ii fcov [5, 12] , suggesting a difference in the geographical distribution of the different serotypes of fcov. to date, almost all fcov strains isolated from china are type i [13] . the goal of our study is to increase the sampling of fcov in china and to also examine the selective pressures acting on the genes of these viruses isolated from different parts of the world. to study this, we obtained ascites samples from 31 cats with suspected fip as well as feces samples from 8 healthy cats. these samples were collected at several pet hospitals in liaoning province, china, during the period october 2017 to may 2019. of these 39 samples, 3 were found to be positive for fcov. the adaptive evolutionary properties of fcov, including selective pressure, were systematically analyzed using paml and datamonkey for the key fcov functional proteins involved in viral entry, 1 replication, and virulence. the aim of this study was to provide insight into the adaptive evolution of fcov, which might provide insight into their pathogenic mechanisms. 2.1. sampling. samples were collected from veterinary hospitals in liaoning province, china, between october 2017 and may 2019. a total of 39 samples were obtained, with 31 being from cats with suspected fip, as they had clinical symptoms such as loss of appetite and weight and increased abdominal girth with peritoneal effusion and/or pleural effusion [11] that are associated with this disease. some, but not all, of the cases were examined for clinical hematologic and biochemical analysis. effusions were collected by needle and syringe puncture guided by ultrasound. in addition, 8 fresh feces samples were collected from healthy cats using anal swabs, which were suspended in pbs and then stored at -80°c. 2.2. viral rna and reverse transcription. viral rna was extracted from 140 μl of effusion or feces suspension with the qiaamp viral rna minikit (qiagen, shenyang, china), following the manufacturer's instructions, and stored at -80°c. extracted rna was used as the template for cdna synthesis with primescript™ ii 1st strand cdna synthesis kit (takara, china) with random hexamers, following the manufacturer's instructions. 2.3. pcr, cloning, and sequencing. to amplify the s gene, we designed primers based on available feline coronavirus sequences. the partial s gene was divided into two segments, with primers designed separately for each part. the best primers and reaction conditions for each pcr reaction were selected using gradient pcr. primer pairs s2b2-f and s2b2-r and sc1-f and sc1-r were used to amplify the 5 ′ and 3 ′ ends of the s gene, respectively. pcr was performed using 2 μl of cdna in a 10 μl reaction containing 1 mmol/l concentration of each primer and 5 μl of 2x es taq mastermix (beijing comwin biotech co., ltd.). to amplify partial nsp12, nsp13, and nsp14 gene sequences, we designed primer pairs 1b3f and 1b1r and 1b6f and 1b6r. to amplify part of the n gene, primer pair n1 and n2 was used [14] . to amplify the 7b gene, the previously designed primer pair 7b-f1 and 7b-r1 was used [3] . all pcr reactions had the same preheating temperature of 94°c and an extension temperature of 72°c. annealing temperatures of the different amplifications are listed in table 1 . products of the amplifications were separated by electrophoresis, with dna from appropriate bands extracted, cloned, and sequenced (sangon biotech, shanghai, china) to confirm virus detection. as previously described [15] , type i fcov is entirely a feline virus; however, type ii fcov is a recombinant of type i fcov and a canine coronavirus (ccov) that resulted in a fcov genome containing the s gene and parts of the adjacent genes from ccov. this results in the recombinant type ii fcov s gene having a different size from the type i s gene. thus, amplification of the s gene allows typing of the fcov virus. studies. in addition to the fcov genomes sequenced in this study, genome sequences that contained all of the 5,895 bases that we amplified in our sequences and represent the diversity found in several select countries (united states, united kingdom, netherlands, and belgium) were downloaded from the genbank database for analysis. the accession numbers for these sequences are listed in supporting information table s1 . to establish the phylogenetic relationships of these viruses, conserved regions of the nsp12, nsp13, nsp14, s, 7b, and n genes were concatenated into a single sequence. from the 24 fcov sequences from mainland china and other countries, an alignment of 5,895 bases was generated using clustalw as implemented in mega 6.0. bayesian phylogenetic trees, based on the nucleotide sequences, which were constructed using mrbayes 3.1 with 5,000,000 generations, sampled every 100 generations, using the commands mcmcp samplefreq=100, a burnin of 20,000 generations, 4 chains, and the best-fit substitution model gtr+i+g applied, which had been selected using jmodeltest [16] . canine cov (ccov) sequences (accession numbers kc175339.1 and jq404410.1) were used as the outgroup to root the trees. since recombination can influence the detection of positive selection, we assessed whether recombination had occurred within our aligned dataset using the gard (genetic algorithm for recombination detection) method [17] . a model selection procedure was run for each gene (the nsp12, nsp13, and nsp14 genes were considered to be a single gene), which sifts through all 203 possible timereversible models in a hierarchical testing procedure combining nested lrt tests with aic selection to pick a single "bestfitting" rate matrix, with site-to-site rate variation accounted for by the β-γ distribution [17] . the best-fitting substitution models for the s gene was trn model; however, there was no name of best model for the other three genes, and serial number of the best-fitting substitution models for nsp12-14, n, and 7b gene was (010230) with aic of 20622.10, (010230) with aic of 5874.10, and (012232) with aic of 5108.58, respectively. to detect the presence of positive selection in the fcov sequences from the different countries, we applied the branch, site, and branch-site tests from the paml suit [18] . values of ω (the nonsynonymous/synonymous rate ratio) greater than 1 suggest positive selection. the p values can be calculated through likelihood ratio tests (lrt), where the null hypothesis would be rejected if the p value is <0.05 when the model allows positive selection. the branch model detects positive selection acting on particular lineages [19, 20] . a variety of models, including one ratio, free ratio, and two ratios (where the foreground lineages should be labeled), were analyzed. comparing the free ratio and one ratio models examines whether the ω ratios differ among lineages. comparing the two ratio and free ratio examines whether the ω ratios are different between the foreground and background lineages. the site models allow different ω ratios among sites [21, 22] . the one ratio model m0 assumes that the same ω exists for all sites across the phylogeny, while the nearly neutral model m1, positive selection model m2, discrete model m3, beta model m7, and beta and ω model m8 assume 2, 3, 3, 10, and 11 classes of codons, respectively, with different ω values, including some that suggest positive selection. if a site class with ω greater than one is found, which suggests positive selection, then sites with evidence of positive selection and a posterior probability p > 95% level were identified. posterior probabilities were calculated by naïve empirical bayes (neb) and the bayes empirical bayes (beb) [21] . selection pressure was also analyzed through the datamonkey suite of programs, including fixed effects likelihood (fel; p < 0:05), random effects likelihood (rel; bayes factor > 50), and mixed-effects model of evolution (meme; p < 0:05). we considered a site to be under positive or negative selection when detected as such by at least two different methods. fcov. these three positive cats were derived from different households. all of the positive cases were classified as type i fcov, as the amplified s gene products had a size that was expected for type i, rather than type ii fcov. symptoms such as fever, anorexia, loss of weight, panting, and abdominal extension were observed in the suspected cases, and the two fip-positive cats subsequently died within 10 days of admission to the hospital, while the healthy cat remained asymptomatic. nsp12, nsp13, nsp14, s, 7b, and n gene clones were obtained from these three positive samples for sequencing and were used in the following analyses. accession numbers for the fcov sequences obtained in this study are provided in supporting information table s2 . analysis. an alignment of 5,895 bases, containing the partial nsp12, nsp13, nsp14, s, and n genes and the complete 7b gene sequence, was used for the phylogenetic analyses, with canine sequences used to root the tree. this analysis showed that our new fcov sequences from mainland china clustered together previously characterized sequences from china, as well as a few sequences from the netherlands as a distinct clade, separate from those of other countries (figure 1 ). the analysis identified two additional clades, one composed of sequences only from the united kingdom and a second composed of sequences from belgium, united states, and the netherlands (figure 1) . apart from the clustering of the sequences from the united kingdom, no clear geographic separation of fcov sequences can be observed for the other two clades. [23] , we tested our fcov sequences for evidence of recombination using gard with kh testing [17] . the results of this analysis for each gene is shown in table 2 . gard detected evidence for one breakpoint within the nsp12-nsp14 genes and 2 within each of the s, n, and 7b genes. however, of these potential breakpoints, only those at locations 1020 (nsp12-nsp14), 360 and 582 (both n), and 434 (7b) were significant supported by the kh test, with p values < 0.05. as only a few recombination breakpoints were reliably detected by gard, this suggests that recombination has little effect on our sequences and should not affect the detection of sites under positive selection [23] . 3.4. measurement of selection pressure. we used 5 methods to identify sites within the fcov sequences with evidence for positive selection. the results of these analysis are shown in table 3 . for the nsp12-nsp14 gene region, four positively selected sites (6, 488, 562, and 631) were identified by at least two methods showing significance at p < 0:05, bayes factors > 50, or posterior probabilities > 95% (table 3 ). site 562 in the nsp12-nsp14 genes showed evidence of positive selection with methods from both paml and datamonkey, while the three remaining sites in this gene fragment were only detected by two or more methods from datamonkey. a total of twelve positively selected sites (22, 43, 101, 149, 151, 166, 167, 172, 175, 229, 245, and 475) were found in the s gene that were identified by at least two methods. sites 151, 172, 175, and 245 showed evidence for positive selection from methods using both paml and datamonkey (table 4) , while the other eight sites were detected by methods from only one platform (either paml or datamonkey) ( table 4) . for the n gene, only one positively selected site, position 21, was identified by methods from both paml and datamonkey, while three others (13, 52, and 195 ) were identified only with methods from paml (table 5 ). three positively selected sites (41, 149, and 187) in the 7b gene were detected by methods from both paml and datamonkey (table 6) , with one site (5) showing evidence for positive selection only with methods from paml (table 6 ). in addition to positive selection, the fel and rel methods can both identify sites with evidence of negative selection sites. using these methods, we identified 106, 168, 25, and 17 negative selection sites in the nsp12-14, s, n, and 7b genes, respectively, which had significant evidence by both methods (supporting information tables s3-s10). when the branch and branch-site models of paml were applied to the alignments, no evidence for positive selection was found for any branch in the phylogenies. to study the evolution of feline cov (fcov) in china, we collected a total of 31 samples from cats, with 3 of these 31 samples testing positive for type i fcov. the identification of type i fcov in cats in liaoning province, china, is in line with a previous study that showed that type i fcov is most prevalent in cats in china [13] . as the occurrence of fip is associated with fecv, which results from fecv mutating into fipv [24] [25] [26] , we selected both fipv and fecv sequences from several other countries for our phylogenetic analysis of our fcov sequences. phylogenetic analysis of the concatenated gene sequences obtained in this study yields some insight into the regionalism of type i fcovs. all fcov sequences from mainland china cluster together with a few sequences from the netherlands. the biomed research international clustered together with other fipv sequences of china; however, it was an early diverging lineage in this clade ( figure 1) ; thus, the other fipv sequences from china possibly contain additional disease-causing mutations. sequences from the united kingdom cluster together as a separate clade. the third cluster of fcov sequences include isolates from belgium, united states, and the netherlands. apart for the united kingdom clade, an obvious geographical separation of the fcov sequences is not observed, which might be a consequence of the trade in cats and tourism between countries. our analysis shows that fcov can be transmitted from one country to another, although a particular geographic position, such as the united kingdom locates in an island, might limit its spread. the datamonkey suite of programs can identify codons under selection as well as the presence of recombinant sequences within a dataset [27] . ideally, we should first determine whether recombination has occurred among the gene sequences, which can be assessed using gard [17] . although a breakpoint was identified within the nsp12-14 gene segment and 2 more within the other three genes examined, the number of breakpoints is less than three and thus likely would have little effect on the identification of sites experiencing positive selection by empirical bayes methods [23] . in addition, it is known that the type ii fcov emerged via double recombination between type i fcov and type ii ccov and that recombination events in type i fcov have rarely been reported [28] . previous studies have shown that the transmission of natural recombinant strain rarely occurs; thus, we should be cautious in concluding putative recombination event based on these computational analyses [29] . although none of the branches of the fcov phylogeny from different countries investigated here showed evidence of positive selection, when site models were applied, four sites in nsp12-14, ten sites in the s gene, 4 sites in the n gene, and 4 sites in the 7b gene were detected as having evidence for positive selection. site 245 of the s gene, which previously had been shown to distinguishes fipv from fecv [30] , was found to be a positive selection site. both the fel and rel methods, from the datamonkey suite of programs, can identify sites experiencing negative selection. from our analyses, 106, 168, 25, and 17 negative selection sites were identified by both fel and rel in the nsp12-14, s, n, and 7b genes (supporting information tables s3-s10). viruses can experience both positive and negative selection. positive selection leads to an increase in the abundance of a specific genetic variant, while negative selection results in genetic conservation [31] [32] [33] . a larger number of sites experiencing negative rather than positive selection were found for all of the genes that we analyzed, which suggests that most sites are conservative, and only a few can be adapted. the proteins encoded by the nsp12-14 genes are responsible for viral genome replication, with the s gene product responsible for viral entry into host cells, the n gene product responsible for the virus nucleocapsid protein, and the 7b gene product is responsible for viral virulence. negative selection in these genes indicates that they are essential to the virus. the few positively selected changes that have occurred likely help these viruses survive and adapt under harmful conditions, with the change at site 245 of the s gene due to positive selection potentially related to the highly virulent fipv of the virus [30] . positive selection at other sites may contribute to the entry into host cells and enhanced virulence of viruses. by analyzing the selective pressure experienced by genes in the fcov genome involved in replication, entry, and virulence, we have identified a few sites that potentially experienced adaptive evolution. as negative selection occurs at a higher rate than positive selection within the fcov genes, this suggests that only a few sites can beneficially adapt to allow greater infectivity in the host and that most sites are under strong negative selection to conserve function. a few of the positively selected sites in fcov might be associated with the occurrence of fipv and lead to these viruses causing a more fatal disease. additional experimental analysis needs to be conducted to better understand the consequences of the positively selected changes identified here. all virus data obtained or analyzed during this study are included in this published article. sequences of the obtained viruses have been uploaded in genbank with accession numbers in supplementary materials table s2 . the authors declare that they have no conflict of interests. the ministry of science and technology of the people's republic of china (the national key research and development program, number 2016yfd0500300) and the educational department of liaoning province of china (climbing scholar). table s1 : other coronavirus isolate sequences used in this study. table s2 : accession numbers of the coronavirus isolate sequences obtained in this study. table s3 : negative selection sites for the nsp12, nsp13, and nsp14 genes based on rel analysis. table s4 : negative selection sites for the s gene based on rel analysis. table s5 : negative selection sites for the n gene based on rel analysis. table s6 : negative selection sites for the 7b gene based on rel analysis. table s7 : negative selection sites for the nsp12, nsp13, and nsp14 genes based on fel analysis. table s8 : negative selection sites for the s gene based on fel analysis. table s9 : negative selection sites for the n gene based on fel analysis. mutations of 3c and spike protein genes correlate with the occurrence of feline infectious peritonitis feline coronavirus 3c protein: a candidate for a virulence marker? genetics and pathogenesis of feline infectious peritonitis virus feline infectious peritonitis virus with a large deletion in the 5′-terminal region of the spike gene retains its virulence for cats isolation and molecular characterization of type i and type ii feline coronavirus in malaysia quasispecies composition and phylogenetic analysis of feline coronaviruses (fcovs) in naturally infected cats persistence and evolution of feline coronavirus in a closed catbreeding colony genotyping coronaviruses associated with feline infectious peritonitis 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protein fusion peptide and feline coronavirus virulence predicting adaptive evolution phylogenetics by likelihood: evolutionary modeling as a tool for understanding the genome codon-substitution models for detecting molecular adaptation at individual sites along specific lineages we thank the staff in the petmate pet hospitals, without whose help, the samples could not be successfully obtained for this study. we also thank dr. xianchun tang for the technological support. this work was supported by grants from hx contributed to the design of research and participated in the analysis and interpretation of data. hx, xl, wz, sj, and xz performed the experiments. hx wrote the draft manuscript and dmi modified the manuscript. sz designed the study and supervised the work. all authors read and approved the final manuscript. key: cord-321953-yql6gpd3 authors: barrera, maritza; garrido-haro, ana; vaca, maría s.; granda, danilo; acosta-batallas, alfredo; pérez, lester j. title: tracking the origin and deciphering the phylogenetic relationship of porcine epidemic diarrhea virus in ecuador date: 2017-12-12 journal: biomed res int doi: 10.1155/2017/2978718 sha: doc_id: 321953 cord_uid: yql6gpd3 in 2010, new chinese strains of porcine epidemic diarrhea virus (pedv), clinically more severe than the classical strains, emerged. these strains were spread to united states in 2013 through an intercontinental transmission from china with further spreading across the world, evidencing the emergent nature of these strains. in the present study, an analysis of pedv field sequences from ecuador was conducted by comparing all the pedv s gene sequences available in the genbank database. phylogenetic comparisons and bayesian phylogeographic inference based on complete s gene sequences were also conducted to track the origin and putative route of pedv. the sequence from the ped-outbreak in ecuador was grouped into the clade ii of pedv genogroup 2a together with other sequences of isolates from mexico, canada, and united states. the phylogeographic study revealed the emergence of the chinese pedv strains, followed by spreading to us in 2013, from us to korea, and later the introduction of pedv to canada, mexico, and ecuador directly from the us. the sources of imports of live swine in ecuador in 2014 were mainly from chile and us. thus, this movement of pigs is suggested as the main way for introducing pedv to ecuador. porcine epidemic diarrhea (ped) is an acute and highly contagious disease, which causes severe enteritis, vomiting, watery diarrhea, dehydration, and high mortality rates in pigs [1] . ped was first described in 1971 in united kingdom, affecting fattening pigs [2] , and the etiological agent was identified in belgium as a new coronavirus, which was designated as ped virus (pedv) [3] . pedv belongs to the genus alphacoronavirus of the family coronaviridae, subfamily coronavirinae, and order nidovirales. pedv genome consists of 28 kb long single-stranded rna with positive polarity and includes seven known open reading frames (orfs). two large orfs, 1a and 1b, occupying two-thirds of the genome, encode two nonstructural polyproteins (pp1a and pp1b) that direct genome replication and transcription. the remaining one-third of the genome encodes four structural proteins, spike (s), envelope (e), membrane (m), and nucleocapsid (n), and one hypothetical accessory protein encoded by the orf3 gene [4] . of all viral proteins, the pedv s protein has a pivotal function regulating interactions with specific host-cell receptor glycoproteins to mediate viral entry [5] . therefore, pedv s protein has been often used to understand the genetic relationships between different pedv strains and the epidemiological status of pedv in the field (reviewed in [4] ). other genes including orf3, e, m, or n have been also utilized for phylogenetic inference [6] [7] [8] [9] and some studies have included pedv full genome sequences to get better phylogenetic resolution [10, 11] . however, realistically, sequencing the full genome of pedv is still expensive, from both computational and laboratory perspectives. moreover, for many computationally intensive analyses, utilizing the full genome is unfeasible. it would be, therefore, beneficial to use only those genomic regions that contain the highest phylogenetic signal to reduce cost without losing valuable information [12] . in fact, phylogenetic markers together 2 biomed research international with powerful bayesian phylogenetic approaches have been successfully applied to track the origin of important viral outbreaks [13, 14] and establish molecular epidemiology links among different viral strains including coronavirus members [15] . for pedv it was recently shown that the s and nsp3 genes contain the lowest phylogenetic noise; therefore both are the highest recommended for phylogenetic analysis and molecular characterization studies [11] . in 2010, new chinese strains of pedv clinically more severe than the classical strains emerged [16] . these strains were spread to united states in 2013 through an intercontinental transmission from china [17] with further spreading to canada [18] , mexico, and other countries into the american continent including colombia and dominican republic (reviewed in [1] ). moreover, in europe, the recent reports in germany [19] , france [20] , and belgium [21] of ped-outbreaks caused by strains closely related to the variants identified in china and the united states evidence the emergent nature of these strains. the epidemiological situation regarding pedv turned more complicated since in december 2013 a second pedv strain oh851 (later named as s-indel strain) emerged in the us [22] . this new pedv strain was also reported in spring 2014 in germany [19, 23] , as a consequence of a probable single or simultaneous introduction [24] and with later reports in other european countries including france [20] , belgium [21] , italy [25] , austria [26] , and spain [27] . even though the pedv s-indel strains have shown lower virulence in the field [28] the clinical manifestations of the disease in the european countries affected have been very variable, with ranges of mortality between 0 and 70% (reviewed in [29] ). this recent global reemergence of ped requires urgent attention and deeper understanding of pedv epidemiological links driving the changes in viral pathogenicity. therefore, this report was conducted using the complete sequence of the s gene and powerful bayesian phylogeographic reconstructions to clarify the putative origin of pedv in ecuador, revealing the wide expansion of the emergent pedv strains, which caused the first pedv outbreak in this country. international standards for animal welfare were used for all animal samples collected, following the regulations for animal sampling of the article number 134 of animal welfare law included into the national constitution of the republic of ecuador. , and processing of samples. on july 2014, a clinical outbreak with epidemiological characteristics compatible with pedv was reported in a pig farm located in the province of cotopaxi, ecuador. in detail, the commercial farm contained a total of 10,909 animals from which 1,341 animals were affected, showing clinical signs compatible with ped, 1,043 died as a consequence of the disease, and 1,401 were slaughtered as control measure. to avoid further spreading of the disease a national contingency plan was applied including restrictions on the animal movement, increasing the biosafety measures, and establishment of epidemiological surveillance at national level. from the ped-outbreak a viral isolation was obtained and named pedv/cotopaxi/2014. a total of five fragments of ileum from three different diarrheic pigs were taken together with the viral isolate pedv/ cotopaxi/2014 for total rna isolation from mucosal scrapings and cell supernatant, respectively. rna isolation was performed using trizol reagent (invitrogen) following the manufacturer's directions with modifications to ensure a high rna yield and quality. a total of thirteen primers were designed using the primer 3plus software [32] to amplify five overlapping fragments of the s gene (table 1 ) covering the complete s gene: 218 bases before the start codon and 213 bases after the termination codon of strain tc pc170-p2 virus ped united states (accession number genbank: km392227). all segments were amplified using superscript5 iii one-step rt-pcr high fidelity system with platinum5 taq dna polymerase (invitrogen) following the manufacturer's directions. the amplicons were visualized in agarose gel and purified by pure link kit quick gel purification (invitrogen) following the manufacturer's instructions. the resulting products were submitted to bidirectional dna sequencing using bigdye terminator cycling conditions by an external laboratory (macrogen, korea). consensus sequences were generated using the software sequencher version 6.1, 2014 (code gene corporation). nucleotide blast analysis (https://www.ncbi.nlm.nih.gov/ blast/blast.cgi) was initially used to verify the identity of the sequences obtained. to perform sequence comparison analyses and to establish the phylogenetic relationships of pedv sequence from ecuador, alignments using the consensus sequence of complete s gene available at genbank database (supplementary information table s1 ) were conducted by the algorithm clustalw included in the program bioedit sequence alignment editor [33] . to remove sequences with a possible recombinant event from the alignment datasets, searches for recombinant sequences and crossover regions were performed using geneconv, rdp, maxchi, chimera, bootscan, siscan, 3seq and lard, all implemented in rdp3 beta 4.69, as previously described in alfonso-morales et al. [12] . the software jmodeltest 2.0 [34] was used to estimate the best-fit model using the akaike (aic) and bayesian information criteria (bic). the bestfit models for the complete s gene were selected. phylogenetic analyses were performed by bayesian inference (bi), neighbour-joining (nj), and maximum likelihood (ml) methodologies as described elsewhere in [35] . all topologies obtained were compared as described by alfonso-morales et al. [12] . to visualize the structure of s protein and denote amino acids changes, a model of the s protein was kindly provided by professor douglas marthaler from department of veterinary population medicine, college of veterinary medicine, university of minnesota, st. paul, mn, usa, and recreated using chimera software package (http://www.cgl .ucsf.edu/chimera). table s1 , sequences denoted by asterisk) as described by alfonso-morales et al. [14] . briefly, the bayesian markov chain monte carlo (mcmc) analysis was performed in two independent runs. the resulting maximum clade credibility (mcc) phylogenetic tree was obtained by treeannotator and summarized using the spread software [36] . a keyhole markup language (kml) file was generated to identify the major routes of geographic diffusion. the bayes factor (bf) test was used to select the most probable routes of transmission. the resulting kml files from spread with a nonzero expectancy that showed a bf > 5 were visualized by google earth (available at: https://earth .google.com/). after the pedv outbreak in the united states in 2013, followed by the fast spreading of the virus to canada, mexico, korea, and taiwan, an important turn in ped research has taken place [1] . thus, a relevant increase of studies about the epidemiology, genetic structure, and characteristics of pedv has occurred to get a better understanding of this disease, which is currently the most fatal in pigs and one of the economic concerns for the pig industry [4] . in the present study, an analysis of pedv field sequences from ecuador was conducted by comparison with all the pedv s gene sequences available in the genbank database. in addition, phylogenetic comparisons and bayesian phylogeographic inference based on complete s gene sequences were conducted to track the origin and putative route of pedv. the s gene of pedv has more than 4000 nucleotides and encodes the s protein, which constitutes the spikes of the viral envelope, responsible for its high variability. the pedv s glycoprotein is known to be an appropriate viral gene for determining the genetic relatedness among pedv isolates [4] . the sequences obtained from the animals selected and the viral isolate pedv/cotopaxi/2014 were assembled, yielding a final fragment of 4414 nt of length for each one, all of them containing the 4160 nt of the complete s gene. the identity of the sequences obtained was 100% between them; therefore, to avoid redundant entries at genbank database, the sequences were submitted as a single entry under the accession number kt336490. the genetic identity of the sequence kt336490 (thereafter mentioned as pedv/cotopaxi/2014) was initially inferred from blastn analysis sharing 99% with more than 94 isolates or strains of pedv from us and korea. the sequence of the pedv usa/colorado/2013 isolate (accession number: kf272920) was selected to compare the sequence pedv/cotopaxi/2014 since it showed the highest score from the blastn analysis. thus, the sequence pedv/cotopaxi/2014 showed four nucleotide changes 1093axg, 2454cxt, 3051cxt, and 3607cxt when both sequences were compared. only two of these mutations led to amino acid changes when the deduced sequence was analyzed. the sequence pedv/cotopaxi/2014 showed the changes 360sxg and 1203lxf compared with the sequence of pedv isolate usa/colorado/2013. these mutations were not located at the neutralizing epitopes (ss2, ss6, or 2c10) [37] . therefore, associations with possible adaptive advantages caused by escaping to the immune response of the host cannot be suggested. the mutation 360sxg was located into the n-terminal domain (ntd) of s1 (figure 1 ). even though this domain has not been directly linked to pedv tropism and functionality as in transmissible gastroenteritis virus and murine hepatitis virus [38] , it is recognized that it can influence virus infectivity [30] . therefore, a mutation of serine in this domain could lead to an increase of the infectivity of the viral strains with this mutation, since serine is recognized as a catalytic residue of the trypsin (enzyme involved in the entry of the virus into the host cell in the digestive tract). nevertheless, further virulence studies will be required to verify this role. [30] ; chimera software v1.6.2 was used for visualization. domains s1 (orange) and s2 (blue) are denoted. the c-terminal rbd of the s1domain is represented in pink; n-terminal rbd of s1 domain is highlighted in yellow. the mutation 360sxg found in pedv/cotopaxi/2014 is denoted and represented in red. the best-fit model and the shape parameter of the gamma distribution (alpha) for the tree are indicated in the upper-left side. the numbers at a node are posterior probability values estimated. all different genogroups are denoted; the different clades ci and cii previously described by vlasova et al. [28] into the genogroup 2a are also denoted. the sequence of pedv/cotopaxi/2014 is highlighted in red. blue rectangles denote pedv strains previously classified as 2a in zhang et al. [31] (see figure 1b in zhang et al. [31] ; the strains were clearly grouped into genogroup 2b but were denoted as 2a). the us indel sequences were also denoted. the phylogenetic relationships among the pedv strains were reconstructed based on complete s gene by means of nj, ml, and bi analyses. all algorithms yielded congruent results showing the same topologies, which was supported by moderate to high confidence values given by the bootstrap percentage and the posterior probability (supplementary material figure s1 ). even though the tree yielded by bi was the best, the statistical support for this tree was not significantly different from the nj or ml trees (supplementary material table s2 ). thus, all topologies obtained showed two highly divergent genogroups (2a and 2b) (supplementary material figure s1 ). for a better visualization of the results, a short tree obtained from 60 pedv strains, representative of all genogroups or clades, was built by bi ( figure 2) . thus, the short tree showed the same distribution than the full tree (supplementary material figure s1 and figure 2 ). in figure 2 , two clades belonging to the pedv genogroups 2a and 2b can be clearly observed. in addition, from the genogroup 2a, two main clades (i and ii) previously described by vlasova et al. [28] were also obtained ( figure 2 ). the sequence from the ped-outbreak in ecuador pedv/cotopaxi/2014 was grouped into the clade ii of pedv genogroup 2a together with other sequences of isolates from mexico, canada, and united states ( figure 2 ). this group was highly supported by 0.93 posterior probability value ( figure 2 ). after the ped us outbreak, the virus rapidly spread to canada and mexico [4] . whilst contaminated food by spray-dried porcine plasma positive to pedv genome is pointed out as a possible way for introducing pedv from us to canada [18] , the legal movement of pigs is suggested as the source of introduction to mexico (http://www.thepigsite.com/swinenews/36693/mexico-reports-83-outbreaks-of-pedv-to-oie/). in the group of sequences in which pedv/cotopaxi/2014 was included, the nearest country to ecuador is mexico, indicating that the movement (legal or illegal) of animals between mexico and ecuador could be a possible source of introduction of the virus to ecuador. however, evidence about this possible link has not been found. phylogeographic reconstruction identified a specific location for the root of the tree with posterior probabilities for state sp = 0.81 for the locality of china (supplementary material figure s2 ). the phylogeographic study revealed the emergence of the chinese pedv strains followed by spreading to us in 2013 (figures 3(a) and 3(b), supplementary material video s1). huang et al. [17] previously observed this result describing the us ped-outbreak as an intercontinental transmission of the chinese strains. after introducing the chinese pedv strains to us the virus spread from us to korea (figure 3 (c), supplementary material video s1). s. lee and c. lee [39] reported the circulation of new pedv strains in south korea that were genetically like pedv strains that affected united states during 2013. this result suggested that the recent strains from south korea might have been originated in the united states, caused by the importation of pig breeding stock during or after the sudden emergence of pedv in the united states [39] . during 2010-2011, more than one-third of the total pig population in south korea was slaughtered as control measure to foot-and-mouth disease outbreaks; thus a huge importation of breeding pigs from us was carried out without a proper implementation of a vaccination policy [39] . the phylogeographic study also revealed the introduction of the virus in canada, mexico, and ecuador directly from the us (figure 3 (d) and supplementary material video s1). this result frames the most probable route of entry of pedv to ecuador from us. the sources of imports of live swine in ecuador in 2014 were mainly from chile and us (http://data.trendeconomy.com/trade/ecuador/import?com-modity=0103). thus, this movement of pigs is suggested as the principal way for introducing pedv to ecuador. because this is the first introduction of pedv in ecuador swine herds, a fast spread of the virus throughout the country is expected, especially because a vaccination policy against pedv has not been implemented yet. therefore, additional studies to decipher how the virus will be disseminated in ecuador and to other south american countries will be required. in this study, a rigorous measurement of the global phylogeographic approach for pedv strains was performed based on complete s gene sequences. the present work is the first study providing evidences that pedv strains are circulating into ecuador swine herds and revealing the molecular characteristic of the pedv isolate in the south american region. the spatial analyses suggested that these strains were introduced to ecuador by an importation from us. the authors declare that they have no conflicts of interest. table s1 . pedv sequences of the complete s gene used in the current study (xls). table s2 . comparison of topologies obtained for the complete s gene using ml, nj, and bi methods. figure s1 . phylogenetic tree obtained from the complete s gene and all sequences collected (table s1 ) (tiff). figure s2 . maximum credibility phylogeographic tree. the posterior probabilities for state were denoted (tiff). video s1. dynamics of spatial pedv diffusion. the most probable temporal distribution of pedv is provided; only rates supported by a bf of >5 were considered significant. 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interfaces bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt jmod-eltest 2: more models, new heuristics and parallel computing phylogenetic networks to study the origin and evolution of porcine circovirus type 2 (pcv2) in cuba spread: spatial phylogenetic reconstruction of evolutionary dynamics bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during 2011-2013 in guangdong, china structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies outbreak-related porcine epidemic diarrhea virus strains similar to us strains key: cord-001566-kkaxha7d authors: zhang, mao-yu; lu, jin-jian; wang, liang; gao, zi-chao; hu, hao; ung, carolina oi lam; wang, yi-tao title: development of monoclonal antibodies in china: overview and prospects date: 2015-02-25 journal: biomed res int doi: 10.1155/2015/168935 sha: doc_id: 1566 cord_uid: kkaxha7d monoclonal antibodies (mabs) have become increasingly important as human therapeutic agents. yet, current research concentrates on technology itself and pays attention to developed countries. this paper aims to provide a comprehensive review of mabs development in china through systematic analysis of drug registry, patent applications, clinical trials, academic publication, and ongoing r&d projects. the trends in therapeutic areas and industrialization process are also highlighted. development and research trends of mabs are analyzed to provide a future perspective of mabs as therapeutic agents in china. over the past three decades, monoclonal antibodies (mabs) have achieved a dramatic development from scientific tools to powerful human therapeutic agents [1] (see figure 1 ). sales of mabs therapies exceeded 40 billion us dollars in 2010 and are expected to reach 70 billion us dollars by 2015 [2] . in 1975, kohler and milstein firstly described the in vitro production of murine mabs from hybridomas [3] , which was an innovative step towards the development of human mabs as therapeutic agents. in the late 1980s, clinical development of murine mabs was initiated but then inhibited by numerous significant drawbacks [4] . later, in attempt to overcome the inherent immunogenicity concerns and reduce effector function of murine mabs in human [5] , chimeric mousehuman antibodies were developed [6, 7] . then, humanization antibodies were developed [8] , which significantly enlarged the clinical usage of mab. in the following research exploration, human antibodies developed by phage display [9] [10] [11] and human ig mice advanced the development of mab greatly [12, 13] . nowadays, human mabs are the fastest growing category of mab therapeutics entering clinical study. development of this class of therapeutic agents started as early as 1980s but achieved no clinical or commercial success until 2002 when adalimumab became the first human mab approved by the us food and drug administration (fda) [14] . clinical development of mabs in china, like many developed countries, started with murine mab [15] . r&d of mabs in china began in the 1980s [16] and the first mab therapeutic agent (murine monoclonal antibody against human cd3 antigen of t lymphocyte for injection) was introduced in 1999 [17] . although mabs development in china has made significant progress over the past 2 decades [18] , all the mabs currently approved by cfda are technologically outsourced from foreign firms, like avastin (roche) [19] . these mabs mainly target cd20 [20] [21] [22] , antitumor necrosis factor (tnf) [23, 24] , vegfr [25] [26] [27] , her 2 [28, 29] , and egfr [30, 31] for the treatment of cancer or immunological disorders [32] . recently, 131i-chtnf and humanized mab h-r3 have been developed and approved as the treatment for solid tumor after panorex [16] . it is anticipated that continuous research on mabs will give rise to an expanding source of therapeutics. stepping into 21st century, the technology level of mabs in china has vastly improved, resulting in great research progress resulting in high expression and specificity. for example, mabs based on research of tnf were developed to treat rheumatoid arthritis (ra) for patients presenting with medium and severe symptoms [33] . these mabs block the interaction between vegf and kdr molecule and exhibit high specificity and activity [34] . this paper aims to provide a comprehensive review of mab development in china through systematic analysis of product registry, patent application, clinical trials, academic publication, and ongoing r&d projects. we used multiple sources for data collection. all mabs products approved for marketing in china were searched in the drug registry maintained by cfda. the united states patent and trademark office (uspto) was the data source for the analysis of patent applications submitted by chinese assignee. all clinical trials involving mabs approved and carried out in china were identified at the chinese clinical trial registry (chictr) database. academic publications about mabs published by researchers in mainland china were searched from thomson reuters' web of science (wos). information about the progress of the ongoing r&d mabs projects was collected from the main research institutes and firms in china. in this research, we focused primarily on the mabs development in mainland china and thus the corresponding data about hong kong, taiwan, or macao was not considered. based on the information from the abovementioned sources, this paper tries to present a comprehensive analysis of mabs development in china which will help provide some perspectives for continuous development of these therapeutic agents. murine monoclonal antibody against human cd3 antigen of t lymphocyte for injection was the only mab approved by cfda since 1999 until 2003 when cfda granted approval to the second mab. up to date, there are a total of eight mab products approved by cfda (see table 1 ). most of the mabs approved as human therapeutic agents are approved for the treatment of cancer or immunological disorders [6, 35] . of the eight mabs launched in china, three target antigens related to antineoplastic diseases, five target antigens related to immunological diseases, and two target antigens related to kidney transplant rejection. as shown in table 1 , there are three products approved for oncological uses including stage iii/iv of nasopharyngeal carcinoma (npc), advanced liver cancer, and non-small cell lung cancer (nsclc). another three products are indicated for immunological diseases, namely, psoriasis vulgaris, subacute eczema, active rheumatoid arthritis, moderate and severe plaque psoriasis, and ankylosing spondylitis. there are also products which are used to prevent rejection after organ transplantation. targets. the target of a therapeutic antibody is a major determinant of its efficacy and safety profile [14] . the eight mabs products which are approved by cfda target a total of seven unique antigens. these include cd147, egfr, intracellular dna for tumor therapy, il-8, tnf for immunological diseases, and cd 3 and cd25 for prevention of organ rejection after transplantation. there is also one mab which target the nucleus of tumor cells. in terms of versions, among the eight mabs approved in china, three are murine, one is chimeric, two are humanized and two are recombinant fusion protein. at present, there is no fully human mab approved for marketing in china. the development of mab technology remains at an early stage in china. overall, the mabs being studied are "me-too" and "me-better" products acquired through internal and external cooperation. on one hand, drug companies and universities with sufficient capacity conducted joint r&d projects. on the other hand, mab producers in china directly purchased the production technology from foreign countries, as demonstrated by the technology purchase actions of shanghai mei' en (collaborate with university of southern california, usa) and shanghai celgen (transfer from condar co., ltd., usa). according to the uspto database, the first mab-related patent in china was granted to monoclonal antibody against hepatitis e virus or its fragment with binding activity and use thereof in 2006. notably, there are a total of nine mab-related patents approved up to date. of these nine patents (see table 2 ), three are related to cancer diagnosis and therapy along with four other mabs targeting tnf, efgr, and vegfr. two patents which are associated with infectious diseases focus on virus or its fragment with binding activity and use thereof. for patent assignee, academic institutions are the most common and powerful controller in china, such as the institute of basic medical sciences of chinese academy of medical sciences, shanghai cancer institute, and xiamen university. table 3 . four candidates are in phase ii (bevacizumab, bimotuzumab and cetuximab), three in phase iii (rituximab, infliximab and bevacizumab), and five under postmarketing surveillance (ranibizumab, metuximab and rituximab). among these, seven were chimeric mabs while the other five were humanized mabs. among these twelve mab candidates, eight are studied as the treatment for cancer while the rest are tested for their use in ophthalmology and immunology. three of the eight mabs for cancer treatment are under review for market reassessment. the technology to develop mabs as antitumor drugs is considered mature. for example, as the first mab product used in non-hodgkin's lymphoma (nhl) therapy, rituxan has demonstrated great achievements in both clinical setting and market share once it has been approved. mabs for immunological diseases are mostly used to treat rheumatoid arthritis and lupus notes: "primary sponsor" is the experiment contract organization, "funding" represents the resource of fund, from which institutions or others. "a" is the independent innovation foundation to universities and colleges by jinan science and technology bureau. "nsclc" is "non-small-cell lung cancer. " "nhl" is short for non-hodgkin's lymphoma. "hcc" is hepatocellular carcinoma. "cpgj" is shanghai cp guojian pharmaceutical co., ltd. source: http://www.chictr.org/en/. myelopathy. rituximab which is indicated in lupus myelopathy has gained a great success in clinical application and it is now being explored further for any new indications. although mabs are not considered as the drug of choice in ophthalmology, they may possibly become the innovative treatment for neovascular glaucoma and diabetic macular edema as shown in table 3 . the following examples are used to provide a holistic view of mabs research development in china. biotech pharmaceutical funded the cancer center in the radiology department of sun yat-sen university to conduct open and multicenter clinical research on the efficacy of cis-platinum combined with nimotuzumab as the first line treatment of npc. this clinical research will help shed light on the possibility of "taixinsheng" combination therapy. similarly, chengdu huasun bio-tech funded fudan university to conduct intervention study to improve clinical outcomes of the combination therapy of "licartin" and transcatheter arterial chemoembolization (tace). these involve eight clinical trials which evaluate the clinical effectiveness of mabs in combination therapies. this is an indication that mabs as part of a combination therapy has become the new and alternative trend in future research. academic publications related to mabs in china were extracted from thomson reuters' web of science (wos) database. this is a powerful database which contains bibliographic data for, and citation to, publications in over 10,000 of the world's most important academic journals dating back to 1900. studies and research about mabs in china began in the 21st century [36] . publications related to mabs between 2000 and 2013 were searched using the following strategies: topic = (monoclonal antibod * ) and title = (monoclonal antibod * ) and address = (china not hongkong not taiwan not macau). "mabs" was used as the keyword in the same way. in the query above, the asterisk ( * ) represents any group of characters or no character and the literature type is limited in "article. " when "monoclonal antibody" was used as the search word, 957 articles were identified. of these, 788 were retrieved after excluding 169 articles that were irrelevant to our research questions. with "mab" as acronym words, 114 articles were identified. of these, 95 articles retrieved after excluding 19 irrelevant records. finally a total of 878 articles were collected after 5 repetitions of literature search (see figure 2 ). the data shows that the number of publications was the most abundant in the last five years (2008-2013) and peaked in 2011, while the decrease in 2012 and 2013 implies the more challenges for chinese researchers to make breakthrough in more innovative mab research. this historical trend of research corresponded with the trends of mabs technology development in china. by using "result analysis, " research areas of mabs in china focused on biochemistry, molecular biology, immunology, biotechnology, applied microbiology, biochemical research methods, chemistry, chemical analytical, and chemistry applied. the data also showed that research in joint forces with international counterparts was a common practice in mainland china especially with the us. this paper described the development and achievement of research in four main areas, namely, approved products, patent, clinical trials, and publications. in addition, research results also showed that academic institutes and enterprises play table 4 . [37] , high expression, mabs purification [38] , and platform construction of phage antibody library technology [39, 40] . as fully human mabs are considered the most promising category of targeted therapeutic agents [41] , china has also shown great interests. academy of military medical science, for instance, is dedicated to advancing the screening techniques of phage antibody libraries and optimization platform establishment necessary for the development of fully human mabs [42] . drug companies in china also play an important role in the development of mabs, especially in biosimilars [43] . being one of the representatives among the numerous creative and energetic mab enterprises in china, biotech pharmaceutical developed and produced the first humanized mab [44] . biotech also formed collaboration with cuba center of molecular immunology, which is yet another example demonstrating the strong interests in international cooperation by chinese enterprises. the r&d institution network in shanghai focused mainly on mabs technology industrialization. one of the r&d companies in the zhangjiang hi-tech park is shanghai cp guojian pharmaceutical co., ltd. (cpgj) which was founded by china international trust and investment corporation (citic) and is now coinvested by shanghai lansheng guojian pharmaceutical co., ltd. contributions have been made to the r&d, pilot plant test, and industrialization of antibody based drugs like cpgj. there are other r&d companies in the park which focus on r&d, manufacturing and marketing of high quality recombinant protein for the treatment of immune disease and neoplastic disease such as shanghai celgen biopharma. to summarize, mabs play an important role as efficient agent for antitumor and immunology disease. eight products in total are launched by cfda currently, mainly are in chimeric and humanized types. the number of total publications has been increasing since 21st century. nevertheless higher quality articles are needed. concerned about the intellectual property, patents applied by chinese assignee grow rapidly. to monitor safety and effectiveness of mabs, enterprises give support to and fund clinical trials for antitumor and other new indications. the future for mabs in china is promising. it should be mentioned that antibody-drug conjugates are becoming an increasingly important subclass of antibodyrelated cancer therapeutics and glycoengineering is being developed as a method to enhance the pharmacological properties of mabs. china may also need to pay much more attention to these kinds of antibodies. this paper found that mabs have experienced the fastest growth among all human therapeutic agents in the past two decades and will continue to do so in coming years [45, 46] and presented a lot of advancements which have been achieved in china [47, 48] . chinese biosimilar antibodies may now be approved in europe [49] ; at the same time, some of the most successful innovated biopharmaceuticals currently in the market (including recombinant insulin, human growth hormone, etc.) will see the expiration of their patents in the us over the next few years. this will provide an opportunistic market share which potentially worth 40 billion dollars for biosimilars including mabs in china [47] . however, there are still some challenges yet to be resolved and at the top of the list would be production capability for china [50] . firstly, there is still a lack of capacity for large-scale cell and perfusion cultivation at present [51] . to address this problem, improving the cells expression is necessary. china should attempt to work with developed countries because, comparing patent information related to us, the patents applied in both countries mainly involve three of these eight sections (section c, section a, and section g) and, as indicated by the patent search results, the mabs research in the us encompasses a lot more fields than in china [52] , especially in protein expression. moreover, according to the data searched in wos, us has been the strategic partner to china in r&d, which as mentioned before indicates that china should be more active to track first-edge technological research to make up the lack of capacity. additionally, data from ims showed that in 2007 mab drugs accounted for 34.4% of the worldwide total sale, but only 1.7% in china [53] , which was far below the global average [16] . while limited by production capability, high price, and low recognition, mabs in chinese market perform not well as other countries. to change the status, actions should be taken by the government. the chinese government is particularly supportive to universities and enterprises by ensuring reliable sources of funding for biopharmaceutical research and development. some of the most important funding sources are national natural science foundation of china, national high technology research and developmental program of china, and national basic research program of china. as universities and institutions account for a significant proportion of patent applications, china is steering towards a research-oriented country in the area of mab development. accordingly, the government has adopted favorable policies to encourage university-industry cooperation [54] . the government also provides financial support to create and maintain a healthy environment for the biopharmaceutical industry. the inclusion of cancer treatment into the national social security would provide prosperous future for mabs development in china once approved [17] . inclusion to the medical insurance catalogues also provides another important incentive for thriving mabs development. for example, basiliximab is listed in the medical insurance catalogues in thirteen provinces while infliximab in nine provinces. last, measures should also be made to attract much more foreign talents to join in the r&d and benefit from technology around the world. with all of these government support and joint efforts of academic and industry, there the mab development in china may contribute to global mabs production and therapeutic innovation. indeed, we strongly believe 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to balkan endemic nephropathy date: 2014-05-15 journal: biomed res int doi: 10.1155/2014/920723 sha: doc_id: 1313 cord_uid: f72hl6du balkan endemic nephropathy (ben) is a familial chronic tubulointerstitial disease with insidious onset and slow progression leading to terminal renal failure. the results of molecular biological investigations propose that ben is a multifactorial disease with genetic predisposition to environmental risk agents. exome sequencing of 22 000 genes with illumina nextera exome enrichment kit was performed on 22 dna samples (11 bulgarian patients and 11 serbian patients). software analysis was performed via nextgene, provean, and polyphen. the frequency of all annotated genetic variants with deleterious/damaging effect was compared with those of european populations. then we focused on nonannotated variants (with no data available about them and not found in healthy bulgarian controls). there is no statistically significant difference between annotated variants in ben patients and european populations. from nonannotated variants with more than 40% frequency in both patients' groups, we nominated 3 genes with possible deleterious/damaging variants—cela1, hspg2, and kcnk5. mutant genes (cela1, hspg2, and kcnk5) in ben patients encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. we suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of ben. balkan endemic nephropathy (ben) is a familial chronic tubulointerstitial disease with insidious onset and slow progression to terminal renal failure. it was first described in serbia and in bulgaria. the disease affects people living in the alluvial plains along the tributaries of the danube river in serbia, bosnia, croatia, bulgaria, and rumania [1] . investigations were directed to the epidemiology, etiology, morphology, and treatment of ben. results of these studies were reviewed elsewhere [2, 3] . ben has an onset of between the 40s and 60s, with a long preclinical period. the disease affects both genders with slight female predominance and often leads to terminal kidney failure. about 30-40% of the affected individuals develop uroepithelial tumours of the upper urinary tract [4] . these are mostly papillar carcinomas and are the most common causes of death in ben patients. different chemical elements, organic and nonorganic compounds, viruses, and microorganisms are implicated in ben development. heavy metals such as mg, mo, cd, pb as, and se can be important for the development of the disease, but there is no clear evidence of their direct toxic effect on the development of the disease. pathomorphologically ben has similarities with chinese herbal nephropathy, which is probably caused by the toxic effect of aristolochic acid, but there is no evidence supporting this theory for ben. other possible agents involved in the etiology of ben are ochratoxin a and some viruses such as picornavirus, polyomavirus, herpes simplex 1 and 2, adenovirus, hepatitis b, cytomegalovirus, and epstein-barr virus [5, 6] . for now there is no unchallenged evidence supporting a viral etiopathogenesis of ben. ben is a multifactorial disease with genetic predisposition to environmental risk agents. familial manifestation of ben implies a polygenic genetic predisposition [7, 8] . previous studies have suggested genes located in chromosome band 3q25-3q26, genes for xenobiotic metabolizing enzymes, tumour-suppressor genes, and protooncogenes. the incidence of rapid debrisoquine metabolizers is higher in ben patients than in healthy controls [9] . cyp2d6 polymorphic variants predisposing to toxic effect of various chemical agents are suspected in ben pathogenesis. lcatdeficient individuals have evidence of renaltubular injury and this defect can be involved in ben [10] . cytogenetic research on lymphocyte cultures from ben patients showed that in vitro higher folic acid inducedchromosomal fragility and more frequent spontaneous chromosomal aberrations. some of the unstable regions contain oncogenes-1p36-c-src, 3p25-raf1, 3q27-fim3, 6q23-myb, 1p13-nras, and 6p11-kras1p [11, 12] . other studies show that environmental factors are very important and can influence genome function without changing the dna sequence itself. the concept of epigenetics was suggested. the major epigenetic modifications include dna methylation, histone modifications, and mirna interference [13] . epigenetic changes over time display familial clustering [14] and could be implicated in transmitting a "predisposition" over generations. epigenetic modifications being heritable and adaptable at the same time may prove to make a significant contribution to ben development and may be the link between the effect of environmental factors and genetic composition in ben progression. in a previous study we have investigated the methylation status across the whole genome in different patient groups, based on gender and endemic region, in comparison to healthy controls from nonendemic regions. differentially methylated regions (dmrs) were determined in ben patient and controls and the commonly presented dmrs were determined to be the most promising methylation alterations in ben. sec61g, il17ra, and hdac11 proved to be differently methylated throughout all patient-control pairs [15] . in the present study we aimed to perform exome sequencing of 22 000 genes with the illumina nextera exome enrichment kit using ngs technology in order to find specific mutations for ben. twenty-two ben patients were selected for ngs exome analysis. informed consent was received from all participants enrolled in the study. we obtained peripheral blood samples from 2 series of patients-11 bulgarian and 11 serbian. clinical assessment was performed according to unified criteria and was applied to all sample cohorts. genealogical analysis was performed to exclude relatives among all study subjects. bulgarian samples were collected by preliminary clinical screening in vratza endemic regions in bulgaria in 2003 [16] . all subjects were born of bulgarian ancestry, born and living in the endemic region. dna was extracted by standard phenol-chloroform extraction procedure and stored at −80 ∘ c. all samples were checked for dna consistency by 1% gel electrophoresis. serbian samples were collected from serbian endemic regions. dna was extracted by dna extraction kit and stored at −80 ∘ c. all samples were checked for dna consistency by 1% gel electrophoresis. the study was approved by the serbian ethics committee of the university of nis, school of medicine, nis, serbia, and the bulgarian commission of medical ethics at the national center of hygiene, medical ecology and nutrition, sofia, bulgaria. the workflow in the nextera enrichment sample preparation guide (revision b) by illumina was followed to prepare the libraries for wholeexome sequencing. a nextera exome enrichment kit was used. the 22 libraries were distributed in 10 enrichment reactions ("pools")-in 6 of them dna from 3 libraries was mixed together, while the other 4 contained dna from only one. the latter were prepared in order to obtain higher mean coverage for these samples. a different dna quantity (500 ÷ 1000 ng) was added to form each pool depending on the quantity of the least concentrated sample in the reaction. after completing the enrichment procedure, dna concentration of the 10 sequencing-ready pools was measured using a kapa library quantification kit. the reactions were run in triplicate on an illumina qpcr eco system. the eco study software was used to calculate the concentrations of the dilutions and then of the stocks. pools 5-10 were denatured and diluted according to the illumina guidelines in preparing dna libraries for sequencing on the miseq (illumina, san diego, usa). due to lower concentrations pools 1-4 were prepared for sequencing following the corresponding chapter in the truseq custom amplicon library preparation guide, which uses heat denaturation instead of denaturation with 0.1 n naoh. miseq system using the miseq reagent kit v2 and a 500cycle 14-tile flow cell. only pool 9 was sequenced using a 300cycle miseq reagent micro kit v2 and a micro 4-tile flow twenty-two bulgarian and serbian ben patients were analyzed by ngs exome sequencing for 22 000 genes. using the softgenetics nextgene software (version 2.3.3) the sequencing data were analyzed. mutation prediction was performed by software provean and polyphen-2. we discovered in total 3666 missense variants with possible deleterious/damaging effect in our patients' groups. among them, 1849 (50%) were not annotated. in total, 980 nonsense and frameshift variants were detected in our study. among them, 541 (55%) variants have not been annotated in human genome data base of genetic variations or no information about their frequency was available. among the annotated variants with possible deleterious/damaging effect we did not find statistically significant difference in the frequency between ben patients and european populations. from nonannotated variants, we selected the variants with frequency of more than 40% in ben patients-hspg2, cela1, and kcnk5 (table 1) . these mutations, alone or in combination, occur in 77% of ben patients. the probable contribution of each of the nominated genes to the pathogenesis of ben (according to their mutation frequency) is represented in figure 1 . the frequencies of each of the nominated mutant genes for bulgarian and serbian ben patients are given in figure 2 . all selected variants occur with similar frequency in the bulgarian and serbian groups of ben patients. the nominated variants are classified according to their function-genes involved in the constitution of basement membrane/extracellular matrix (ecm) (hspg2 and cela1) and gene for renal potassium transport and membrane potential (kcnk5). figure 3 represents the distribution of the three mutant genes in all ben patients. kcnk5 variant c.1397a>c occurs only in combination with one or two of the other variants (hspg2 c.5239a>c and cela1 c.9 10delc), while cela1 variant is present as a single aberration in 6 out of 10 positive cases. in 5 of ben patients none of these variants were found. this is probably due to the presence of other rare mutations, which did not pass the threshold criteria in our study (presented in more than 40% of the patients). balkan endemic nephropathy has been traditionally described as an end-stage kidney disease, characterized by bilaterally and symmetrically contracted kidneys of a very small size and reduced weight, coinciding with multiple upper urinary tract tumors in 8-48% of cases [17] . basic histomorphological changes include tubular atrophy and interstitial fibrosis with sclerosed glomeruli usually of collapsing obsolescence and sclerotic changes in blood vessels. the hypothesis for ben multifactorial pathogenesis [8] proposes that inherited genetic defects predispose the kidney to damage after exposure to different agents-toxic, immune, or infective. the familial clustering of the disease has prompted genetic investigations [12, 18, 19] . this is the first analysis by ngs sequencing in ben patients aimed at discovering mutations associated with the disease. despite intensive molecular studies in ben, the etiopathogenesis of the disease is still not elucidated and there is no biomarker for disease predisposition. most of the molecular studies so far have focused on single genes/polymorphisms and very limited information has been provided. because gene-by-gene analysis by sanger sequencing is too laborious and expensive, genetic testing has been the exception until recently. now, next-generation sequencing (ngs) allows for simultaneous and efficient analysis of all known genes for a given trait. here we applied exome sequencing (comprised of 22 000 genes) in 22 ben patients in order to detect the most prominent genetic variants with highly probable pathological effect. recently poon et al. have applied whole-genome and exome sequencing for analysis of aristolochic acid-(aa-) associated upper urinary tract urothelial cell carcinoma (utuc) [20] . aa is a carcinogen that can cause nephrotoxicity as well. authors observed a high frequency of somatic mutations in chromatin modifiers, particularly kdm6a, in aa-utuc, demonstrated the sufficiency of aa to induce renal dysplasia in mice, and reproduced the aa mutational signature in experimentally treated human renal tubular cells. our study was looking for germ-line mutations predisposing to another specific nephropathy-balkan endemic nephropathy. three genes (hspg2, cela1, and kcnk5) were nominated as related to the pathogenesis of ben based on their mutation frequency, their similar incidence in both bulgarian and serbian patients' groups, lack of information about the established variants in european population, and nonincidence in healthy bulgarians. analysis of their function sheds light on the possible pathophysiology in ben, which we discuss here. the first two genes hspg2 and cela1 are evidently involved in the process of angiogenesis. the gene kcnk5 encodes a protein for potassium channel, which could also be involved in vascular disease and complications. the number of patients requiring renal replacement therapy due to end-stage renal disease (esrd) is increasing worldwide [21] . the prevalence of chronic kidney disease (ckd) and the importance of ckd as a risk factor in development of esrd have been confirmed. in recent years, the involvement of angiogenesis-related factors in the progression of ckd has been studied, and the potential therapeutic effects on ckd of modulating these factors have been identified [22] . a number of angiogenic growth factors are involved in the development of the kidney and in the maintenance of glomerular structures and the glomerular filtration barrier function in adults. our study revealed significant candidate gene-hspg2, which encodes perlecan protein, a major component of basement membranes, where it is involved in the stabilization of other molecules important for glomerular permeability to macromolecules and for cell adhesion [23, 24] . it binds to and crosslinks many extracellular matrix components and cellsurface molecules [25] [26] [27] [28] [29] . it has been shown that this protein interacts with laminin, prolargin, collagen type iv, tenascin-c, fgfbp1, fbln2, fgf7, transthyretin, and so forth, and plays essential roles in multiple biological activities. cukuranovic et al. have intensively studied the pathological changes in the kidneys of ben patients and presented evidence that renal vascular changes occur early in balkan nephropathy [30] . they detected by ihc a marked overexpression of laminin in renal interstitial capillaries. the pattern of laminin staining in glomeruli corresponded to focal and segmental glomerular sclerosis present in the advanced stages of balkan nephropathy. later stages were characterized by an intensive expression of laminin in atrophic tubules, much more in proximal than in distal ones. the coexpression of vimentin and cytokeratin in proximal tubular cells was also demonstrated. the changes described, particularly those taking place at the level of interstitium, bear the key responsibility for ben progression. cela1, the second nominated gene, is also involved in the process of angiogenesis [31] . the gene encodes elastase-1, which degrades elastin in the vascular matrix. tumor angiogenesis, chicken angiogenesis, and mesenteric angiogenesis data suggest that elastin and elastin degradation products play a key role in vascular morphogenesis. cela1 was expressed in vascular cells in the embryonic lung and in a fetal mesenchymal cell line with angiogenic properties [32] . degraded elastin causes deposition of hydroxyapatite-like mineral and osteogenic transformation of vascular smooth muscle cells (as they lose the specific -sma), resulting in vascular calcification [33] . in contrast, collagen-1 levels in areas of calcification are increasing [34] . changes in both -sma and elastin inversely correlate with the hemodynamic parameters such as pulse wave velocity (pwv) and lead to media remodeling. this was associated with the increased arterial stiffness observed in ckd rats with vascular calcification. dysregulation of normal anticalcification factors and elastin degradation represent a pattern of vascular injury existing in patients with end-stage renal diseases [35] . the third gene nominated in our study was the potassium channel gene kcnk5. potassium channels in the kidney play an essential role in controlling and maintaining plasma potassium levels in the normal range, as well as exerting very different functions such as cell volume control, membrane potential stabilization and excitability, or regulation of hormone or ion secretion [36] [37] [38] [39] . in addition, potassium ion (k + ) channel activity is a major regulator of vascular muscle cell membrane potential and is therefore an important determinant of vascular tone. there is growing evidence that the function of several types of vascular k + channels is altered during major cardiovascular diseases, such as chronic hypertension, diabetes, and atherosclerosis [40] . defects in potassium channels cause abnormal vasodilation responses reflecting a gradual deterioration of vascular mechanisms during the progression of diabetic nephropathy [41] . enhanced dilator responses and basal activation of k + channels may occur in the renal circulation early during diabetes. an increased k + channel activity may therefore reflect a very high metabolic state of vascular smooth muscle cells [42] . the molecular mechanisms leading to interstitial fibrosis and chronic kidney disease are complex and are probably related to the primary processes leading to renal injury. as blood vessels nourish all the tissues and organs in the body, abnormal formation and remodeling of blood vessels probably contribute to the pathogenesis of renal fibrosis. neoangiogenesis is a complex process of recruitment, migration, proliferation, and apoptosis of stem/progenitor cells, endothelial cells, vascular smooth muscle cells, and other mural cells. the extracellular matrix plays important roles in vessel development via providing a supportive matrix scaffold and growth factors for cells. close interactions between vascular cells and their ecm is crucial in blood vessel formation and remodeling. our results suggest three new genes for predisposition to ben pathology, related to angiogenic alterations. we hypothesize that mutations in hspg2, cela1, and kcnk5 participate in extracellular matrix modifications, arterial media remodeling, and regulation of vascular tone, all these events leading to interstitial vessel remodeling, connected to renal interstitial fibrosis in ben (figure 4 ). cohort of patients. herein we suggest a possible mechanism between the three candidate genes and ben. the main pathological characteristic of ben kidney is interstitial fibrosis. abnormal angiogenesis and vascular remodeling probably contribute to pathogenesis of renal fibrosis. interactions between endothelial cells, vascular smooth muscle cells and progenitor cells with an extracellular matrix (ecm) play an important role in these processes. scattered glomeruli showing an obvious segmental or global thickening of the capillary walls with a double outline of the glomerular basement membrane were found in early ben patients. interstitial sclerosis could result from the overproduction of extracellular matrix by injured proximal tubular epithelium and interstitial capillary endothelial cells-this could be the pathogenic role of mutated hspg2 gene. the increase of the cortical interstitial volume results in resistance of the postglomerular capillary network with impairment of the glomerular flow [43] . this impairment leads to chronic rise in hydrostatic pressure. the increase of the cortical interstitium additionally leads to an increase in the length of diffusion between the tubules and the intertubular and peritubular capillaries. this increase in the length of diffusion subsequently results in the atrophy of the tubules, reduction of reabsorbtion, and therefore impairment of the effective filtration pressure. the haemodynamic changes inversely correlate with -sma and elastin in vessels and here could be the additional pathogenic role of mutated cela1 gene. the mutated kcnk5 gene is probably a factor stimulating the haemodynamics-driven vascular remodeling. oxford textbook of clinical nephrology balkan nephropathy balkan endemic nephropathy: a need for novel aetiological approaches urinary tract tumors and balkan nephropathy in the south morava river basin nature of the virus associated with endemic balkan nephropathy isolation of a coronavirus from urinary tract tumours of endemic balkan nephropathy patients genetic predisposition to balkan endemic nephropathy etiology of balkan endemic nephropathy: a multifactorial disease genetic predisposition to balkan endemic nephropathy: ability to hydroxylate debrisoquine as a host risk factor partial lecithin: cholesterol acyltransferase (lcat) deficiency in balkan endemic nephropathy cytogenetic studies in balkan endemic nephropathy spontaneous and induced chromosome aberrations in balkan endemic nephropathy the epigenetic conductor: a genomic orchestrator in chronic kidney disease complications? intraindividual change over time in dna methylation with familial clustering whole genome methylation array analysis reveals new aspects in balkan endemic nephropathy etiology the role of some polymorphisms of detoxicating enzyme for predisposition to balkan endemic nephropathy pathology of balkan endemic nephropathy-a correlation with established kidney disease entities mdr1 haplotypes modify ben disease risk: a study in bulgarian patients with balkan endemic nephropathy compared to healthy controls identification of nqo1 and gsts genotype frequencies in bulgarian patients with balkan endemic nephropathy genome-wide mutational signatures of aristolochic acid and its application as a screening tool cellular pathophysiology of ischemic acute kidney injury angiogenesis and chronic kidney disease perlecan maintains microvessel integrity in vivo and modulates their formation in vitro a central function for perlecan in skeletal muscle and cardiovascular development heparanase cleavage of perlecan heparan sulfate modulates fgf10 activity during ex vivo submandibular gland branching morphogenesis endothelial cells provide feedback control for vascular remodeling through a mechanosensitive autocrine tgf-signaling pathway the protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7 binding of perlecan to transthyretin in vitro fibroblast growth factor-binding protein is a novel partner for perlecan protein core immunohistochemical localization of laminin in renal lesions of balkan nephropathy chymotrypsin like elastase-1 (cela1) regulates pulmonary vascular morphogenesis the role of platelet factor 4 in local and remote tissue damage in a mouse model of mesenteric ischemia/reperfusion injury hydroxyapatite and calcified elastin induce osteoblast-like differentiation in rat aortic smooth muscle cells vascular remodeling and media calcification increases arterial stiffness in chronic kidney disease clinical detection, risk factors, and cardiovascular consequences of medial arterial calcification: a pattern of vascular injury associated with aberrant mineral metabolism simvastatin reverses podocyte injury but not mesangial expansion in early stage type 2 diabetes mellitus proximal renal tubular acidosis in task2 k + channel-deficient mice reveals a mechanism for stabilizing bicarbonate transport physiology and pathophysiology of potassium channels in gastrointestinal epithelia invalidation of task1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis potassium channel function in vascular disease modification of vasodilator response in streptozotocin-induced diabetic rat exaggerated impact of atp-sensitive k + channels on afferent arteriolar diameter in diabetes mellitus damage to kidney in balkan endemic nephropathy: initial lesion, target structures and pathomorphogenesis this study was funded by bnsf grant dmy o3/35, snsf-scopes grant iz73zo 127949, and bnsf grant dtk 02/76. the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord-296682-ugffeegr authors: rahimi, hoda; tehranchinia, zohreh title: a comprehensive review of cutaneous manifestations associated with covid-19 date: 2020-07-05 journal: biomed res int doi: 10.1155/2020/1236520 sha: doc_id: 296682 cord_uid: ugffeegr the novel coronavirus (sars-cov-2), the cause of coronavirus 2019 disease (covid-19) pandemic, is associated with some cutaneous manifestations. although the cutaneous presentations of covid-19 are infrequent, it is of great importance for all clinicians to be aware of these manifestations, as it may contribute to sooner and better diagnosis and management of the disease, even in asymptomatic or paucisymptomatic patients. the reported cutaneous manifestations of covid-19 are various, dispersed, and sometimes confusing. in this article, all reported cases to date were collected and classified under 6 major groups: maculopapular rash, urticaria, chilblain, vesicular lesions, livedo reticularis, and petechiae. different characteristics of each group were discussed in detail as well. on 31 december 2019, a newly emerged pneumonia caused by a novel coronavirus, named sars-cov-2, was announced by china [1] . it spread so rapidly until who announced coronavirus 2019 disease (covid-19) as a pandemic condition on march 11. the firstly reported presentations of covid-19 were like other viral respiratory infections, including high fever and dry cough. however, it might lead to acute respiratory distress syndrome and the mortality rate was quite high [2] . since then, a wide spectrum of clinical manifestations have been described, ranging from the absence of any symptoms to fever, cough, dyspnea, diarrhea, ageusia, anosmia, and even cutaneous lesions [3, 4] . although the cutaneous manifestations of covid-19 are infrequent, it is of great importance for all clinicians to be aware of these presentations, as they may contribute to sooner and better diagnosis and management of the disease, even in asymptomatic or paucisymptomatic patients. this could be a valuable help for epidemiological control of the disease, especially in regions where diagnostic kits are limited [5] . on the other hand, the reported cutaneous manifestations of covid-19 are various, dispersed, and sometimes confusing. thus, we aimed to review and summarize the different skin lesions, which have been reported in association with covid-19 to date, in this article. pubmed and cochrane were searched with the search terms "skin", "cutaneous", and "dermatology", each in combination with "covid-19" or "sars-cov-2". all articles including case reports and original articles from the emergence of the disease (31 december 2019) to the submission of the article (9 may 2020) were included except for one article in which all 6 cases had neither positive pcr test nor common symptoms of covid-19, and the authors presumed that their cutaneous manifestations may be related to sars-cov-2 without any documented evidence [6] . different cutaneous lesions have been reported in 451 patients with covid-19 to date (table 1) . however, it is predictable that the cutaneous lesions have been undoubtedly 5 biomed research international underdiagnosed due to the lack of dermatology consultations in this pandemic situation. the age of patients ranged between 2 months and 89 years. patients consisted of 244 females and 178 males, while the gender of 29 patients was not reported. most reported cases were from spain (379 pts; 84%), followed by italy (51 pts; 11.3%), france (10 pts; 2.3%), usa (4 pts; 0.9), thailand (3 pts; 0.7%), belgium (2 pts; 0.4%), and kuwait (2 pts; 0.4%). it is obvious that these data are not compatible with the real prevalence of the disease in different countries, since many dermatological cases might not be reported in the literature due to different reasons. table 1 shows detailed characteristics of reported patients. although the cutaneous presentations of covid-19 are various, they could be categorized in 6 major groups (tables 1 and 2) . (1) maculopapular rash. maculopapular rash, with or without pruritus, was the most common cutaneous manifestation of covid-19, presented in 200 patients (44.4%). the mean age of the patients in this group was 60.4 years (range: 6-89 6 biomed research international years). more than half of the patients were female, and the most common localization of the lesions was the trunk. although 35% of patients were asymptomatic, the most frequent symptom was itching which presented in 57% of cases. this kind of rash is mostly observed in the active phase of the disease. (2) urticaria. the second most frequent skin rash in covid-19 patients was urticaria which appeared in 84 patients (18.6%). the mean age of the cases was 47.6 years, while the youngest patient aged 2 months and the eldest 71 years. again, females were dominant (66%), spain reported the most number of cases, and the rash appeared mostly within the active phase of the infection. (3) chilblain. chilblain occurred in 81 patients (18%) without any history of exposure to cold. this group had some especial characteristics which made it different from the others; it mostly presented in younger patients with a mean age of 31.7 years, and females were involved significantly more than males (68% vs. 32%). moreover, in contrast to other cutaneous manifestations, which appeared mostly in the active phase, chilblain often presented later in the course of the disease (after the complete recovery, in half of the patients). furthermore, approximately 10% of the patients were asymptomatic carriers of covid-19 who had only chilblain and positive pcr test, without any symptom of covid-19. none of the cutaneous manifestations was reported in asymptomatic carriers, except for chilblain. its distribution was, as usual, in acral parts. however, the heels and toes were affected significantly more than the fingers (97.5% vs. 2.5%). the most frequent symptom was pain (32.1%). (4) vesicular lesions. vesicular lesions, reported in 61 patients (13.5%), usually manifested like chicken pox, consisting of pruritic papulovesicular rashes involving the trunk mostly. however, three patients presented with localized vesicular lesions which was herpetiform [7] . vesicular rashes were reported only from italy and spain, often in the active phase of the disease. (5) livedo reticularis. livedo reticularis was reported in 23 patients (5.1%) as generally asymptomatic lesions, affecting both genders almost equally. in two cases, it was unilateral, resolving in few hours without any treatment [8] . they were in contrast with the usual bilateral distribution of the common livedo reticularis which does no subside spontaneously. (6) petechiae. to date, only 2 cases (0.4%) of petechiae were reported in association with covid-19 [9, 10] , one of which was firstly misdiagnosed and mistreated as dengue [10] . as it may be noticed, cutaneous lesions of covid-19 may manifest in different forms, in every age and sex, and involve every part of the body. however, the involvement of mucosa has not been reported yet, except for a case of oral herpes simplex virus-1 reactivation in an intubated patient, which seemed to be secondary to the intubation rather than a presentation of the virus [13] . in all groups, pruritus was the most common symptom, except for chilblain which was more associated with pain than pruritus. generally, the skin lesions were observed in the active phase of infection (61% of cases) and subsided within few days without any treatment. although the appearance of skin rash in the prodromal phase or asymptomatic carriers was scarce, it is of great importance for all clinicians to keep in mind that cutaneous lesions might be the only symptom of covid-19, as it would contribute to sooner diagnosis and management of the patients/carriers and better control of the disease spreading. from 451 reported patients, only 296 cases were confirmed by polymerase chain reaction (pcr) test. due to the lack of the diagnostic kits in this pandemic period, they were performed only for hospitalized or severe cases in most countries. that is why all cases did not have a documented diagnosis and suspected cases were included as well. however, it should be noticed that (1) many of these suspected cases had other typical signs and symptoms of covid-19; (2) many of them had positive family history of covid-19 or close contact with infected patients; and (3) almost in all of these cases there was no other explanation for their cutaneous lesions except for covid-19. another important point which remained controversial is that whether these cutaneous presentations have any correlation with the severity of the disease or could be used as a prognostic factor. although galván casas et al. reported that chilblain was associated with less severe disease, and livedo reticularis was associated with the most severe forms of the disease [17] , the role of confounding factors such as age should be noticed, as well. however, recalcati reported no correlation between cutaneous presentations and disease severity [6] . although these data do not prove that covid-19 was the definite cause of these skin lesions, they demonstrate that cutaneous lesions should be considered in the spectrum of presentations potentially associated with this infection. in particular, some cutaneous manifestations such as chilblain without any explanation may warn about asymptomatic virus carriers. however, further investigations should be carried out to evaluate the relation between skin lesions and covid-19. the novel coronavirus originating in early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the epidemiology and pathogenesis of coronavirus disease(covid-19) outbreak olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study covid-19: learning from experience cutaneous manifestations in covid-19: a first perspective cutaneous manifestations in covid-19: the experiences of barcelona and rome a dermatologic manifestation of covid-19: transient livedo reticularis cutaneous lesions in a patient with covid-19: are they related? covid-19 can present with a rash and be mistaken for dengue a distinctive skin rash associated with coronavirus disease 2019? cutaneous lymphoid hyperplasia associated with leishmania panamensis infection comment on "cutaneous manifestations in covid-19: a first perspective " by recalcati s morbilliform exanthem associated with covid-19 a case of covid-19 pneumonia in a young male with full body rash as a presenting symptom manifestaciones cutaneas en contexto del brote actual de enfermedad por coronavirus 2019 classification of the cutaneous manifestations of covid-19: a rapid prospective nationwide consensus study in spain with 375 cases comment on: cutaneous manifestations in covid-19: a first perspective. safety concerns of clinical images and skin biopsies urticarial eruption in covid-19 infection acute urticaria with pyrexia as the first manifestations of a covid-19 infection diffuse cutaneous manifestation in a new mother with covid-19 (sars-cov-2) two cases of covid-19 presenting with a clinical picture resembling chilblains: first report from the middle east skin signs resembling vascular acrosyndromes during the covid-19 outbreak in italy varicellalike exanthem as a specific covid-19-associated skin manifestation: multicenter case series of 22 patients varicella-like exanthem associated with covid-19 in an 8-year-old girl: a diagnostic clue? acral cutaneous lesions in the time of covid-19 the authors declare that they have no conflicts of interest. key: cord-262468-7ddgegb2 authors: deng, jianqing; liu, jie; cao, long; wang, qun; zhang, hongpeng; liu, xiaoping; guo, wei title: the association between hyperhomocysteinemia and thoracoabdominal aortic aneurysms in chinese population date: 2020-07-28 journal: biomed res int doi: 10.1155/2020/4691026 sha: doc_id: 262468 cord_uid: 7ddgegb2 objective: to shed light on the association between hyperhomocysteinemia (hhcy) and thoracoabdominal aortic aneurysms (taaas). methods: from july 2013 to march 2017, we conducted a matched case–control study involving individuals who presented to the chinese people's liberation army general hospital and underwent thoracoabdominal magnetic resonance angiography or computed tomography angiography. a total of 73 patients with taaas were enrolled in the case group, and 219 sex-matched subjects without taaas were included in the control group. we then examined the relationship between hhcy and taaas by logistic regression models and subgroup as well as interaction analyses. results: serum total homocysteine (thcy) concentrations and the proportion of hhcy were significantly higher in the patients with taaas than in those without taaas (p < 0.001). furthermore, the multivariate logistic regression models indicated that participants with hhcy had a 2.14-fold higher risk of taaas than those with a normal serum thcy level (adjusted odds ratio (or), 2.14; 95% confidence interval, 1.00–4.56). similarly, each 1 μmol/l increase in the serum thcy concentration was associated with a 4% higher risk of taaas (adjusted or, 1.04; 95% confidence interval, 1.00–1.07). subgroup analyses indicated that hhcy tended to be associated with a greater risk of taaas in all stratified subgroups (adjusted ors > 1). furthermore, the interaction analyses revealed no interactive role in the association between hhcy and taaas. conclusions: the present case–control study suggests that hhcy is an independent risk factor for taaas. larger prospective cohort studies are warranted to validate these findings. a thoracoabdominal aortic aneurysm (taaa) is defined as a permanent and continuous dilatation of the descending thoracic aorta and abdominal aorta [1] . although taaas reportedly have a low incidence of 5.9 cases per 100,000 inhabitants annually [2] , surgical repair of taaas is associated with a perioperative mortality rate ranging from 7.5% to 46.1% [3, 4] . in addition, one study showed that the 5-and 10-year survival rates of patients with taaas undergoing open repair were 63.6% and 36.8%, respectively [3] , indicating that the prognosis of patients with taaas is far from satisfactory. consequently, it is essential to decrease the incidence of taaas by discovering and controlling their risk factors. many patients with taaas are asymptomatic, and these taaas may remain undiagnosticated for years [5] . the risk of rupture increases with the aortic diameter, and the risk dramatically increases at diameters > 7 cm [1, 5] . most ruptured taaas are catastrophic. among patients with ruptured taaas who are able to undergo surgical repair, the operative mortality rate is reportedly as high as 46.1%, which is significantly higher than that of unruptured taaa repair (15.9%) [4] . therefore, the natural history of taaas causes many patients to easily miss the optimal timing of intervention. identifying the biomarkers that indicate the presence of taaas might benefit early detection and intervention and thus improve the prognosis. the interaction between media degeneration of the aortic wall and hemodynamic tension leads to the majority of taaas [1] . during the last few decades, hypertension, hyperlipidemia, obesity, smoking, a family history, and connective tissue diseases have been described as risk factors for taaas [1, 6] , indicating that many risk factors for taaas are similar to those for atherosclerosis. hyperhomocysteinemia (hhcy), defined as an elevated serum total homocysteine (thcy) level, is associated with atherosclerotic diseases and intracranial aneurysms as well as abdominal aortic aneurysms (aaas) [7] [8] [9] [10] [11] [12] [13] . however, no study has been performed to investigate the association between hhcy and taaas. therefore, we performed a matched case-control study to explore the relationship between the serum thcy level and taaas. we also performed subgroup and interaction analyses to detect the interactions of hhcy with other conventional factors in the association with taaas. from july 2013 to march 2017, we conducted a matched case-control study involving individuals who presented to the chinese people's liberation army general hospital and underwent thoracoabdominal magnetic resonance angiography or computed tomography angiography. consecutive patients who were newly diagnosed with taaas in the department of vascular and endovascular surgery were enrolled in the case group. these patients were matched 1 : 3 by sex with controls without taaas in the health management center and were recruited during the same period (within 3 months). patients with taaas caused by aortic dissection, trauma, or infection were excluded from the case group. the other exclusion criteria for all subjects were a history of vitamin b, folate, or antiepileptic medication use; a history of contraception and/or hormone replacement therapy; a chronic history of connective tissue disease (such as the marfan syndrome, loeys-dietz syndrome, or ehlers-danlos syndrome), takayasu arteritis, or aortitis; a history of serum thcy-associated disease such as a malignant tumor or hypothyroidism; a history of mental disorders or diseases; and pregnancy. our study was approved by the ethics committee of the chinese people's liberation army general hospital. we obtained a written informed consent from all participants, and all procedures in our study adhered to the principles of the declaration of helsinki. finally, 292 participants met the criteria (73 in the case group and 219 in the control group) during the study period and were recruited in our study. all subjects underwent a medical consultation, standard physical examination. a uniform questionnaire was administered to record the participants' demographic characteristics and medical history, including age, sex, body mass index (bmi), smoking and drinking habit, hypertension, diabetes mellitus, hyperlipidemia, ischemic stroke, and coronary artery disease (cad). the subjects filled out the questionnaire according to the guidance provided by trained doctors. a smoking habit was defined as consuming ≥100 cigarettes throughout life, and a drinking habit was defined as drinking ≥50 ml of alcohol per week for 6 months [8] . hypertension was defined as receiving antihypertensive treatment or having a systolic blood pressure of ≥140 mmhg or diastolic blood pressure of ≥90 mmhg. diabetes mellitus was defined as a glycated hemoglobin level of ≥6.5% or current treatment with medication for diabetes. moreover, hyperlipidemia was defined as either the use of hypolipidemic agents or a serum total cholesterol concentration of >5.7 mmol/l, triglyceride concentration of >1.7 mmol/l, low-density lipoprotein cholesterol (ldl) concentration of >3.4 mmol/l, and high-density lipoprotein cholesterol (hdl) concentration of <1.0 mmol/l [8] . the medical histories of cad and ischemic stroke were self-reported and confirmed by the subjects' families. all subjects underwent blood biochemical tests for either a preoperative evaluation or health check-up. we retrieved the value of the serum thcy, glucose, creatine, uric acid, total cholesterol, triglyceride, hdl, and ldl concentrations from the medical records. the blood biochemical test was performed as follows. after an 8-hour overnight fast, blood samples were withdrawn from the subjects and placed in a refrigerator at 4°c. the serum was centrifuged for separation within 1 hour and then stored at −80°c until analysis. all biochemical parameters were measured using a roche modular chemistry analyzer (roche diagnostics, basel, switzerland) at the clinical laboratory of our hospital. the intra-and interassay coefficients of variation were <5% for all of the assays performed. the estimated glomerular filtration rate (egfr) was calculated by the chronic kidney disease epidemiology collaboration equation based on the serum creatinine concentration [14] . to minimize selection bias that might occur if we had excluded participants with missing data on bmi, ldl, hdl, glucose, uric acid, or creatine (<1%), we used multivariate imputation by chained equations to impute missing values. five datasets were generated, and the results were pooled according to rubin's rules. categorical variables are summarized as frequency and percentage, and continuous variables are presented as mean and standard deviation. the chi-squared test or mann-whitney test was used to compare the distributions of demographic and clinical characteristics between the taaas and control groups. next, we performed a univariable logistic regression analysis to detect the relationship between taaas and conventional risk factors including age, sex, smoking and drinking habit, diabetes, hypertension, cad, ischemic stroke, hyperlipidemia, egfr, and bmi. continuous parameters including age, egfr, and bmi were transformed into categorical variables based on the recognized clinical cutoff points or laboratory reference ranges in the logistic regression models. hhcy was defined as a circulating thcy level ≥ 15 μmol/l [15] . next, the independent association between the serum thcy level (as either a categorical variable or a nontransformed continuous variable as well as a log(e)transformed continuous variable because the distribution of the thcy level was found to be skewed toward the left) and taaas was determined by two different multivariable logistic regression models. adjusted odds ratios (ors) with corresponding 95% confidence intervals (cis) were estimated. the minimally logistic regression model was adjusted for sex and age. the fully adjusted model was adjusted for sex, age, smoking habit, hypertension, bmi, and egfr (variable changing the coefficient of the continuous thcy level by ≥10% when adding it into the basic logistical model including only the serum thcy level or when deleting it from the complete logistical model comprising all variables in the abovementioned univariable logistic regression analysis). finally, interaction and subgroup analyses were performed, including sex, age, smoking habit, hypertension, bmi, and egfr. all statistical analyses were performed with the software packages r (http://www.r-project.org, the r foundation, vienna, austria) and empowerstats (http://www .empowerstats.com, x&y solutions, inc., boston, ma, usa). a two-tailed p value of < 0.05 was considered statistically significant. characteristics. the subjects' baseline characteristics are summarized in table 1 . generally, patients with taaas were slightly older (64:59 ± 12:73 years vs. 59:29 ± 7:92 years, p < 0:001), and there were higher proportions of smokers (57.53% vs. 19.18%, p < 0:001), drinkers (34.25% vs. 18.72%, p < 0:01), those with hypertension (68.49% vs. 32.42%, p < 0:001), and those with a chronic medical history of cad(23.29% vs. 9.59%, p < 0:01) compared with individuals in the control group. the rates of patients with a chronic medical history of diabetes, hyperlipidemia, and ischemic stroke were not significantly different between the two groups. additionally, a significantly lower bmi (24:22 ± 3:78 kg/m 2 vs. 26:07 ± 3:33 kg/m 2 , p < 0:001), serum hdl concentration (1:12 ± 0:32 mmol/l vs. 1:21 ± 0:32 mmol/l, p < 0:05), and egfr (97:40 ± 30:21 ml/min per 1.73 m 2 vs. 112:05 ± 22:53 ml/min per 1.73 m 2 , p < 0:001) were observed in patients with taaas, whereas there were no statistically significant differences in the blood pressure, serum concentrations of cholesterol, triglycerides, ldl, glucose, or uric acid between the two groups. 3.2. hhcy and thcy levels. hhcy was found in 57 of 73 patients with taaas (78.08%) and 116 of 219 participants without taaas (52.97%), as is shown in figure 1 (a). the proportion of hhcy was significantly greater in the case than the control group (p < 0:001). likewise, the serum thcy level was significantly higher in the patients with taaas (median, 19.40; interquartile range, 16.50-23.10) than in those without taaas (median, 15.20; interquartile range, 11.60-19.90) with a p value of < 0.001 (figure 1(b) ). the univariate logistic regression analyses indicated that age, hypertension, cad, smoking and drinking habit, bmi, and egfr were significantly associated with the presence of taaas ( table 2 ). the results of the multivariate logistic regression models are shown in table 3 . after adjustment for confounders, the serum thcy level was independently associated with the risk of taaas in different multivariate logistic regression models (as either a categorical variable or continuous variable). subjects with hhcy had a 2.14-fold higher risk of taaas than those with a normal serum thcy level (adjusted or, 2.14; 95% ci, 1.00-4.56). similarly, each 1 μmol/l increase in the serum thcy concentration was associated with a 4% higher risk of taaas (adjusted or, 1.04; 95% ci, 1.00-1.07). the relationship remained significant when the log(e)-transformed thcy level was used in the multivariate logistic regression model (adjusted or, 2.98; 95% ci, 1.36-6.53). the results of the stratified and interaction analyses are presented in table 4 . the stratified analysis indicated no statistically significant association between hhcy and taaas in all stratified subgroups. nevertheless, hhcy tended to be associated with a greater risk of taaas in all stratified subgroups (adjusted ors > 1). furthermore, the interaction analysis revealed no interactive role in the association between hhcy and taaas. results and implications. this paper describes the first case-control study to address the association between the serum thcy level and risk of taaas. our main finding is that an elevated serum thcy level is independently associated with a higher risk of taaas. this association was robust when the serum thcy concentration was included as either a nontransformed or log(e)-transformed continuous variable or a categorical variable in the same multivariate logistic regression model and when different potential confounders were adjusted in two multivariate logistic regressions. the stratified analyses revealed that hhcy was associated with a greater risk of taaas in all stratified subgroups (adjusted ors > 1), although not statistically significant, which may have been a result of the limited sample size after stratification. furthermore, no interactive role was found in the association between hhcy and taaas. our main finding might have certain implications in identifying individuals at higher risk of developing taaas and tailoring more aggressive prevention. in addition, our results may help to detect higher risk among patients who already have taaas to provide earlier diagnostic strategies and treatments that can improve their prognosis. homocysteine is a thiol-containing nonessential amino acid derived from the metabolism of the essential amino acid methionine [13] . homocysteine can be degraded via the remethylation pathway in which 5methyltetrahydrofolate is the substrate, employing vitamin b 12 and folate as cofactors. it can also be converted into cysteine via the transsulfuration pathway catalyzed by cystathionine β synthase and vitamin b 6 [16] . consequently, the homocysteine level is affected by the folate and vitamin status. previous studies have proven that an elevated serum thcy level is associated with atherosclerotic diseases and intracranial aneurysms as well as aaas [7-10, 13, 3 biomed research international 17]. in the present study, we showed a similar association between the thcy level and taaas. the rarity of taaas has made the investigation of the molecular mechanisms underlying the association between hhcy and taaas difficult. however, basic research exploring the mechanisms underlying the association between hhcy and aaas (the more common and closely related aortic aneurysms) has been more comprehensively conducted. these studies have provided important clues for us to explain and understand the association mechanically. from a molecular perspective, taaas might be associated with the same pathological features as aaas, including degradation of the extracellular matrix, accumulation of reactive oxygen species, dedifferentiation or apoptosis of smooth muscle cells, and activation of various inflammatory cells [5, 18, 19] . hhcy may also mediate the formation of taaas through some biomed research international of these pathogenetic pathways. previous in vitro studies have shown that hhcy can increase serine elastase synthesis in aortic smooth muscle cells by 5-to 6-fold. elastase leads to fragmentation of elastin, which provides the aorta's elasticity [20] . elevated levels of thcy also reportedly increase the secretion of elastolytic metalloproteinase-2 and metalloproteinase-9 in human endothelial cells [21] . these phenomena account for, at least in part, the degeneration of extracellular matrix in taaas. previous studies have also shown that hhcy mediates formation of aaas through recruitment of monocytes and macrophages to the aortic wall and activation or polarization of macrophages into inflammatory m1 cells [18, 22, 23] , with subsequently aggravation of the aortic inflammation. furthermore, homocysteine also directly interacts and activates the angiotensin ii type i receptor to aggravate the vascular injury and then the development of aaas [24] . it is reasonable to consider that the abovementioned mechanisms are also implicated in the association between hhcy and taaas. however, the pathological features of taaas and aaas might be different to a certain extent [1] . basic researches should be carried out to identify the specific mechanisms underlying the association between hhcy and taaas. additionally, longstanding atherosclerosis is thought to be the most frequent cause of degenerative aneurysms [6] . hhcy promotes atherosclerosis by causing vascular injury and adversely affecting several cellular functions, including lipid dysregulation, programmed cell death, and inflammation [25] . hhcy might also lead to taaas by aggravating atherosclerosis. it might be effective to lower the serum thcy level by folic acid and vitamin b 12 or b 6 supplementation to decrease the risk of taaa formation in patients with hhcy. future prospective studies are warranted to address this issue. however, a previous randomized controlled trial with a large population was performed to investigate the effect of thcylowering therapy on the risk of major cardiovascular events in patients with vascular disease. the results showed that the thcy-lowering treatments did not decrease the incidence of myocardial infarction, death of any cause, or adverse events compared with placebo. only the incidence of stroke was reduced by thcy-lowering interventions [26] . one possible reason for the failure to demonstrate certain clinical benefits of the thcy-lowering interventions is that the study included patients who already had chronic thcy elevation with vascular alterations that could not be reversed by vitamin supplementation [25] . another reason may be that individuals with hhcy have an "hcy memory effect" as a result of epigenetic alterations, which could continue to promote pro-gression of cardiovascular complications even after the thcy level is lowered [13] . therefore, therapies targeting the epigenetic alterations should be developed to lower the risk of taaa formation and atherosclerosis together with folic acid and vitamin b 12 or b 6 supplementation. this study has several limitations. first, our study was retrospective in nature, and recall bias cannot be totally avoided. for example, the participants' self-reported histories of chronic disease might have affected the accuracy of estimation of the disease prevalence. second, the serum thcy level was measured after the diagnosis of taaas; therefore, the causality of the association between an elevated thcy level and taaas cannot be determined. third, our study had a limited sample size, and only a few patients were included in each subgroup after stratifying by conventional risk factors, yielding limited statistical power. this limitation especially concealed some meaningful results in the stratified and interaction analyses. fourth, this study mostly focused on the association between the thcy level and taaa occurrence, while further analysis involving the type, length, or maximum diameter of taaas was not performed. fifth, the c677t polymorphism in methylenetetrahydrofolate reductase (mthfr) closely affects the enzymatic activity of homocysteine metabolism. however, data on that genetic background was lacking, precluding assessment of the role of mthfr c677t polymorphism in the association between serum thcy concentration and taaa formation. finally, the serum levels of vitamin b 6 , vitamin b 12 , and folic acid were not measured, preventing us from assessing their interaction with the serum thcy level. despite the aforementioned limitations, this is the first attempt to explore the association between the serum thcy level and taaas and suggests that hhcy is independently associated with a higher risk of taaa formation. we believe that our study can provide a foundation for subsequent larger prospective cohort studies. thoracoabdominal aortic aneurysm thorakoabdominelles aortenaneurysma outcomes of 3309 thoracoabdominal aortic aneurysm repairs editor's choice -hospital incidence, treatment, and in hospital mortality following open and endovascular surgery for thoraco-abdominal aortic aneurysms in germany from 2005 to 2014: secondary data analysis of the nationwide german drg microdata epidemiology and natural history of thoraco-abdominal aortic aneurysms imaging of thoracoabdominal aortic aneurysms hyperhomocysteinaemia is an independent risk factor of abdominal aortic aneurysm in a chinese han population hyperhomocysteinaemia is an independent risk factor for peripheral arterial disease in a chinese han population hyperhomocysteinemia is an independent risk factor for intracranial aneurysms: a case-control study in a chinese han population homocysteine level and risk of abdominal aortic aneurysm: a meta-analysis abdominal aortic aneurysm and its correlation to plasma homocysteine, and vitamins plasma total homocysteine is associated with abdominal aortic aneurysm and aortic diameter in older men the potential role of homocysteine mediated dna methylation and associated epigenetic changes in abdominal aortic aneurysm formation a new equation to estimate glomerular filtration rate homocysteine and mthfr mutations the controversial role of homocysteine in neurology: from labs to clinical practice high prevalence of mild hyperhomocysteinemia in patients with abdominal aortic aneurysm b cell-derived anti-beta 2 glycoprotein i antibody contributes to hyperhomocysteinemiaaggravated abdominal aortic aneurysm immune cells and molecular mediators in the pathogenesis of the abdominal aortic aneurysm homocysteine induces synthesis of a serine elastase in arterial smooth muscle cells from multi-organ donors homocysteine modulates the proteolytic potential of human vascular endothelial cells hyperhomocysteinemia exaggerates adventitial inflammation and angiotensin ii-induced abdominal aortic aneurysm in mice macrophage inflammasome mediates hyperhomocysteinemia-aggravated abdominal aortic aneurysm homocysteine directly interacts and activates the angiotensin ii type i receptor to aggravate vascular injury evaluation of mild hyperhomocysteinemia during the development of atherosclerosis in apolipoprotein e-deficient and normal mice homocysteine lowering with folic acid and b vitamins in vascular disease we thank angela morben, dvm, els, from liwen bianji, edanz editing china (http://www.liwenbianji.cn/ac), for editing the english text of a draft of this manuscript. j.d. appreciates the company and support from his parents and wenhan cai during the covid-19 pandemic. hhcy: hyperhomocysteinaemia taaas: thoracoabdominal aortic aneurysms cad:coronary artery disease aaas: abdominal aortic aneurysms hdl:high-density lipoprotein cholesterol ldl:low-density lipoprotein cholesterol egfr: estimated glomerular filtration rate bmi:body mass index. all of the data supporting the findings in this study are available from the corresponding author upon request. the authors have no competing interests. key: cord-001525-b7kbyp3s authors: zadrazilova, iveta; pospisilova, sarka; pauk, karel; imramovsky, ales; vinsova, jarmila; cizek, alois; jampilek, josef title: in vitro bactericidal activity of 4and 5-chloro-2-hydroxy-n-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides against mrsa date: 2015-01-15 journal: biomed res int doi: 10.1155/2015/349534 sha: doc_id: 1525 cord_uid: b7kbyp3s a series of nine substituted 2-hydroxy-n-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant staphylococcus aureus (mrsa) and s. aureus atcc 29213 as the reference and quality control strain. the minimum bactericidal concentration was determined by subculturing aliquots from mic determination onto substance-free agar plates. the bactericidal kinetics of compounds 5-chloro-2-hydroxy-n-[(2s)-3-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (1f), n-{(2s)-1-[(4-bromophenyl)amino]-3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide (1g), and 4-chloro-n-{(2s)-1-[(3,4-dichlorophenyl)amino]-3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide (1h) was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x mic; aliquots were removed at 0, 4, 6, 8, and 24 h time points. the most potent bactericidal agent was compound 1f exhibiting remarkable rapid concentration-dependent bactericidal effect even at 2x mic at 4, 6, and 8 h (with a reduction in bacterial count ranging from 3.08 to 3.75 log(10) cfu/ml) and at 4x mic at 4, 6, 8, and 24 h (5.30 log(10) cfu/ml reduction in bacterial count) after incubation against mrsa 63718. reliable bactericidal effect against other strains was maintained at 4x mic at 24 h. the antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems [1] . methicillin-resistant staphylococcus aureus (mrsa) has become the most common clinically relevant multiresistant pathogen [2] causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% [3] . the prevalence of mrsa is increasing worldwide and, according to the latest information of the european centre for disease prevention and control from 2012 [4] , can be considered alarming in some european countries, especially in portugal and romania, where ≥50% of all s. aureus isolates from invasive infections were identified as mrsa in 2012 (although, e.g., in romania the prevalence of mrsa was 25-50% in 2010), followed by italy, greece, and poland with 25-50% isolates being mrsa in 2012 (for comparison, in poland mrsa isolates constituted 10-25% from all s. aureus isolates in 2010). the treatment failure of vancomycin, the therapeutic anti-mrsa agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration (mic) values (i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism) within the susceptible range was described previously [5, 6] . thus, the emergence of mrsa (and vancomycin-resistant s. aureus in the recent years as well [7] ) makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria [8] . however, for the treatment of s. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs [9] . this fact should be considered during the development of effective and safe treatment options for mrsa infections. the history of clinical usage of salicylanilides (2-hydroxy-n-phenylbenzamides) dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s [10] . nowadays, salicylanilides (sals) are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic [11] , antibacterial [12, 13] , antimycobacterial [13] , antifungal [14] , and antiviral [15, 16] , among others. despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. sals have been found to inhibit the two-component regulatory systems (tcs) of bacteria [17] . the latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis [18] . furthermore, salicylanilides have been recognised as inhibitors of some bacterial enzymes, such as sortase a from s. aureus [19] , d-alanine-d-alanine ligase [20] , or transglycosylases from s. aureus (but not from m. tuberculosis) [12] . these enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: (i) methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and (ii) isocitrate lyase, which is essential for the metabolism of fatty acids [21] . thus, sals seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. such new agents could be a solution to the resistance challenges. this study is a follow-up paper to a recently published article [13] . the synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-n-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides, called diamides due to their skeleton (for general structure see table 1 ), was described previously [13, 22] , and their antimycobacterial and antibacterial activities against various bacterial species were reported [13] . as these compounds expressed very significant antibacterial activity with low mic values against clinical isolates of mrsa as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against mrsa. the aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of mrsa as representatives of multidrug-resistant bacteria. to the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of sal analogues and revealing their bactericidal effect. the synthetic pathway of the series of novel diamides was described recently [13, 22] , and their structures (see table 1 ) were confirmed by ir, nmr, and ms spectrometry, and the purity of the compounds was checked by chn analysis [13, 22] . [27] ; and mrsa sa 3202 [27] (national institute of public health, prague, czech republic) both of human origin. suspected colonies were confirmed by pcr; a 108 bp fragment specific for s. aureus was detected [28] . all isolates were tested for the presence of the meca gene encoding methicillin resistance [29] . these three clinical isolates were classified as vancomycin-susceptible (but with higher mic of vancomycin equal to 2 g/ml (va2-mrsa) within the susceptible range for mrsa 63718) methicillinresistant s. aureus (vs-mrsa). for the mics of vancomycin, see table 1 . vancomycin-susceptible methicillin-susceptible staphylococcus aureus (vs-mssa) atcc 29213, obtained from the american type culture collection, was used as the reference and quality control strain. the bacteria were stored at −80 ∘ c and were kept on blood agar plates (columbia agar base with 5% ovine blood) between experiments. (mbcs) . the mbcs (i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium) were determined by subculturing aliquots (20 l) from wells with no visible bacterial growth and from control wells of mic determination onto substance-free mueller-hinton agar (mha) plates. the plates were incubated aerobically at 37 ∘ c for 24 h for colony count. the mbc was defined as the lowest concentration of substance, which produced ≥99.9% killing table 1 : chemical structures and in vitro mic and mbc [ g/ml] values of tested 5-and 4-chloro-2-hydroxy-n-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides (bactericidal effect of individual compounds against particular strains marked in bold). after 24 h of incubation as compared to the colony count of the starting inoculum [30] . to ensure reproducibility, each mbc assay was performed in at least triplicate on separate occasions. n h o h n o oh 1 2 r 1 r 3 r 2 comp. r 1 r 2 r 3 mic [ g/ml] mbc [ g/ml] 1 2 3 4 1 2 3 4 1a 5-cl 4-ch 3 (s)-ch 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-cl 4-ch 3 (s)-ch(ch 3 ) 2 >256 >256 32 32 >256 >256 128 >256 1c 5-cl 4-ch 3 (s)-benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-cl 4-ch 3 (r)-ch 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-cl 4-och 3 (s)-ch(ch 3 ) 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-cl 4-cf 3 (s)-ch(ch 3 ) 2 4 2 2 2 4 4 8 4 1g 4-cl 4-br (s)-ch(ch 3 ) 2 8 4 4 4 1 6 8 8 8 1h 4-cl 3,4-cl (s)-ch(ch 3 ) 2 2 1 1 1 4 1 4 2 1i 4-cl 3,4-cl (s)-benzyl 1 1 0.5 0.5 8 1 8 1 amp >16 >16 >16 0.25 >16 >16 >16 0.25 cpx >16 >16 >16 0.5 >16 >16 >16 0.5 van 2 1 1 1 2 1 1 1 time-kill assays were performed by the broth macrodilution method according to previously described methodology [30] with some modifications. briefly, flasks containing sterile fresh mueller-hinton broth (mhb) with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 cfu/ml (actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 cfu/ml) and a final concentration of the antibiotic equal to 1x, 2x, and 4x mic in 10 ml volume. for the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots (20 l) were plated on mha plates in duplicate. colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. the plates were incubated at 37 ∘ c for 24 to 48 h, and the number of colonies was determined. to ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. the growth control without the addition of antimicrobial agents and the control containing dmso without any antimicrobial agent to exclude antibacterial activity of this solvent were included. time-kill curves were constructed by plotting the log 10 cfu per millilitre versus time (over 24 h), and the change in bacterial concentration was determined. the results were analysed by evaluating the numbers of strains that yielded δ(log 10 cfu/ml) values of −1 (corresponding to 90% killing), −2 (99% killing), and −3 (99.9% killing) at 4, 6, 8, and 24 h compared to counts at 0 h. bactericidal activity was defined as a reduction of at least 99.9% (≥3 log 10 ) of the total count of cfu/ml in the original inoculum. diamides seem to be promising candidates for antibacterial agents with very strong anti-mrsa activity, as it was published recently [13] . in the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of mrsa, and staphylococcus aureus atcc 29213 (methicillin-susceptible) as the reference and quality control strain. since sals and their analogues are known as compounds with bacteriostatic effect [31] , this is the first study where sal-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. recently mic values of diamides expressed as molar concentrations in mol/l were published [13] . to allow comparison with mbc values of the present study, mics in g/ml were calculated and are recorded in table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. potential bactericidal activity of diamides was assessed using mbc assay [26] . mbc values of all tested compounds are recorded in table 1 as well. based on the obtained results, all compounds assessed as active according to mic values in our previous study (1f-i) showed low or moderate mbc values against all four strains. the mbc values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/ml. in all cases, there were comparable mbc values for the clinical isolates of mrsa and the s. aureus reference strain. bactericidal activity is defined as a ratio of mbc to mic of ≤4 [32] . table 1 bactericidal activity is expressed in bold. as mentioned above, sals are known to exhibit a bacteriostatic effect [31] , so it was very interesting to discover that diamides possess bactericidal activity. the amide bond (-conh-) can cause interactions with a variety of enzymes [33] ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against mrsa. the activity of sals and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that sals are capable of inhibiting transglycosylases in later stages of s. aureus (including mrsa) cell wall biosynthesis [12] . these enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid ii, which occurs at the outer face of the membrane [12] . since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents [30, 34] , the failure in the cell wall biosynthesis due to the inhibition of transglycosylases could be responsible for bactericidal activity of diamides against mrsa. based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time. 1-oxobutan-2-yl}-2-hydroxybenzamide (1h) were tested in time-kill studies at 1x, 2x, and 4x mic against all mrsa isolates and the s. aureus reference strain. the antibacterial effect of dmso [35] used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. the extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log 10 cfu/ml in viable cell count at different times after incubation. a summary of these data is presented in table 2 . based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. no bactericidal activity (i.e., ≥3 log 10 cfu/ml decrease) was observed at 1x mic for any strain and time after incubation tested. at 4x mic from the four strains, compounds 1f, 1 g, and 1h killed 2, 1, and 2 strains, respectively, at 8 h after incubation and 4, 2, and 2 strains, respectively, at 24 h after incubation. the findings of time-kill studies for each of the four staphylococci strains at exposure to compounds 1f, 1g, and 1h are summarized in table 3 . bactericidal activity (i.e., ≥3 log 10 cfu/ml decrease) is expressed in bold. for compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of mrsa 63718. time was not the predictive factor influencing the antibacterial activity because log 10 differences in cfu/ml from the starting inoculum were the same for 4x mic (with the highest efficiency with a reduction in bacterial count of 5.30 log 10 cfu/ml) or very similar for 2x mic (with a moderate regrowth after 24 h causing a loss of bactericidal activity) over 24 h. the bactericidal effect was maintained even at 2x mic at 4 h after incubation for this strain (reduction of 3.08 log 10 cfu/ml). for the remaining strains, clinical isolates of mrsa sa 630, mrsa sa 3202, and s. aureus atcc 29213, reliable bactericidal effect was recorded at 4x mic at 24 h after incubation for all these strains with a reduction in bacterial count of 3.22, 3.30, and 3.65 log 10 cfu/ml, respectively. for compound 1g bactericidal effect against mrsa 63718 was noticed at 2x mic at 6 and 8 h after incubation and at 4x mic at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 cfu/ml. the most effective killing was achieved at 6 h for both concentrations. as in the case of compound 1f, a regrowth was observed after 24 h after incubation. for the remaining isolates of mrsa, sa 630 and sa 3202, bactericidal effect occurred only at 4x mic at 24 h after incubation with a reduction in bacterial count of 3.38 and 4.01 log 10 cfu/ml, respectively. the highest bactericidal effect was recorded for mrsa sa 3202 at 4x mic at 24 h after incubation. a reduction consistent with bacteriostatic effect (0.03 to 2.37 log 10 cfu/ml) was observed at other concentrations over time for both isolates. no bactericidal effect was observed for the s. aureus reference strain; compound 1g demonstrated a pattern of bacteriostatic activity against this strain with a reduction in bacterial count ranging from 0.07 to 2.33 log 10 cfu/ml at 4x mic over time. in other cases, a slight increase in bacterial counts (i.e., overgrowth) compared with the starting inoculum was observed with values ranging from 0.10 to 1.57 log 10 cfu/ml for this reference strain. for compound 1h bactericidal effect against mrsa 63718 was maintained at 4x mic at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 cfu/ml, respectively. the same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation. regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. the reason for regrowth of the test organism at 24 h in the experiment is unknown. most probably, selection of resistant mutants is responsible for this phenomenon [30] ; degradation of the drug in the growth medium is not assumed, as regrowth was number of strains showing the following log 10 cfu/ml decrease a at the designated incubation time not observed for any other tested strain. for mrsa sa 630 concentration-dependent killing was recorded at 4x mic at 6, 8, and 24 h after incubation with log 10 differences in cfu/ml from the starting inoculum being very similar over time (ranging from 3.18 to 3.39 log 10 cfu/ml). for mrsa sa 3202 reliable bactericidal effect was maintained only at 4x mic at 24 h after incubation with a reduction in bacterial count of 3.02 log 10 cfu/ml. as for compound 1g, bacteriostatic activity against s. aureus reference strain was observed with a reduction in bacterial count ranging from 0.34 to 2.62 log 10 cfu/ml at 2x and 4x mic. overgrowth (values ranging from 0.04 to 1.43 log 10 cfu/ml) was recorded at 1x mic for this strain. it is of note that in all staphylococci strains with similar mics and mbcs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied with clinical strains of mrsa being effectively killed and the reference strain remaining unaffected at 4x mic. there is a discrepancy between bactericidal results of mbc assay compared with time-kill kinetics. this difference could be caused by comparing microtiter (mbc assay) to macrobroth (time-kill assay) dilutions [36] . moreover, although time-kill assays are more labour intensive and time consuming than mbc assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents [37] . concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then [34] . however, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load [38] . moreover, in the case of an immune system disorder (e.g., immunosuppressive therapy, aids patients, etc.) bactericidal agents are unequivocally indicated. considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds [30] . the clinical outcome of mrsa bacteraemia is significantly influenced by vancomycin mic. treatment failure exceeding 60% for s. aureus with vancomycin mic of 4 g/ml resulted in the change of susceptibility breakpoint from 4 g/ml to 2 g/ml by the clinical and laboratory standards institute (clsi) in 2006 [23] as well as by the us food and drug administration (fda) in 2008 [39] . it has been recommended that for infections caused by mrsa strains with elevated vancomycin mics (2 g/ml), alternative therapy should be considered [40] . it is of note that based on time-kill assays in the present study, all tested diamides (particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x mic) were most effective against isolate mrsa 63718, which is the strain with elevated vancomycin mic of 2 g/ml. the activity against the remaining isolates with vancomycin mic of 1 g/ml was lower. considering the emergence of decreasing vancomycin susceptibility of mrsa isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against mrsa in vitro. based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. the present study is the first evidence of bactericidal effect of sal analogues. 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dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-ninth edition a timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant staphylococcus aureus evaluation of different methods to detect oxacillin resistance in staphylococcus aureus and their clinical laboratory utility species-specific and ubiquitous-dna-based assays for rapid identification of staphylococcus aureus antimicrobial susceptibility testing protocols multiple mechanisms of action for inhibitors of histidine protein kinases from bacterial two-component systems in vitro biofilm formation and bactericidal activities of methicillin-resistant staphylococcus aureus clones prevalent in korea acetylcholinesterase-inhibiting activity of salicylanilide -alkylcarbamates and their molecular docking in vitro antimicrobial activity of dimethylsulfoxide activity of telavancin compared to other agents against coagulase-negative staphylococci with different resistotypes by time kill methods for determining bactericidal activity of antimicrobial agents; approved guideline mrsa bacteraemia fda lowers vancomycin breakpoints for staphylococcus aureus vancomycin therapeutic guidelines: a summary of consensus recommendations from the infectious diseases society of america, the the authors would like to thank marie slavikova for her help and excellent laboratory cooperation. they thank also helena zemlickova from national institute of public health, prague, czech republic, for providing clinical isolates of mrsa. this study was financially supported by iga vfu brno, projects nos. 65/2012/fvl and 52/2014/faf, and by project "ceitec-central european institute of technology" (cz.1.05/1.1.00/02.0068) from european regional development fund. the authors also wish to acknowledge the institutional support of the faculty of chemical technology, university of pardubice to the ministry of education, youth and sports. the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord-309319-si5c14e8 authors: cao, chunxiang; chen, wei; zheng, sheng; zhao, jian; wang, jinfeng; cao, wuchun title: analysis of spatiotemporal characteristics of pandemic sars spread in mainland china date: 2016-08-15 journal: biomed res int doi: 10.1155/2016/7247983 sha: doc_id: 309319 cord_uid: si5c14e8 severe acute respiratory syndrome (sars) is one of the most severe emerging infectious diseases of the 21st century so far. sars caused a pandemic that spread throughout mainland china for 7 months, infecting 5318 persons in 194 administrative regions. using detailed mainland china epidemiological data, we study spatiotemporal aspects of this person-to-person contagious disease and simulate its spatiotemporal transmission dynamics via the bayesian maximum entropy (bme) method. the bme reveals that sars outbreaks show autocorrelation within certain spatial and temporal distances. we use bme to fit a theoretical covariance model that has a sine hole spatial component and exponential temporal component and obtain the weights of geographical and temporal autocorrelation factors. using the covariance model, sars dynamics were estimated and simulated under the most probable conditions. our study suggests that sars transmission varies in its epidemiological characteristics and sars outbreak distributions exhibit palpable clusters on both spatial and temporal scales. in addition, the bme modelling demonstrates that sars transmission features are affected by spatial heterogeneity, so we analyze potential causes. this may benefit epidemiological control of pandemic infectious diseases. in recent years, emerging infectious disease of severe acute respiratory syndrome (sars) has become one of the most egregious public health problems in 21st-century china. sars is caused by a new pathogen that was finally identified as a coronavirus (sars-cov) [1, 2] . sars-cov is regarded as a person-to-person infectious disease that infects suspected individuals through droplet transmission [3] . after infection, patients go through a 2-to-10-day incubation period before typical symptoms (fever, cough, and body aches) appear. during the incubation period, the patients are contagious to surrounding people [4] . to date, there is no vaccine for sars, no reliable diagnostic test, and no specific treatment. this leads to the need for refinement of public health controls to be effective [5, 6] . much loss of life, a high mortality rate, and substantial wealth damage were associated with sars throughout a wide part of china in 2002 and 2003. the first case of sars was identified and confirmed by the national laboratory of china in foshan, a city in guangdong province, south china, on november 16, 2002 . with rapid development over 3 months, sars became a disease that was difficult to control, and it spread to other cities through regular population movement [7] . according to the world health organization (who), after nearly 9 months of spread (november 16, 2002 16, , through july 13, 2003 , 29 countries had been affected by sars. in this period, 8096 persons were infected and 774 died, which caused domestic panic in countries of east and southeast asia [8, 9] . the evidence shows patterns of vast geographic transmission that distinguish sars from other epidemic infectious diseases. traditionally, epidemiologists focus on the temporal epidemic evolution and describe it through various types of mechanical models [4, 10, 11] . riley models have been unable to effectively explain its dynamics. as a result, the pandemic cannot be explained as a combination of endemics. complex spatial transmission networks [12] , compound period epidemics, and overwhelming government controls inspired epidemiologists to research this type of person-to-person infectious disease more profoundly with respect to its geographic features. dye and gay [13] suggested that doing so could lead to variations in average contact rate. lloyd-smith et al. simulated sars dynamics in a complex network based on an epidemiological mechanism with full consideration of population, contact rate, control strategies, and spatial diffusion [14, 15] . however, their approach requires the assumption of numerous parameters, which in turn require the support of detailed data. the approach makes it difficult to explain overall spatial transmission without geographic epidemic analysis. wang et al. [7] unveiled the broad existence of spatial clusters during the beijing epidemic periods, influencing government intervention [16] . this work inspired us to investigate spatial principles in sars transmission, which could include significant indications for pandemics within a more general perspective. a study that combines both a temporal epidemic study and spatial transmission is required to understand sars dynamics. recent advances in geostatistical analysis, which have been effectively used in infectious disease mapping, health risk assessment, and epidemiological studies, could be applied in sars research for spatiotemporal analysis [17, 18] . the bayesian maximum entropy (bme) approach of modern geostatistics incorporates higher-order statistical estimation for space-time epidemic phenomena and has shown more accurate mapping results than those derived from linear kriging geostatistics [19] . we benefited from such advantages in investigating statistical features of the sars epidemic and in simulating geographic transmission aspects on a day-byday basis. we focused on exploring and understanding spatial heterogeneity impacts of sars transmission and simulating and mapping the disease temporally. under the increasing threat of pandemics in 21st century, the present study illustrates actual epidemic spread and provides useful information toward establishing government intervention to preventing pandemics. the surveillance sanitary system of the chinese centre for disease control and prevention (chinese cdc) monitored all sars infections in mainland china and confirmed them in the national microbe laboratory. the infections were recorded cautiously and totaled 5318 cases. the data were updated and include cases from november 2002 through june 2003. a confirmation system slightly different from that of who statistics raised the number of cases to 5327. the recorded information includes basic patient information (e.g., name, sex, age, and occupation; figure 1 ), spatial information (including registered permanent residence location, company, first symptom onset location, current resident location, and hospital location), and temporal information (e.g., dates of first symptom onset, hospital treatment, recovery, or death). the daily number of sars infections and their temporal characteristics were analyzed and are shown in figure 2 . in mapping all cases, the location of infection onset is an elaborate process. even though some cases were recorded at locations using various scales, the records were eventually sorted at county level to facilitate processing. considering that population movement is one of the effects on sars diffusion, the following order was observed in the geocoding process: (1) registered permanent residence; (2) temporary residence (work address); (3) onset location; and (4) reporting unit location. to maintain rigorous containment, sanitary departments collected minute information for spatial indexing. thus, the registered permanent residence was selected as the first index for the benefit of data integrity and for monitoring the impact of mass population movement. on february 1, 2003, during the period of sars outbreaks, the spring festival took place. people commonly travel to join family for this traditional chinese festival, returning to their permanent residence after 1-4 weeks. this population movement increased the probability of cross infection among family members and travellers. consequently, with some exceptions, the temporary residence, onset, and reporting unit locations could be additional spatial information in geocoding the infections. some cases lacked records of both permanent residence and workplace, and the only available addresses for these were syndrome onset locations. if all the above addresses were void, the hospital address was used as the only thread to locate the patient, although this was rare in our research. we focused on outbreak regions for convenience and clarity in the process. there are 194 counties in the focus area, which is described in greater detail in subsequent sections. among them, beijing, guangzhou, and taiyuan experienced the most severe sars outbreaks. we used a 2002 county-level digital map to illustrate the epidemic ( figure 3 ). in temporal mapping, we also sorted the cases chronologically. all records were sorted at daily level. the onset date of patients was set as the optimal time index. prior to this date, there was a 2-8-day incubation period [8, 20] , which is generally intractable. the period from onset to hospitalization, including the incubation period, was considered the contagion period. because it is impossible to know the infection date accurately for a patient, incubation period data are unavailable. therefore, onset date was the first temporal index in our research. if this date was unavailable, then the hospitalization date was used as a secondary index. typical modes of investigation in health research focus on discovering spatial patterns or establishing a time series to study the principles that govern health phenomena. separation of the space-time domain into spatial and temporal components permits analysis of those subdomains individually but ignores principles and correlations that may exist because of the composite spatiotemporal structure [21] . in infectious disease research, and especially the study of person-to-person contagious diseases, epidemic conditions are closely related to both spatial and temporal dimensions. thus, proper relationships must be constructed to combine and account for the space-time continuum. bme provides an effective stochastic method, based on a cogent theoretical and technological strategy, to analyze relationships of sars outbreaks in the composite space-time domain. it comprises not only epidemiological knowledge bases but also spatiotemporal statistics and dynamic modelling aspects for study. it also offers a software framework for modelling and prediction of epidemic conditions across space-time [22, 23] . details include the following. (1) bme integrates knowledge from epidemiology into geographic information system science by assimilating epidemic laws, empirical relations, and statistical calculations in sars studies. (2) bme considers spatial heterogeneity in sars outbreaks, which broadens the traditional epidemic research field from the temporal to space-time domain. (3) bme simulates sars transmission and predicts diffusion tendency. the resultant study of these processes can reflect interesting underlying principles behind sars spread. for stochastic representation of the outbreaks, we considered the daily number of outbreaks as a random variable within a three-dimensional space-time random field (s/trf) [24] . then each of the sars records is a single realization out of all possible values that can be observed at a specific space-time location. two of the s/trf dimensions correspond to geographic coordinates of the records, and the third dimension axis represents the temporal dimension. in this sense, we studied the sars outbreaks in a composite spatiotemporal domain, in which each observed record is uniquely represented by the space-time vector = ( , ). bme takes these records as individual s/trf points, to which each is assigned a spatial location and temporal instance. to illustrate the composite spatiotemporal approach, consider a spatial-only map at the time instance 0 , represented by where [ the bme method operates in three successive stages to analyze the stochastic epidemic process. in the first stage, structural characteristics of a space-time random field are incorporated in the analysis by means of all available epistemic information about the s/trf. this information comes from theoretical or empirical sources related to the procedure and is known as the general knowledge base or g-kb. at the end of the first stage, the input allows the computation of probability density functions (pdfs) that describe the s/trf based on the g-kb. in the case of the sars outbreaks, we used the observations to explore general structural characteristics of the sars s/trf, that is, the existence of mean (or surface) trends in the space-time domain, and to explore the underlying temporal and spatial structure of the s/trf with suitable covariance functions. the second stage relates to the selection of case-specific information so that bme can assess characteristics and perform inference given the particular s/trf realization facilitated by the recorded information. this information is known as the specific knowledge base or s-kb. the sars sampling dataset that was described in the previous section consists of sampling points ( 1 , 2 , . . . , ) and comprised the s-kb in this study. in the final stage, bme integrates the g-kb and s-kb to compute updated prediction pdfs at selected space-time locations. the prediction or posterior pdfs provide a complete statistical description of the health attribute distribution in space-time, and they enable selection of a predictor of choice for assessment of the sars outbreak distribution. in traditional analysis, health attributes exhibit higher similarity for closer occurrences. consequently, spatial distance is typically considered as the sole substantial factor that describes and demonstrates autocorrelation and the underlying disease field structure. however, the epidemic time is also important in the study of disease spread, which could extend further with rapid transportation and population movement. the previous discussion leads us to define a composite spacetime distance, , as which depends on both spatial and temporal distances ( and , resp.), connected through an appropriate spatiotemporal metric . hence, the distance combined with temporal effects is more representative in epidemic analysis. in our research, = 1 − 2 and = 1 − 2 for two s/trf points 1 ( 1 , 1 ) and 2 ( 2 , 2 ). once distances are defined within the s/trf, permissible functions can be used to describe correlations in space-time. the covariance function that yields spatial and temporal autocorrelation between two points, and , is given by in this function, ( ) and ( ) represent a pair of realizations (possibilities) at and in the sars outbreak s/trf. it is expected that the observation data sample statistics should be the same as the s/trf. for example, the data mean model should coincide with the average statistic, and the empirical covariance model should follow the covariance statistic of the field. let be the arithmetic covariance. is presumed to be a theoretical function that is the same as the traditional covariance models in kriging analysis, such as the spherical, exponential, and nugget models. following this initial setup, the classic bayesian model is then incorporated, as shown by is a normalization constant and is the domain field of . in (4), note that the posterior pdf on the left side is conditioned upon the bme prior pdf, which is given in more detail by with the support of bme, we can compute various statistical quantities, such as the mean, covariance, semivariogram, and higher-order statistics, because bme estimates the posterior pdf according to the constraints. for instance, we sought the most probable value for and so computed the differential coefficient for that gives the prediction pdf mode, that is, where = 0 is a constant, ( ), = 0, . . . , are functions representing the stochastic g-kb information, and , = 0, . . . , are the so-called lagrange coefficients that are in spatiotemporal coordinates. the latter serve as weights for the corresponding ( ) functions in (7). bme theory develops a system of equations for each value of ( ), from which the coefficients are calculated. subsequently, their values are inserted in (4) and (6) to evaluate the posterior pdf, whose maximum gives the prediction mode estimate. a cross-validation procedure is followed to assess bme mapping accuracy. specifically, we use a set of ref validation data that were randomly selected as references for accurate estimation. we take turns to exclude each validation datum and calculate its value at the corresponding spatiotemporal coordinates, and then we compute the mean square error (mse), given by thus, the mse provides a measure of the uncertainty of bme estimation. in (8) , ref, is the bme estimate and is the th validation observation value. implementation. bme computations were performed with the specialized matlab-based interactive software interface known as the spatiotemporal epistemic knowledge synthesis graphical user interface, or seks-gui [21] . seks-gui is a rich-featured intuitive visual interface that provides a variety of g-kb and s-kb information types, adjusts model parameters for bme mapping, performs the analysis, and visualizes the predictions [25] . all types of data were input to the seks-gui software. the original observations were sorted in a file, in which each row corresponds to a certain spatial location, date, and outbreak condition. for computing, outbreak cases recorded at county level had their spatial index converted to longitude and latitude of the corresponding county centroid. this conversion typically introduces additional uncertainty into the result, because we effectively approximate the spatial reference for each observation. but it was used in our case because these uncertainties were considered acceptable, for the following two reasons. first, all counties with sars outbreaks were in the south and east of mainland china. these locations have high population densities and relatively small county areas. second, the sars infections were overwhelmingly observed in towns near their county centroids. there were 950 outbreak cases observed in 194 locations. in addition to the previous activities, we had to define the study area. west china has only ∼1% of the total national population but covers nearly half the country's area. for statistical homogeneity, the data of outbreak numbers should have a normal distribution. in our study, the observation cumulative density function (cdf) showed as much as 36.99% deviation from the normal cdf distribution (figure 4(a) ). thus, the original data were transformed using the normal scores method, which forces the original data distribution into the shape of the normal distribution (figure 4(b) ). following estimation, the data are back-transformed to the original value space. for structural correlation analysis of the spatiotemporal epidemic spread, we incorporated information from the empirical second statistical moment of the sars s/trf. specifically, we computed the empirical spatiotemporal covariance and then fit a suitable permissible mathematical model to describe it for bme computation. figure 5 depicts both the empirical and fitted theoretical spatiotemporal covariances characterizing the sars s/trf. figure 5 shows normalized covariance values, in which darker colors correspond to higher correlation. note that spatial correlation has some fluctuation close to zero distance. that is, covariance in space decreased rapidly and then briefly rebounded. this behavior indicates a potentially smallerscale correlation that we could not detect with our method. correlation in the temporal axis decreases more smoothly from its maximum value to a minimum point far from the temporal origin. given this behavior of empirical covariance, we fit the following theoretical covariance model, which has a sine hole spatial component and exponential temporal component: where is distance and sin( ) indicates a sine hole spatial component; the sill coefficient 0 is estimated at ∼1.0068; the spatial lag coefficient = 0.45 and the temporal lag coefficient = 60. using the aforementioned covariance model, we obtained bme estimates representative of the pandemic-spread behavior. the approach above allowed for a different possible explanation for the rapid decline and recovery in spatial correlation in figure 5 , as follows. a spatial lag coefficient = 0.45 is in good agreement with the first geographical law; that is, the numbers of outbreak events that were <0.45 degrees in longitude (∼50 km) apart had greater similarity to corresponding numbers of closer events. inversely, there was little to no spread relationship between numbers of outbreak events >2 degrees (∼220 km) apart. however, from a different perspective, two locations >220 km apart possibly showed increased correlation because of population movement between more remote locations. the accurate fit of the exponential model to empirical temporal covariance suggests that sars outbreak events belong in the category of traditional person-to-person epidemics. accordingly, the specified temporal coefficient of 60 days is based primarily on the duration observed in the most severely affected cities, such as beijing, guangzhou, and taiyuan. it is technically possible to limit the number of observations that are simultaneously used for bme estimation at each output grid. this is a necessary step to balance between adequate numbers of neighbors that should be used for estimation at a specific point. this is based on the assumed spatiotemporal correlation ranges on the one hand and the pragmatic need to maintain reasonable computation time for estimation, on the other. in our case study, we used up to 50 of the closest pieces of data for each estimation location. based on the spatial lag and temporal coefficients in (9) and considerations in the previous subsection, we defined the maximum spatial and temporal ranges for neighbor search to be 15 degrees in longitude and 20 days, respectively. the parameters were set to refine calculations within reasonable estimation ranges, as follows. first, given the limited period of spread, the sars infection presumably could not reach a location 15 degrees away within mainland china, even considering rapid population transportation by air. then, the observed data suggest that taking the 50 pieces of data nearest an estimation point essentially considers average temporal ranges of 5 days and distances ∼10 degrees. the mse of 0.69 indicates that the bme estimation in this case was effective and the accuracy was acceptable. finally, we estimated the outbreaks by choosing a spatiotemporal metric parameter value of 0.3. this parameter was used to convert the space-time coordinates of any location into a common spatiotemporal distance in the continuum of sars outbreak s/trfs. for instance, with the above choice, the spatiotemporal distance of two outbreaks occurring at the same location 10 days apart is roughly equal to that of two simultaneous outbreaks three degrees apart. the aforementioned parameter value was based on correlation considerations and the spatiotemporal covariance in (9). according to our analysis, the sars epidemic was divided into two distinct phases, namely, endemic spread and pandemic spread. the result, refined to 220 km and 60 days, shows that the sars outbreaks were effectively captured by spatiotemporal autocorrelation. sars is a typical personto-person, rapidly spreading infectious disease, and it takes about 60 days to spread from an affected area to adjacent ones. the process of outbreak spread to the adjacent areas is a stochastic one, owing to the synergistic effect of three factors. first, the sars outbreaks were related to population density. urban areas have higher population density than rural and residential areas, so for an outbreak in a city, it is more likely that it will spread within the urban limits than outside those limits. for example, among the total 5318 cases, 1934 were recorded in beijing. this number is much higher than the number of outbreak cases observed in nearby cities and entire counties. second, cities typically have convenient transportation to nearby cities and counties. thus, commuting and population exchange between cities and nearby locations is likely to be much greater than corresponding activity between those cities and more distant areas. consequently, suspected individuals from the nearby countryside are more likely to have travelled to a city and contacted infected people, and vice versa. this would increase the probability that the disease spreads in urban areas and their environs. the third and final factor is that control measures limit population transport, so the disease is likelier to have greater spread over nearby areas. following the sars outbreaks, the chinese government established strict control measures for quarantine of the suspect population. people travelling from affected areas were mandated to undergo several diagnostic tests during their trip. as a result, the entire transportation system worked as a rigorous diagnostic tool for potential sars cases, and all train, airplane, and long-distance bus passengers were surveilled for the epidemic. among the measures, individual travellers had their temperature taken. if this was above 37.5 ∘ c, the individual would be forced into a state of quarantine in isolation wards until the temperature recovered to normal levels. such strict measures were effective in restricting outbreak spread to limited areas for each city [26, 27] . bme estimation led to informative results, shown in the plot of figure 6 and the maps of figure 7 . this output reveals higher temporal correlation and relatively low spatial correlation for the sars outbreaks and could be summarized in the following three priority rules of spreading: first, spreading occurs within the city; second, stochastic spreading occurs between the city and the towns closest to it; finally, stochastic spreading takes place across different cities through quick transportation. the population density is an explanatory factor for the fact that sars spread is mostly in urban areas. approximately 19% of people working in health services were infected by their patients. these professionals were able to obtain rapid treatment, which means that it was unlikely that they would contribute to sars spread outside their areas of residence. housewives and retired people are groups that were extremely susceptible to infection by their families. however, these population groups typically have no desire to move to distant places. the most probable suspects for disease spread were labourers and businessmen, but these groups constituted only ∼7% of total infection cases. the randomness of sars spread among various cities is a characteristic that has been explained via the theory of super spreaders. the starting point of this theory is that the sars virus always evolves and mutates. most virus strings evolve slowly with similar infectivity, but a small fraction evolve into extremely infective virus types. people affected by such virus strings are so-called super spreaders. these individuals were comparatively very contagious, and they transmitted the virus to dozens of other people. for instance, five super spreaders in singapore were found to have infected 103 people [28, 29] . the randomness of virus mutation to high infectivity indicates considerable randomness in the way people became infected. this randomness also extended to the relatively random directions in which the epidemic spread among cities. sars generated one of the most egregious public health events in china of the beginning of 21st century, causing great loss of life and a grave threat to human survival and development. in the present study, spatial and temporal aspects of this person-to-person contagious disease were explored, and its spatial and temporal transmission dynamics were simulated through the bme method. based on these analyses, it is concluded that sars transmission varies in its epidemiological characteristics. moreover, there was a high temporal correlation and relatively low spatial correlation of sars outbreaks. in addition, the bme modelling demonstrated that sars transmission features are affected by spatial heterogeneity. therefore, we analyzed potential causes, including infected populations and transportation modes. our findings can benefit epidemiological control of pandemic infectious diseases and public health protection in the future. structure, mechanism, and evolution of the mrna capping apparatus single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction contact tracing and disease control transmission dynamics and control of severe acute respiratory syndrome infectious diseases of humans: dynamics and control sars outbreak spatial dynamics of an epidemic of severe acute respiratory syndrome in an urban area severe acute respiratory syndrome epidemic in asia severe acute respiratory syndrome-singapore transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions severe acute respiratory syndrome: temporal stability and geographic variation in casefatality rates and doubling times network theory and sars: predicting outbreak diversity modeling the sars epidemic curtailing transmission of severe acute respiratory syndrome within a community and its hospital modelling strategies for controlling sars outbreaks understanding the spatial diffusion process of severe acute respiratory syndrome in beijing spatiotemporal information systems in soil and environmental sciences modern spatiotemporal geostatistics a simple approximate mathematical model to predict the number of severe acute respiratory syndrome cases and deaths on the physical geometry concept at the basis of space/time geostatistical hydrology bayesian maximum entropy analysis and mapping: a farewell to kriging estimators? soil salinity mapping using spatio-temporal kriging and bayesian maximum entropy with interval soft data random field models in earth sciences interactive spatiotemporal modelling of health systems: the seks-gui framework evaluation of control measures implemented in the severe acute respiratory syndrome outbreak in beijing different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures sars outbreaks in ontario, hong kong and singapore: the role of diagnosis and isolation as a control mechanism transmission of severe acute respiratory syndrome in dynamical small-world networks state key laboratory of remote sensing science is jointly sponsored by the institute of remote sensing and digital earth of chinese academy of sciences and beijing normal university. the authors declare that they have no competing interests. chunxiang cao and wei chen were the main researchers and responsible for the data analysis and paper writing. wei chen and sheng zheng performed data processing and model implementation. jinfeng wang commented on idea selection. wuchun cao provided sars data and supervised the modelling details. jian zhao and chaoyi chang greatly helped in result analysis and gave excellent advice on paper preparation. key: cord-004269-g6ki6vyy authors: de rooij, doret; belfroid, evelien; eilers, renske; roßkamp, dorothee; swaan, corien; timen, aura title: qualitative research: institutional preparedness during threats of infectious disease outbreaks date: 2020-01-23 journal: biomed res int doi: 10.1155/2020/5861894 sha: doc_id: 4269 cord_uid: g6ki6vyy background: as demonstrated during the global ebola crisis of 2014–2016, healthcare institutions in high resource settings need support concerning preparedness during threats of infectious disease outbreaks. this study aimed to exploratively develop a standardized preparedness system to use during unfolding threats of severe infectious diseases. methods: a qualitative three-step study among infectious disease prevention and control experts was performed. first, interviews (n = 5) were conducted to identify which factors trigger preparedness activities during an unfolding threat. second, these triggers informed the design of a phased preparedness system which was tested in a focus group discussion (n = 5) were conducted to identify which factors trigger preparedness activities during an unfolding threat. second, these triggers informed the design of a phased preparedness system which was tested in a focus group discussion (n = 5) were conducted to identify which factors trigger preparedness activities during an unfolding threat. second, these triggers informed the design of a phased preparedness system which was tested in a focus group discussion ( results: four preparedness phases were identified: preparedness phase green is a situation without the presence of the infectious disease threat that requires centralized care, anywhere in the world. phase yellow is an outbreak in the world with some likelihood of imported cases. phase orange is a realistic chance of an unexpected case within the country, or unrest developing among population or staff; phase red is cases admitted to hospitals in the country, potentially causing a shortage of resources. specific preparedness activities included infection prevention, diagnostics, patient care, staff, and communication. consensus was reached on the need for the development of a preparedness system and national coordination during threats. conclusions: in this study, we developed a standardized system to support institutional preparedness during an increasing threat. use of this system by both curative healthcare institutions and the (municipal) public health service, could help to effectively communicate and align preparedness activities during future threats of severe infectious diseases. e four pandemics (sars, influenza a/h1n1, mers, ebola) that have emerged since the beginning of this century [1] underpin the necessity of global awareness and optimal control strategies. ese outbreaks showed the potential for the worldwide spread of such severe diseases [2] and led to social unrest and large economical consequences for the affected countries [3] . during the spread of the ebola viral disease (evd) outbreak in west africa, the likelihood of imported cases in europe increased, and european countries advised their healthcare institutions to prepare for patients with suspicion of evd. admission of a patient suspected for evd, or another transmittable viral hemorrhagic fever (vhf), requires a large pool of trained healthcare workers and of specialized medical facilities [4, 5] . erefore, in the netherlands, the care for these patients was designated to a few highly specialized hospitals. an evaluation by the harvard-lshtm independent panel on the global response to ebola concluded that international response to evd was inadequate [6] . a multidisciplinary national evd outbreak evaluation in the netherlands concluded that better guidance on preparedness during threats of outbreaks was needed for diseases, such as evd, where patients only can be admitted to highly specialized hospitals [6] . in the netherlands, as in other countries [7, 8] , it had been unclear among curative and public health institutions which preparations during a developing threat of evd were necessary, and for which preparations, the institutional or national level should have guidance [9] . while many studies describe preparedness for outbreaks, preparedness during a remote threat is not discussed separately in literature. e european center for disease control defines preparedness for infectious diseases as "the knowledge and capacities […] to effectively anticipate, respond to, and recover from, the impacts of a likely, imminent or current crisis" [10] . preparedness includes the development of institutional, national or international plans, communication and collaboration among different (types of) healthcare institutions, training and simulation, and surge capacity. e evd outbreak, however, showed that successful preparedness during a threat requires flexibility and adaptations to be able to respond to differences in the probability of occurance of the disease. what these adaptations should be, has been unclear. ere is a need to clarify what preparedness entails in healthcare institutions during threats of severe infectious diseases whose patients can, due to the severity of the disease, only be admitted to designated, highly specialized hospitals (from now on described as diseases "which require centralized care"). erefore, the aim of this study was to define preparedness during an unfolding threat of an infectious disease that requires centralized care. second, we aimed to exploratively develop a standardized preparedness system describing preparedness activities for healthcare institutions in different preparedness phases. we conducted a qualitative three-step study with an iterative design of in-depth interviews (steps 1 and 3) and a focus group (step 2), in order to identify the key elements of a preparedness system. e system includes (a) the triggers for healthcare institutions to initiate extra preparedness activities during different levels of a threat, which define preparedness phases, and (b) preparedness activities for each preparedness phase. we aimed at finding generic triggers for preparedness, applicable to different types of healthcare institutions, such as hospitals, ambulance services, general practitioners and the municipal health services. e outline of the preparedness system is shown in figure 1 . in the first round of in-depth interviews, we explored the phases and triggers of the preparedness system, which we validated in the consensus meeting. in the second round of interviews, we aligned the preparedness activities per phase and grouped them per topic. we obtained ethical approval from the medical ethical committee of the umc utrecht (wag/mb/17/028319). all participants provided informed consent and were informed that their responses would be used for research purposes. for all three steps, we invited professionals working at various levels and in various healthcare institutions and public health organizations. included healthcare institutions were academic and peripheral hospitals, ambulance services, general practitioners, and municipal health services. figure 2 shows how healthcare institutions are involved in the case of a potential patient requiring centralized care [11] . e municipal health service (mhs) was additionally involved because of their coordinating role between all partners at the regional level. professionals with the following backgrounds were invited: (i) academic hospitals: microbiologists and infectious disease specialists; (ii) peripheral hospitals: infection preventionists; (iii) ambulance services: medical managers at the regional and national level; (iv) general practitioners: respresentattives of the national association for general practitioners (lhv) and dutch college of general practitioners (nhg); (v) municipal health services: regional communicable disease control consultants and infectious disease control specialists. we used purposeful sampling by approaching the key players in the netherlands, from the above-mentioned healthcare and public health organizations, who were involved in preparedness and/or response during the evd outbreak. when the invited key player was not able to participate, we asked him or her to nominate a colleague in the same type of healthcare institution with comparable expertise. all professionals had, in this way, expertise in evd preparedness or response. for step 1 and 3 we aimed for one participant per healthcare institution. for step 2, we aimed for 1-3 participants per healthcare institution. participants were invited by e-mail and a consecutive telephone call during september-november 2017. collection. data collection comprised three steps: individual interviews, a focus group and another individual interview round. a hypothetical scenario of a vhf outbreak was used for data collection, which was developed by experts on outbreak control of the national institute for public health and the environment (rivm). e scenario described a fictitious marburg virus outbreak in uganda that spread towards neighboring countries and continuously led to exported cases throughout the world. e outbreak scenario consisted of three stages, each hypothetically representing a preparedness phase of the preparedness system, as shown in figure 1 . based on the identified triggers, preparedness phases for the preparedness system were developed. for each of these preparedness phases, again a corresponding scenario of the marburg virus outbreak was developed to discuss in the focus group of step 2. e preparedness system was adjusted based on the results from step 2 and was sent by e-mail to the participant to discuss in step 3. e results of step 3 were used to finalize the preparedness system by grouping preparedness activities into overarching topics, and in institutional and collaborative activities. step 1 consisted of individual, in-depth, semistructured interviews. to ensure that collected data was as reliable and consistent as possible, an interview guide was developed beforehand (additional file 1). e interview guide for step 1 was piloted with a microbiologist at a dutch academic hospital. all interviews were conducted by one researcher (ddr), to safeguard inter-observer reliability. interviews took between 35 and 50 minutes. during the interviews, the interviewer presented the outbreak scenario to the participant. per preparedness phase, the participant was asked if and why preparedness activities would be necessary for their healthcare institution. in this way, the triggers for preparedness activities were explored. alongside, questions covered terminology for preparedness during a threat, responsibility for preparedness during a threat, and collaborative preparedness activities carried out together with other healthcare institutions. step 2. in step 2, a mixed focus group discussion with 1-3 representatives per type of healthcare institution was organized to validate the concept preparedness system. a focus group guide was developed beforehand (additional file 1). e focus group was guided by two researchers (ddr and cs), and supported by an expert in guiding focus groups (re). e focus group took place at the rivm and lasted 2 hours and 15 mintues. per preparedness phase, the corresponding scenario was presented. participants were asked if and why preparedness would be necessary within their institutions, what they would expect the other healthcare institutions to do, and where cooperation and support between healthcare institutions was needed. in the second phase of the focus group, preparedness activities identified in step 1 were presented to representatives of each type of healthcare institution separately. representatives of one type of healthcare institution debated if and in which preparedness phase these preparedness activities were needed. subsequently, terminology for preparedness during a threat was discussed among all participants, since the interpretation of terminology had shown to be different. step 3 consisted of individual, in-depth, semistructured interviews. we included one participant per type of healthcare institution out of the participants in step 2. an during the first interview round, "scaling up" and "enhanced preparation" were used as synonyms by the interviewer for different preparedness phases. in the focus group, we observed differences in interpretation between curative and municipal health services. according to the curative partners, the dutch term for upscaling that was used, applied to the response phase "with the presence of an actual potential patient". in contrast, for the mhs, the term could also be used for preparedness during an increasing threat. e need for congruent language was highly stressed by the participants, and consensus was reached on the definition of "enhanced preparedness" to describe preparedness activities during a threat. during the first interview round several factors that trigger preparedness activities were identified for different healthcare institutions. e microbiologist at an academic hospital reported that they were at all times ready for such cases. however, they would enhance preparations if the likelihood of admitting a vhf patient increases, such as or with repatriated staff from the outbreak area. municipal health services started with preparedness activities as soon as the outbreak somewhere in the world occurred and would be further enhanced when health institutions in their region were likely to become involved. for academic hospitals, general hospitals and ambulance services, (1) the likelihood that an unexpected potential patient was presented to their healthcare institution, and/or (2) unrest among the general population and staff, triggered preparedness activities. for general practitioners, only an outbreak in their would lead to preparedness activities. in the focus group, trigger 1 and 2 were accepted as main triggers distinguishing between preparedness phases. besides, a third trigger was the situation of several (potential) patients hospitalized within the country, conceivably leading to different referral pathways between healthcare institutions. not all triggers would lead to the same intensity of extra preparations in all healthcare institutions, but all healthcare institutions would be involved in these phases. and most importantly, they all agreed upon the need for univocal communication. e final preparedness system based on these three triggers consists of four preparedness phases and is shown in figure 4 . preparedness phase green is a situation without the presence of the infectious disease threat that requires centralized care, anywhere in the world. preparedness phase yellow is the occurrence of the disease somewhere in the world but without triggers one and two. in preparedness phase orange, trigger one or two applies, and in preparedness phase red trigger number three applies. e preparedness activities as derived from step 1 and 2 and tested in step 2 and 3, were divided by topic as shown in the institutional preparedness column in figure 4 . all participants needed preparedness activities on infection prevention, such as the right type and stock of personal protective equipment, donning and doffing procedures, and waste management. regarding diagnostics, academic hospitals described preparedness activities. ese consisted mostly of extra checks whether interview guide was developed beforehand (additional file 1). interviews were conducted by telephone and were all conducted by the same researcher (ddr). interviews took between 15 and 20 minutes. e preparedness system was reviewed during the interview by discussing preparedness activities per phase. e participants discussed specific needs and adaptations per preparedness phase of the preparedness system. further analyses included comparing preparedness activities between healthcare institutions, to see whether their expectations matched. each interview and focus group was voice-recorded, with permission from the participants, and transcribed. transcription started directly a er the first interview and continued parallel to further data collection. data were processed anonymously using a coding system. a summary of every interview and focus group was sent to the participants to verify their input. all interviews and focus group sessions were coded using content analysis. a coding guide (additional file 2) was developed beforehand, based on the structure of the interview guide. for each step, the guide was expanded and adapted. coding was done by two researchers independently (ddr, and dr), using atlas. ti [12] , and differences were discussed until consensus was reached. data collected from each step of the study were analyzed and interpreted before the beginning of the subsequent step. in step 1, we invited 8 participants, of whom 3 could not be included. five experts participated in the interview round: a microbiologist of an academic hospital, an infection preventionist in a general hospital, a medical manager of the national ambulance service with extensive experience as an ambulance nurse, a practicing gp and representative of the lhv, and a regional communicable disease control consultant of a municipal health service. in step 2, we invited 24 participants of whom 13 could not be included. eleven experts participated in the focus group: 1 microbiologist and 1 infectious disease specialist of two different academic hospitals, 3 infection preventionists of different general hospitals, 2 medical managers of different regional ambulance services, 1 gp who also was representative of the nhg, and 3 regional communicable disease control consultants of different municipal health services. in step 3, we re-invited 7 participants from step 2, of whom only 3 accepted participation: one of the infection preventionists, the gp and representative of the nhg, and one of the regional communicable disease consultants. all representatives of academic hospitals and medical managers of the national ambulance services were either not responding or not able to participate due to time constraints. for all steps, reasons why professionals could not be included were the absence of reaction to the invitation (푛 = 7), unavailability during the data collection period (푛 = 11), completeness of inclusions (푛 = 2). figure 3 provides an overview of the different steps, the number of included participants and their backgrounds. step 1: individual interviews (n = 5) 1x general practitioner at the nhg (gps) step 3: individual interviews considerations start in preparedness phase orange as well, but policy on this should ideally be made in the green phase. (v) for mhs, diagnostics and training are required from preparedness phase yellow on, depending on the type of pathogen. in preparedness phase orange and red, they start to prepare their internal communication and personnel capacity. considerations. during step 1 and 2, ethical considerations were mentioned by representatives of all institutions except the municipal health services. e considerations of gps, academic hospitals and ambulance services described how to deal with suspected cases in life-threatening situations. ey need guidance on when concerns for their own safety would overrule their duty as a care provider, and based on which criteria. another aspect named several times by representatives of ambulance services, general hospital and academic hospitals was the priority of care: to respectively transport, temporarily accommodate, or care for one evd suspected patient, meant that many other patients could not receive care because an ambulance, an emergency department or large parts of intensive care had to close due to cleaning procedures, panic reduction or lack of staff or resources. participants explicitly stated that these were dilemmas they faced during the latest evd outbreak, and they would still face them should a case be admitted today. in addition to institutional preparedness activities, collaborative preparedness activities were discussed in all interview rounds. ese are activities that overarch individual healthcare institutions or should be performed by multiple healthcare organizations together. we identified collaborative preparedness activities in information, training and simulation, and coordination, as shown in the differential diagnoses for these patients could run. academic hospitals, peripheral hospitals and ambulance services named preparedness activities for patient care. academic hospitals and peripheral hospitals discussed the need to prepare their personnel for extra working hours, or the need for extra supplies. general practitioners and municipal health services needed preparedness for controlling unrest among their staff and the population, e.g., by informing staff and the availability of a telephone line for questions. in additional file 3a, an overview of identified preparedness activities that resulted from step 2 and 3 are shown per type of healthcare institution. we identified the following trends: (i) academic hospitals start with all preparedness activities from preparedness phase yellow on. in preparedness phase orange, a sub-commission on preparedness for the admittance of patients needs to be installed, and in preparedness phase red, there will be a need to consider more ethical challenges related to the threat and challenges related to a shortage of staff. (ii) peripheral hospitals inform triage staff and professionals at the gate during preparedness phase yellow. in preparedness phase orange, preparedness activities should start, except for diagnostics and patients' care/cure. in preparedness phase red, mainly a more intense communication among hospital departments and healthcare institution in the region is needed. (iii) for ambulance services, preparedness activities start in preparedness phase orange and no clear difference in preparedness activities were identified between preparedness phase orange and preparedness phase red. (iv) for general practitioners, phase orange is most important. e preparedness activities of general practitioners are limited to triage and primary infection prevention in all preparedness phases. ethical phases reflect the preparedness activities at a global, international and national level, rather than the institutional level within a country. e need for such specific phases for frontline institutions emerged during the evaluation of the ebola threat [16] , since all types of healthcare institutions experienced the need to perform extra activities to stepwise increase their level of operational response as the threat evolved. healthcare institutions need thus to adapt their preparedness activities to the level of a threat. e identified triggers for enhanced preparedness match with other studies and theory. schol et al. identified higher fear among dutch healthcare workers during the threat of ebola and identifies a relation between fear and the need for information. is study provides support for our finding that unrest is a trigger for enhanced preparedness (in this case by providing additional information) [17] . e founding risk classification theory of kinney and wiruth (1976) [18] states that risk is the chance that something happens times the impact of that event. within this formula, the presentation of an unexpected patient is the event that could happen, and the unrest among healthcare workers and the population represents impact. together they define the risk, which is then translated in the urge to prepare. in this way, the phase system in this study builds upon the literature on risk classification. studies showed that extra preparedness was needed for countries during threats with increasing severity of outbreaks elsewhere in the world [7, 8, 19, 20] . however, these studies report on disease-specific preparedness activities and, therefore were, not necessarily applicable to other threats. is study used marburg virus disease in the scenario and included experts with evd outbreak experience. we worked in the a ermath of the evd outbreak, but used a case of another disease. hereby, we strongly aimed to work towards a generic preparedness system. while specific preparedness activities differ between types of healthcare institutions and threat phases, in this study, a uniform enhanced preparedness system has been developed. during interviews, the focus group, healthcare institutions expressed the need to communicate explicitly and uniformly about preparedness activities. it became clear that there is no uniform terminology among experts from different healthcare institutions. for example, the term "scale-up" applies in curative care to the act of responding to an actual patient, while in public healthcare, the term could also be used during the preparedness. absence of uniform terminology impedes communication between public and curative health care, while smooth communication between the two is a must, especially during threats or outbreaks. with clear definitions of phases, our system offers this uniformity both within institutions, as well as among institutions. it could therefore be used to effectively arrange communication about the required specific enhanced preparedness. although this specific study was conducted in the netherlands, the results are also applicable in other countries with a comparable organization of healthcare. centralized care in dedicated health centers for patients suspected for an infectious disease requiring centralized care, is described in israel [7] , the united states (new york state) [19] , and in canada [20] . besides, they can be of value in other countries, because columns headed "collaborative preparedness" in figure 4 . e expectations of the different types of healthcare institutions regarding information and coordination match well between healthcare institutions. is implies that information exchange between organisations is reported to be adequate. furthermore, information on case definitions and information on the current preparedness phase is expected from the national centre for disease control. what did not match were the expectations of the different types of healthcare institutions regarding training and simulation exercises. e need to perform training or exercises together was mentioned by ambulance services and academic hospitals towards each other. but for peripheral hospitals, the need to practice together with ambulance services varied among participants. an overview of collaborative preparedness activities per preparedness phase is shown in additional file 3b. participants of all types of healthcare institutions stressed that aligned preparedness activities are preferred over institutional autonomy. however, healthcare institutions with a specific function should be able to deviate from the preparedness system activities. examples are healthcare institutions serving points of entry or those with national tasks such as the academic hospital with the reference laboratory. e aim of this study was to define preparedness during an unfolding threat of an infectious disease that requires centralized care. second, we aimed to develop a standardized system describing preparedness activities per preparedness phase for healthcare institutions. we developed this standardized system by defining phases of preparedness during a threat and their corresponding preparedness activities, within both the perspective of individual healthcare institutions and of the collaborative network in which these institutions need to function. e four identified preparedness phases were based on (a) the likelihood of presentation of an infected patient to one of the healthcare institutions and (b) the unrest among the general population and staff. phases ranged from no outbreak to the situation in which several potential or confirmed patients were hospitalized, conceivably leading to other referral pathways in the country. is system could be used for any future threat from an infectious disease requiring centralized care. using phases in preparedness to threats is not new. for terrorist attacks, for example, a level system using numbers 1-5 is common in several european countries, with 1 being considered a low threat, and 5 being a critical one [13] . in the netherlands, a code system using colors is used in the weather forecasting, ranging from green ("business as usual"), through yellow and orange, to code red ("high impact on society") [14] . and the who announced a pandemic phase system during the influenza outbreak (h1n1) in 2009, reaching from 0 to 6 [15] . however, by our knowledge, explicit preparedness phases following an unfolding threat caused by an infectious disease that offers concrete measures for frontline institutions have not been identified in literature. certainly, we acknowledge the existence of the pandemic phases of the who [15] . ese is study investigated preparedness during threats of infectious diseases requiring centralized care. is is the first study that explicitly defines preparedness activities during a threat for different frontline healthcare institutions. we reached consensus that a standardized preparedness system is required. a phased preparedness system has been developed, which can be used for improving institutional preparedness in curative healthcare institutions, and collaborative preparedness among curative healthcare institutions and the public health services. ebola virus disease gp: general practitioner lhv: national association for general practitioners md: medical doctor mhs: municipal health service n: number nhg: dutch college of general practitioners ppe: personal protective equipment rav: region of ambulance services rivm: national institute for public health and the environment vhf: viral hemorrhagic fever who: world health organization. data availability e datasets generated and/or analyzed during the current study are not publicly available due to the privacy protection of the participants, but are available from the corresponding author on reasonable request. we obtained ethical approval from the medical ethical committee of the umc utrecht (wag/mb/17/028319). all participants provided informed consent and were informed that their responses would be used for research purposes. past experience with outbreaks has shown that presentation or even the likelihood of imported patients, indeed led to unrest among the general population and hospital staff [21, 22] . our study has several limitations. ere was a high attrition rate between the focus group and last interview round, leading to a lack of representation of academic hospitals and ambulance services. is has led to gaps in the completeness of the review of preparedness activities per stakeholer and per phase. however, to increase validity the outcomes of this study were presented and discussed in a 1,5-hour slot during the regular national meeting on evd preparedness. during this meeting with national representatives of academic and peripheral hospitals, ambulance services and mhs who had been involved with preparedness and response during the evd outbreak of 2013-15, the findings of this study were endorsed. is strongly supports further generalizability for both institutional as well as collaborative preparedness. another limitation is that data collection was only through interviewing, whereby direct observation of preparedness activities might yield additional findings. also conducting a simulation exercise might lead to other insights. moreover, we need to acknowledge the chance of recall bias. although we used a new scenario, participants referred to activities they had performed two years before, during the evd outbreak. is could have resulted in the identification of preparedness activities in phase yellow, orange and red that should not be performed in that phase. participants might have reported from previous evd experience where sometimes activities were performed in phase yellow, organge or red, whereas ideally these should be tackled in the green phase. an example are the ethical considerations, which indeed turn up during higher phases, but which should be covered in standard guidelines or procedures. an additional limitation is that most participants worked in the most urbanized parts of the netherlands. although regional organization might be different in the more rural regions, we have chosen to approach healthcare institutions with most experience with infectious diseases requiring centralized care. e expertise of the participants can be mentioned as a strength. since there was a strong need for a system that identifies different phases of a threat and the corresponding activities, this preparedness system could be used as a communication tool on a national or regional level. future research should focus on identifying all activities for each phase. e completed preparedness system can be used by healthcare instiutions as a checklist of all preparedness activities they should perform during unfolding threats. in addition, it can be used as an agenda-setting for regional meetings to discuss the collaboration between healthcare institutions for unfolding threats. finally, we recommend investigating the applicability of this system to other severe infectious diseases, not requiring centralized care. examples could be, the recent outbreak of plague in madagascar [23] or the ongoing threat of the middle east respiratory syndrome-coronavirus [24] . roles and responsibilities among types of healthcare institutions, in case of outbreaks of these diseases, vary and it is possible that other triggers and preparedness activities are required. our phased preparedness system may also be applicable in these situations. hospital preparations for viral hemorrhagic fever patients and experience gained from admission of an ebola patient clinical management of ebola virus disease in the united states and europe will ebola change the game? ten essential reforms before the next pandemic. e report of the harvard-lshtm independent panel of the global response to ebola preparing for imported ebola cases in israel european centre for disease prevention and control, public health emergency preparedness for cases of viral haemorrhagic fever (ebola) in belgium: a peer review -16 evaluatie van de ebolapreparatie in nederland united nations international strategy for disaster reduction rijksinstituut voor volksgezondheid en milieu. lci richtlijnen en draaiboeken atlas.ti gmbh. version 7.5.17. scientific so ware development terrorism and national emergencies kleurcodes current who phase of pandemic alert for pandemic (h1n1) 2009 ebola preparedness in the netherlands: the need for coordination between the public health and the curative sector knowledge, perceptions and media use of the dutch general public and healthcare workers regarding ebola practical risk analysis for safety management preparing the health system to respond to ebola virus disease how the 4 biggest outbreaks since the start of this century shattered some long-standing myths world health organization infectieziekten en veiligheid. toekomstige uitdagingen voor maatschappij en beleid implementation of the canadian contingency plan for a case of suspected viral hemorrhagic fever ebola virus disease cluster in the united states e first case of ebola virus disease acquired outside africa emergencies preparedness, response: revamp of the plague detection in madagascar yields quick and sustainable wins middle east respiratory syndrome corona virus (mers-cov) acknowledgments is research was supported by the utrecht university medical center. we thank our colleague joyce l. browne, md, ph.d., for assistance with the methodology of this study, for her insights regarding the clinical applicability of our research, and for here feedback on an earlier version of the manuscript.we would also like to show gratitude to our participants, for their efforts, insights and feedback during the course of this research. we thank jim van steenbergen md, ph.d., for sharing his insights on the connection of this study with earlier performed research. additional file 1a: guide for individual, semi-structured interviews, belonging to step 1 (written in dutch). additional file 1b: guide for the mixed focus group, belonging to step 2 (written in dutch). additional file 1c: guide for individual, semi-structured interviews, belonging to step 3 (written in dutch). additional file 2: coding guide used for qualitative content analysis of data in step 1-3. (written in dutch). additional file 3a: institutional preparedness activities per type of healthcare institution and per preparedness phase (written in dutch). additional file 3b: collaborative preparedness activities per preparedness phase. (supplementary materials) e authors declare that they have no conflicts of interest. ddr collected, analyzed and interpreted the data and has dra ed the manuscript. eb has been involved in designing the data collection and in revising the manuscript critically for important intellectual content. re has been involved in the conception of the data collection and guided the focus group. dr has performed coding of the data. cs has made substantial contributions to the design of the study and interpretation of data and had supervision over the research project. at has critically revised the manuscript and has given final approval of the version to be published. all authors read and approved the final manuscript. key: cord-001572-ap4ro5me authors: oosterhoff, dinja; van de weerd, gerard; van eikenhorst, gerco; de gruijl, tanja d.; van der pol, leo a.; bakker, wilfried a. m. title: hematopoietic cancer cell lines can support replication of sabin poliovirus type 1 date: 2015-02-28 journal: biomed res int doi: 10.1155/2015/358462 sha: doc_id: 1572 cord_uid: ap4ro5me viral vaccines can be produced in adherent or in suspension cells. the objective of this work was to screen human suspension cell lines for the capacity to support viral replication. as the first step, it was investigated whether poliovirus can replicate in such cell lines. sabin poliovirus type 1 was serially passaged on five human cell lines, hl60, k562, kg1, thp-1, and u937. sabin type 1 was capable of efficiently replicating in three cell lines (k562, kg1, and u937), yielding high viral titers after replication. expression of cd155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed cd155. furthermore, we showed that passaged virus replicated more efficiently than parental virus in kg1 cells, yielding higher virus titers in the supernatant early after infection. infection of cell lines at an moi of 0.01 resulted in high viral titers in the supernatant at day 4. infection of k562 with passaged sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. altogether, these data suggest that k562, kg1, and u937 cell lines are useful for propagation of poliovirus. vaccines are pharmacological formulations that incorporate the disease-causing agent or an antigen derived from this agent, which are capable of inducing an immune response once administered to a healthy individual, without causing the disease itself. licensed vaccines can be divided into viral and bacterial vaccines, and viral vaccines can be further classified into four categories: live attenuated viruses, inactivated viruses, subunit vaccines, and virus-like particles. for the production of the first two categories, large amounts of viral particles are needed, and most of these viral vaccines are produced by infecting susceptible cell lines. since there is no standard cell line that can be used for the replication of every virus, a whole panel of different cell lines has been used for vaccine production processes throughout the years. cell lines that have historically often been used for the production of viral vaccines are mrc-5 and wi-38 [1, 2] . these two cell lines are human diploid cell lines derived from fetuses, and these cells were used for the manufacture of a number of vaccines, for example, hepatitis a, polio, and rubella [3] [4] [5] . diploid cell lines have a finite lifespan and in these cell lines the chromosomes are paired. often these cells retain many characteristics of the cell types from which they originate. the disadvantage of diploid cell lines lies in the fact that the cells can only be cultured for a limited number of passages before the cells die of senescence. in general, diploid cells grow as adherent cells and require serum-containing growth media to grow efficiently. the major benefit of diploid cells is the fact that the cells are nontumorigenic and therefore are considered safe to use for the production of vaccines (reviewed by hayflick et al. [6] ). given the high demand of vaccines and the restrictions associated with the use of diploid cell lines, in the last decades, continuous cell lines were introduced in vaccine production processes. from a vaccine production point of view, the characteristic of continuous growth is beneficial, since such cells have the potential for an infinite lifespan, and characterized and approved master and working cell banks can be established. a thorough understanding of the cell substrates with respect 2 biomed research international to identity, stability, purity, tumorigenicity, and the presence of adventitious and endogenous agents is, however, essential for the production of quality assured vaccines [7] . the first continuous cell line approved for the production of vaccines was the vero cell line, originating from african green monkeys and developed at the chiba university in japan. the mechanism of immortalization of vero cells is unknown. it has been described that vero cells at passages 140 to 165 are not tumorigenic in immunocompromised mice [8] [9] [10] and at those passages vero cells are currently used for the manufacturing of viral vaccines. a recent paper, however, concluded that the transition from nontumorigenic to a tumorigenic phenotype of vero cells did not occur until passage 185 [11] . vero cells have, over the years, proven to be safe, since millions of vaccine doses produced on vero cells have been given to healthy individuals. a major advantage of vero cells is that the cells are sensitive to infection with many different viruses [12] , meaning that vero cells can be used for the production of a number of different vaccines [13] [14] [15] [16] . this wide infectivity may be the result of a defective antiviral interferon response of susceptible cells, which was demonstrated in general for cells that are permissive for poliovirus replication [17] . however, not all viruses are capable of replicating on vero cells and the consensus is that the current repertoire of cell substrates is inadequate for the manufacture of certain types of (new) vaccines. to address this limitation, the vaccines and related biological products advisory committee meeting (vrbpac) recognized in 2012 that (human) tumor-derived cell lines could be an important addition to the repertoire of cell substrates for the production of viral vaccines (http://www.fda.gov/downloads/ advisorycommittees/committeesmeetingmaterials/blood-vaccinesandotherbiologics/vaccinesandrelatedbiological-productsadvisorycommittee/ucm319573.pdf). in some cases, even the only susceptible cell available to propagate specific viruses for which vaccines are needed could be tumor cells. therefore, currently several tumor cells lines are being explored for their capacity to propagate viral vectors, like the madin-darby canine kidney (mdck) cell line [18] , hela cell line [19, 20] , and the per.c6 cell line of which the latter was immortalized by transfection with adenoviral e1 proteins [21] . at the present time though only a limited number of vaccines that were produced in tumorigenic cell lines have entered clinical trials or were registered [22] [23] [24] [25] [26] . overall, it can thus be stated that the regulatory opinion on cell lines that are used for the production of viral vaccines has changed radically at the last 30 years with respect to the risks and benefits of immortalized tumorigenic cell lines [7, 26] . this is most likely also due to the development of techniques that can detect adventitious viruses or host cell dna in vaccines with a very high sensitivity [27] . in the future, a potential use of human tumor cell lines for the production of viral vaccines can be foreseen. a characteristic of viruses is that viruses evolve, and due to mutations in their genetic material or recombination with other viruses, outbreaks of dangerous and potential lethal new viruses can occur. in these cases, fast development of vaccines is essential for global health. it would be of great importance if cell lines that are needed for the production of such vaccines could be selected upfront, at the start of a viral outbreak, based on scientific understanding instead of trial and error. in this study, we have made a start with the characterization of human tumor cell lines and their capacity to support viral replication. five human cell lines, capable of growing in suspension and often used in research, but not currently qualified for vaccine production purposes, were selected, hl60, k562, kg1, thp-1, and u937 [28] [29] [30] [31] [32] . these cell lines are all cancer cell lines and originally derive from patients with leukemia or lymphoma. all different cell lines originate from blood cells that were abrogated in their development at different promonocytic stages. depending on the mix of cytokines and/or growth factors added to the cells, the cells can differentiate towards several endstages. since the stage of differentiation of a cell can have an effect on susceptibility of the cell for replication of specific viruses, this could be an interesting feature of the selected cell lines [33] [34] [35] . possibly, the cells can become infected as progenitor cells, whereas infection is not possible when the cells are differentiated or vice versa. interestingly, k562 cells are currently used as a vaccine for patients with lung cancer. irradiated k562 cells, transfected with the gene encoding gm-csf and cd40 ligand, were mixed with allogeneic tumor cells and this vaccine was tested in a phase ii trial in lung cancer patients [36] . the overall aim of this study is to generate data that will facilitate decision making on which substrate may be the best for the production of novel vaccine strains. as a first step for this, we investigated whether poliovirus, as a representative of the picornaviridae family of viruses, can be propagated in the human suspension cell lines. conditions. the human hematopoietic progenitor tumor cell lines hl60, u937, k562, kg1, and thp-1 were cultured in imdm (hl60, u937, k562, and kg1) (invitrogen) or rpmi (thp-1) supplemented with 10% fetal bovine serum (fbs, paa) and 10 u/ml penicillinstreptomycin (gibco) at 37 ∘ c in a 5% co 2 humidified atmosphere. the vero cell line was taken along as a positive control cell line and these cells were cultured in virus production serum-free medium (vp-sfm, invitrogen) supplemented with 2 mm glutamine (life technologies) at 37 ∘ c in a 5% co 2 humidified atmosphere. cho suspension cells, which served as a negative control, were cultured in ex-cell 302 medium (sigma) in shaker flasks (corning) gently rotating at 50-100 rpm at 37 ∘ c in a 5% co 2 humidified atmosphere. to determine whether k562 cells were capable of growing in bioreactors, cultibags (sartorius stedim) were inoculated with cells at a concentration of 0.3 × 10 6 cells/ml in 1 l of culture medium. cultibag cultures were performed at 37 ∘ c, dissolved oxygen concentration of 50%, ph 7.2 at a constant rocking speed of 10 rpm, and an angle of 7 ∘ . tumor cell lines. to serially passage sabin poliovirus type 1 on the hematopoietic cell lines, 1 × 10 6 cells were infected biomed research international 3 in t25 flasks with an moi of 1. cells and supernatants were harvested if full cytopathic effect (cpe) was observed or after 1 week of culture. after freeze-thawing three times to lyse the cells, samples were centrifuged at 3000 rpm to remove cellular debris and half of the supernatant was used to reinfect new hematopoietic cells. this was repeated for 4 times to allow the virus to adapt to the new cell lines in 5 passages. in the last infection round, 1 × 10 7 cells in a t175 flask were infected and samples were harvested at day 3 (vero and u937) or day 6 to obtain small viral stocks that were used for subsequent experiments. to determine replication kinetics, the susceptible tumor cell lines were infected with sabin poliovirus type 1 from the parental virus or virus that was passaged for 5 times on the hematopoietic cell lines at moi 1 or moi 0.01, and samples of the supernatant and cellular lysates were harvested at different time points. after harvesting, the supernatant was centrifuged at 1000 rpm for 4 minutes to remove cells floating in the medium that did not release their progeny virus yet, and this fraction was added to the cellular fraction. cells still attached to the flask were harvested by scraping the cells from the flask with a cell scraper (becton dickinson) in pbs. after centrifugation at 1000 rpm, pbs was removed and the cell pellet was resuspended in 1 ml of pbs. the cellular samples were freeze-thawed three times to release virus from the cells, and after centrifugation at 3000 rpm supernatant was transferred to a new tube. the virus titer in all the samples was determined by end-point titration on vero cells. k562 cells grown in cultibags were infected at an moi of 0.01 with passaged sabin poliovirus type 1 when cells reached a concentration of 1.2-1.5 ×10 6 cells/ml, and samples of the culture medium were taken at days 3, 4, 5, and 6 after infection. samples were filtered (0.22 m filter) to remove viable cells and the virus titer and d-antigen levels in these samples were determined. to determine the virus titer of sabin poliovirus type 1 that replicated in the different cell lines, the virus titer was determined by end-point titration. in short, adherent vero cells were seeded at a concentration of 1 × 10 4 cells/100 l in 96-well flat bottom plates in m199 medium containing 10% serum. serial 10-fold dilutions were prepared from cellular lysates or supernatant from hematopoietic cells infected with sabin poliovirus type 1 in serum-containing m199 medium, and 50 l of these dilutions was added to 6 separate wells. after 7 days, all the wells were scored for the presence or absence of cytopathic effects (cpe), and the virus titer was determined using the reed and muench method, thereby calculating the 50% cell culture infective dose (ccid50)/ml. to calculate the plaque forming unit titer (pfu) from the ccdi50 value, the ccid50 was multiplied with 0.69 to generate the pfu titer/ml. to be able to compare the number of infectious sabin type 1 polioviruses in the supernatant samples with the cellular lysate samples, the total amount of pfu in the samples was determined by multiplying the pfu/ml titer with the total amount of supernatant or cellular lysate that was harvested. to determine the presence of dantigen in the virus samples, a sandwich elisa was performed as described previously [37] . briefly, plates were coated with an anti-sabin type 1 caprylated bovine antiserum diluted 1 : 1600 in pbs (gibco) overnight at 4 ∘ c. after washing, samples were added for 30 minutes at 37 ∘ c. after washing, a mixture of a sabin type 1 specific mouse monoclonal antibody and an hrp-labeled goat anti-mouse antibody were added for 30 minutes at 37 ∘ c. after four washing steps, the signal reagent highlite was added and the emitted light was detected with a luminometer. to determine the expression of viral receptors on the different cell lines, facs analyses were performed. pe-labeled antibodies directed against human cd155 (ebioscience), cd54 (bd pharmingen), and car (millipore) and fitc-labeled anti-human cd81 (bd pharmingen) were used for flow cytometric analyses, with fitc-or pe-labeled igg1 antibodies (bd pharmingen) as controls. antibody staining was performed in pbs supplemented with 0.1% bsa and 0.02% sodium-azide for 30 min at 4 ∘ c. after washing of the cells, the stained cells were analyzed on a guava facs (merck millipore) using cell flowjo software. to determine whether hematopoietic cell lines can support replication of sabin poliovirus type 1, cells were infected with an moi of 1 and cells together with supernatant were harvested at day 3 (for all virus passages in vero cells and for passages 3-5 on u937 cells) or day 6 after infection. after lysis and centrifugation of the cells, half of the supernatant was used for the reinfection of new cells. this procedure was repeated for 4 more times, and virus derived from passage 5 was used for additional experiments. in the samples derived from all 5 passages, the virus titer (ccid50/ml) was determined by a virus titration assay. as shown in figure 1(a) , replication was efficient in the control cell line vero, grown in serum-free medium, yielding high titers of more than 1 × 10 7 ccid50/ml in all 5 passages. in cho cells, sabin poliovirus type 1 could only be detected in the first passage, and this is most likely due to the fact that cells were not washed after primary infection, suggesting that the virus titer is the result of virus that remained present in the culture medium, which was unable to infect the cells. in samples from passages 2-5 of sabin poliovirus type 1 added to cho cells, as expected no virus could be detected. a comparable pattern was observed after infection of thp-1 and hl60 cells with sabin poliovirus type 1, suggesting that these two cell lines were refractory for the virus. the k562 and u937 cell lines were highly permissive for sabin poliovirus type 1, resulting in high virus titers in all 5 viral passages. kg1 cells were also fully permissive for sabin poliovirus type 1, but the amount of virus obtained after infection of kg1 cells was slightly lower compared to sabin poliovirus type 1 that replicated in the other permissive cell lines. microscopically it was observed that u937 cells infected with virus from passages 0-2 did not show clear cytopathic effects (cpe) after 6 days of culture, whereas cells infected with virus from passages 3, 4, and 5 were harvested after 3 days because full cpe was observed. a photographic overview of cells infected with virus from passage 4 is shown in figure 1(b) . also, in k562 cells, clear cpe was visible at day 6 after infection. the amount of virus produced after replication of sabin poliovirus type 1 in k562 cells did seem to increase after the first passage, suggesting that replication of the passaged virus is more efficient than replication of the parental virus. to be able to compare the d-antigen level per virus, the specific d-antigen level per infectious virus particle was calculated for the fifth viral passage in the permissive cell lines and is shown in figure 1(c) . for vero cell derived sabin poliovirus type 1, the d-antigen per infectious virus particle was 4 du/10 7 ccid50. in general, it was observed that passaged virus had a d-antigen level per virus that was slightly lower compared to virus replicated in vero cells, varying between 1.2 and 2.3 du/10 7 ccid50 depending on the cell line used. hematopoietic cell lines correlated with the capacity to propagate sabin poliovirus type 1, human cd155 expression was determined using facs analysis. in figure 2 it can be seen that all hematopoietic cell lines, as well as vero cells, express cd155. vero, u937, and k562 had the highest level of expression, whereas the expression level of thp-1, kg1, and hl60 cells was lower. also, not all hl60 cells were positive for cd155. since sabin poliovirus type 1 was not capable of replicating in thp1 and hl60 cells whereas replication in kg1 cells was efficient, expression levels of cd155 thus cannot fully predict the capacity of sabin poliovirus type 1 to replicate in these cell lines. supernatant at day 1 is slightly higher if cells are infected with the passaged virus compared to control virus, although the differences are not significant. it could be that in these cell lines also the replication speed of the passaged virus is slightly enhanced, but differences remain limited because after 2 days the maximal viral titer has been obtained, due to lysis of all the cells. furthermore, it can be concluded from these data that, for all three hematopoietic cell lines tested, the virus titer of the passaged virus at day 2 in the supernatant is comparable to the virus titer obtained 2 days after replication of sabin poliovirus type 1 in vero cells. also, the virus titer in the culture medium remained stable during the 7 days of the experiment. altogether, these data indicate that, with respect to the viral kinetics, all three human hematopoietic cancer cell lines are efficiently producing poliovirus particles. , or k562 with a low moi. for vaccine production purposes it is important that, after infection of cells with a low moi, a high viral yield in the culture medium can be achieved. to investigate this, cells were infected with an moi of 0.01 of passaged sabin poliovirus type 1, and supernatant and cellular lysates were harvested separately at day 4 and day 7. the total viral titer in these samples was determined and is shown in figure 4 . in the supernatant of all cell lines tested, at day 4, a high virus titer, comparable to sabin poliovirus type 1 replicated in vero cells, was observed in the culture medium, indicating that virus replication was efficient during multiple rounds of replication. in k562 cells, the virus titer was moderately further increased at day 7 after infection, whereas in the other cell lines the virus titer at day 7 in the culture medium slightly decreased compared to the titer at day 4. interestingly, at both day 4 and day 7 after infection, a high amount of virus was still present in the viable cells. this was also observed in vero cells, and it suggests that not all cells were lysed at day 4 or day 7 by sabin poliovirus type 1. sabin poliovirus type 1. to determine whether it is possible to produce sabin poliovirus type 1 at a larger scale, k562 cells were grown in cultibags. cells were inoculated at a concentration of 0.3 × 10 6 cells/ml in 1 l culture medium. after 4 days, in which the k562 cells grew to a concentration of 1.2-1.4 × 10 6 cells/ml, the cells were infected with the passaged sabin poliovirus type 1 at an moi of 0.01. at days 3, 4, 5, and 6, the cell viability was determined and samples of the culture medium were taken, in which the virus titer and d-antigen concentration were determined. as can be seen in figure 5 (a), the virus replicated efficiently, resulting in titers of 1 × 10 9 ccid50/ml in the culture medium already at day 3 after infection, which remained stable for at least the following 3 days. the d-antigen level at day 3 after infection ( figure 5(b) ) was somewhat lower than that at later time points. also, a large variation in the d-antigen level between the 3 experiments was observed at day 3, suggesting that not all viruses expressed d-antigen yet. however, at days 4, 5, and 6 after infection, d-antigen levels were high and comparable in all three experiments. hematopoietic tumor cell lines. to investigate whether the human suspension cell lines express multiple viral receptors, surface staining of cd54, car, and cd81 was performed. cd55, car, and cd81 are the receptors for rhinovirus, coxsackie, adenovirus, and hepatitis c virus, respectively. in figure 6 , the percentage of cells expressing the specific receptor is shown. all cell lines, except hl60, highly expressed cd54 and cd81. hl60 cells did express high levels of cd81, but cd54 was only expressed by 50% of the cells. with respect to car expression, differences between the cell lines were observed. a minority of k562 cells expressed car, whereas, for the other cell lines tested, 50-90% of the cells did express car. whether the expression of these receptors predicts the capacity of the virus to replicate in these cells remains to be determined. high vaccination rates have helped to prevent many infectious diseases and resulted in less sickness and millions of lives saved (reviewed by rappuoli et al.) [38] . among the greatest success stories are the eradication of smallpox virus, rinderpest, and the polio eradication program, where poliovirus type 2 was already eradicated in 1999 [39] [40] [41] . complete eradication of poliovirus is, however, more difficult than what has been initially anticipated, because of persistence of wild type poliovirus transmission and recurring outbreaks in polio-free countries (reviewed by wassilak et al.) [42] . the capacity to develop vaccines that induce efficient immune responses in a short period of time, together with a large global immunization rate, is thus essential to prevent viral outbreaks or further spread of viruses. in the last decades, many different cell lines have been used in vaccine production processes, and new cell lines are still being developed. since regulatory views are changing with respect to the risks and benefits of cell lines (reviewed by hess et al.) [7] , it is important to compare different cell lines for their capacity to propagate specific viruses. with such an approach, using viruses that belong to different viral groups, it might be possible in the future to select producer cell lines based upon scientific understanding instead of trial and error, and timelines needed to produce viral vaccines might be shortened. in this study, we performed a first step for such a comparison, with a focus on human suspension cell lines. these cell lines have an infinite lifespan and this, together with the fact that these cells grow in suspension, could facilitate vaccine production processes. all cell lines described in this study have been used extensively in experimental research. the disadvantage of these cell lines, obviously, is that these cell lines consist of tumor cells that are grown in serum-containing medium and are currently thus not qualified as suitable vaccine substrates. however, with changing regulatory opinions, it is foreseen that continuous cell lines will be accepted for the production of viral vaccines in the (near) future. until then, studies, like this, can be used to gain knowledge about the interaction of cell lines with viruses and/or the development of new producer cell lines that are approved for the production of viral vaccines. because of the experience that our group has with poliovirus production processes [43] [44] [45] , it was decided to perform this study with sabin poliovirus type 1 as the first model virus. first, it was determined whether the five selected cell lines were susceptible for poliovirus infection. k562, kg1, and u937 cell lines appeared to be capable of supporting replication of sabin poliovirus type 1, whereas hl60 and thp-1 were not. all five cell lines did express cd155, the receptor for poliovirus entry. this means that receptor expression does not predict the capacity of a virus to replicate in a cell. possible explanations for the lack of replication in the cd155 expressing hl60 and thp-1 cells could be that (i) cd155 expressed on these cell lines is nonfunctional, (ii) the nonsusceptible cell lines lack the expression of a coreceptor on the cellular membrane, or (iii) hl60 and thp-1 express an intracellular viral restriction factor. since the tested cell lines have the capacity to differentiate towards several end-stages and it has been shown for other viruses that the differentiation stage of a cell can affect the susceptibility for a virus [33] [34] [35] , it would also be interesting to determine permissiveness of hl60 and thp-1 for sabin type 1 after differentiation of the cells towards different end-stages. in the literature it has been described that human blood cell lines were susceptible for infection with poliovirus [46] , and that study demonstrated that well-differentiated human blood cell lines are more susceptible to cytopathic effects of poliovirus than the less-differentiated blood cell lines. from the selection of cell lines we used in this study, k562 cells were the least differentiated, but with respect to viral replication we did not observe a difference between k562 cells and the more differentiated cell lines. another study compared the replication capacity of mahoney poliovirus on k562 and u937 cells to hela cells [47] . compared to hela cells, poliovirus replicated less efficiently in both k562 and u937 cells yielding a 20-fold and a 50-fold reduced virus output. finally, a study by benton et al. showed differences between k562 clones in their response to poliovirus infection, because two out of four k562 cell lines were killed by the poliovirus, whereas in the two other cell lines persistent infections were established [48] . in our study, we did not observe a persistent infection of k562 cells, nor that the poliovirus replicated less efficiently in u937 cells or the less-differentiated k562 cells. the discrepancies between the observed replication characteristics in these studies with our study could possibly be explained by the fact that in our study the replication characteristics of passaged sabin type 1 were studied, meaning that the virus has had 5 passages to adapt to the new cell line. we did, however, observe already after the first passage of sabin type 1 in k562 or u937 cells a high virus titer. this was the amount of virus present in culture medium together with the virus in the cellular lysates, so the majority of the detected virus could have still been present in the cellular fraction, whereas the other studies determined the amount of virus present in the culture medium only. another difference between these studies was that benton et al. and lopez-guerrera et al. used mahoney strain of type 1 poliovirus, and in our study attenuated sabin poliovirus type 1 was used. in two more recent papers, the replication capacity of sabin types 1, 2, and 3 or wild type polioviruses was studied on a panel of different suspension cell lines, deriving from humans, avians, or canines. in both studies the virus was not adapted to the new cell lines, but experiments were directly performed with virus produced on vero cells. vlecken et al. compared a number of adherent and suspension cell lines for the capacity to replicate sabin poliovirus types 1, 2, and 3. of the 5 cell lines tested (bhk-21, cho-k1, cap, smdck, and age1.cr.hs), only the cap cell line was capable of propagating sabin type polioviruses, whereas all the other suspension cell lines were not capable of supporting viral replication [49] . in the second study, published by sanders et al., the capacity of per.c6 to support replication of brunenders, mef-1, and saukett poliovirus was determined in comparison to vero cells [50] . per.c6 is an immortalized human cell line capable of supporting the replication of a number of viruses, like influenza virus and west nile virus [51, 52] . the poliovirus was capable of replicating efficiently in the per.c6 cell line, resulting in a high virus yield, which can be attributed to the fact that per.c6 cells can be cultured under optimized conditions to very high cell densities. altogether, these two studies suggest that, for efficient replication of poliovirus, human or primate cells are needed, since, also in the adherent cell lines tested by vlecken et al., viral replication was not observed in cell lines with another origin [49] . this observation underscores the need of a wider panel of human/primate cell lines for the production of viral vaccines for human diseases. in our study, we have identified three additional cell lines that are susceptible for infection and replication of poliovirus that can also be grown at a larger scale in a disposable bioreactor system (only tested for k562 cells). it is important to realize that, by passaging a virus on a new cell line, the virus can adapt to this new cell line to optimize its replication cycle. this can also be accompanied by changes in antigenicity or virulence of the adapted virus. a new combination of cell line and virus should thus always be analyzed thoroughly, before the new cell line can be used for the production of viral vaccines. in this study, we did observe a difference in replication speed between original virus and virus passaged on kg1 cells, but sequencing is needed to determine whether the virus truly adapted to the cell line. since we already observed that also other sabin poliovirus types are capable of replicating in the permissive three cell lines (oosterhoff et al., unpublished data) and that all cell lines express other viral receptors, needed for efficient infection with rhinovirus, coxsackie, adenovirus, and hepatitis c virus, it would thus be interesting to determine the capacity of these viruses to replicate in these cell lines. ultimately both the panel of (human) cell lines and viruses need to be expanded, in order to be able to predict upfront the suitability of cell lines for the production of specific viral vaccines, based on scientific understanding, thereby reducing timelines and costs in vaccine production processes. in conclusion, these data demonstrate that k562, kg1, and u937 cell lines are efficient in supporting the replication of sabin poliovirus type 1. all five human hematopoietic cell lines expressed cd155, which thus did not explain susceptibility to viral replication, although we did not study the functionality of cd155. furthermore, we have shown that in kg1 cells the passaged sabin poliovirus type 1 replicated more rapidly than the control virus. infection of k562, kg1, and u937 at an moi of 0.01 resulted in a high viral titer in the culture medium after 4 days. also, k562 cells grown and infected with adapted sabin poliovirus type 1 in a disposable bioreactor system yielded high viral titers in the culture medium. altogether, it is concluded that k562, kg1, and u937 cell lines can be used for the propagation of poliovirus and in follow-up studies the capacity of other (entero)viruses to replicate in these cell lines will be determined. characteristics of a human diploid cell designated mrc-5 preparation of poliovirus vaccines in a human fetal diploid cell strain preparation and immunogenicity of an inactivated hepatitis a vaccine inactivated poliovirus vaccine and test development at connaught laboratories ltd a comparison of the reactivity and immunogenicity of ra 27/3 strain rubella vaccine prepared in wi-38 or mrc5 human diploid cells history of the acceptance of human diploid cell strains as substrates for human virus vaccine manufacture regulatory, biosafety and safety challenges for novel cells as substrates for human vaccines tumorigenicity of vero cells assessing the tumorigenic phenotype of vero cells in adult and newborn nude mice characterization of vero cells micrornas as potential biomarkers for vero cell tumorigenicity biological characteristics and viral susceptibility of an african green monkey kidney cell line (vero) industrial-scale production of 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dendritic cells but not monocytes differential effects of bovine viral diarrhoea virus on monocytes and dendritic cells cellular microrna expression correlates with susceptibility of monocytes/macrophages to hiv-1 infection phase ii trial of a gm-csf-producing and cd40l-expressing bystander cell line combined with an allogeneic tumor cell-based vaccine for refractory lung adenocarcinoma development of a fast elisa for quantifying polio d-antigen in in-process samples vaccines, new opportunities for a new society rinderpest. driven to extinction smallpox: gone but not forgotten reaching the last one per cent: progress and challenges in global polio eradication progress toward global interruption of wild poliovirus transmission, 2010-2013, and tackling the challenges to complete eradication inactivated polio vaccine development for technology transfer using attenuated sabin poliovirus strains to shift from salk-ipv to sabin-ipv multivariate data analysis on historical ipv production data for better process understanding and future improvements next generation inactivated polio vaccine manufacturing to support post polio-eradication biosafety goals poliovirus infection of established human blood cell lines: relationship between the differentiation stage and susceptibility or cell killing restriction of poliovirus rna translation in a human monocytic cell line k562 cell strains differ in their response to poliovirus infection comparison of initial feasibility of host cell lines for viral vaccine production per.c6 cells as a serum-free suspension cell platform for the production of high titer poliovirus: a potential low cost of goods option for world supply of inactivated poliovirus vaccine the human cell line per.c6 provides a new manufacturing system for the production of influenza vaccines safety and efficacy in geese of a per.c6-based inactivated west nile virus vaccine the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord-336177-p7b7yw28 authors: selvi, valeria title: convalescent plasma: a challenging tool to treat covid-19 patients—a lesson from the past and new perspectives date: 2020-09-22 journal: biomed res int doi: 10.1155/2020/2606058 sha: doc_id: 336177 cord_uid: p7b7yw28 on march 11(th), 2020, the world health organization declared covid-19 infection as a pandemic. since it is a novel virus, there are basically no proven drugs or therapies; although many laboratories in different countries are working to develop a vaccine, it will take time to make it available. passive immunization is the therapy born from the intuition of behring and kisato in the late 19(th) century. it was widely used for the treatment of bacterial infections until the discovery of antibiotics, as well as during the viral pandemics of the 20(th) century and of the beginning of the 21(st); it still has clinical applications (e.g., tetanus prevention). this paper summarizes the basic principles of passive immunization, with particular reference to convalescent plasma. the literature concerning its use during past epidemics and the results of the first clinical studies concerning its use during the current pandemic are discussed too. a large section is dedicated to the analysis of the possible, although rare, side effects. recently, in 2017, the who blood regulators network (brn) published a position paper, recommending convalescent plasma as the first-choice treatment to be tested in the absence of authorized drugs; however, this strategy has not been followed. in the current epidemic, the principle of passive immunization through convalescent plasma has been applied in several circumstances and particularly in patients with serious complications. the first reported results are encouraging and confirm the effectiveness of plasma therapy and its safety. also, the fda has proposed plasma treatment in order to face the increasingly complex situation and manage patients with serious or immediately life-threatening covid-19 disease. several studies and clinical programs are still ongoing. on march 11 th , 2020, the world health organization (who) declared covid-19 infection as a pandemic [1] . the virus causing covid-19 infection is a coronavirus called sars-cov-2; it began to scare the world since the first days of 2020 during its initial outbreak in china, because of the characteristics of contagion (high rate of contagiousness associated with high lethality) [2] . since it is a novel virus, there are basically no proven drugs or therapies. in hospitals all over the world, there are many ongoing clinical studies. many attempts have been made to treat seriously sick patients, using off-label drugs already known; nevertheless, to date, there is no effective targeted antiviral therapy. in most cases, drug administration has been authorized for a compassionate purpose [3] . in fact, who management of covid-19 has been mainly focused on infection prevention, case detection, and monitoring; supportive care and nonspecific anti-sars-cov-2 treatment have been recommended [4] . extensive vaccination is the only strategy to prevent pandemic transmission of sars-cov-2. major efforts are currently being made by many laboratories in several countries to develop a vaccine; however, it will still take time before the vaccine is widely available to the population [5] . encouraging news about passive immunization arrived from china at the end of february, and some authors reported them in their scientific publications. cai et al. cited two official sources (national health and health commission, health bureau of the logistics support department of the central military commission; chinese society of blood transfusion) reporting significant improvements in patients affected by covid-19 and treated with plasma donated by recovered patients [5] . anecdotal experiences are reported by keith et al. [6] and from cunningham et al. [7] ; in particular, the latter group reported that biotec group co. announced that 10 seriously ill patients, treated with immunoglobulin therapy, demonstrated improved oxygenation and reduced inflammation and viral load [7] . in addition, casadevall and pirofski, referring to the news from the xinhua news source, reported that convalescent serum was used for the therapy of 245 patients with covid-19 in china during the first outbreak. although few details are available and published studies involve a small number of patients, the authors concluded that convalescent serum is safe and reduces viral load [8] . additionally, convalescent plasma could potentially be used to prevent disease in high-risk cases (vulnerable individuals with underlying medical condition, health care providers, and individuals exposed to confirmed cases of covid-19 [8] . the literature about convalescent plasma is rapidly growing. the uniqueness of this work is that it presents all the main aspects about convalescent plasma in a single body. the already published review articles often focused on single aspects of convalescent plasma, and to the best of the author's knowledge, there are no available works covering all the main topics concerning convalescent plasma use. the present manuscript is intended to be a complete and update guide for doctors and institutions. a systematic search was conducted in major electronic databases (pubmed and medline) and google scholar; the applied query was "plasma or convalescent plasma" and "covid-19 or sars-cov-2". virus neutralization by antibodies is the principle behind the functioning of plasma of patients recovered from sars-cov-2; high-titer-specific antibodies bind to sars-cov-2 neutralizing the viral particles, blocking access to cells, and activating potent effector mechanisms, such as complement activation and phagocytosis [7] . there are several ways to achieve passive immunization. antibodies can be delivered to the recipient by (i) human whole blood, (ii) human or animal plasma or serum, (iii) pooled human immunoglobulin for intravenous (ivig) or intramuscular (ig) use, (iv) high-titer human immunoglobulin for intravenous or intramuscular use from immunized or convalescing donors, and (v) monoclonal antibodies (mab) [9, 10] . transfusion of whole blood to provide convalescent plasma should be avoided unless its use is clinically indicated; collection of convalescent plasma should be performed only by apheresis to avoid unnecessary red cell loss in the donor [11] . plasma administration is the preferred method to provide passive immunity in pandemic scenarios at least in the immediate term; usually, immunoglobulins are prepared by fractionating large pools of human plasma collected from approximately 10,000-40,000 donors [12] . moreover, plasma administration represents a rapid and effective therapy, has lower costs than other methods [13] , and presents a broader spectrum response. studies suggest that it not only neutralizes the pathogen but also provides passive immunomodulatory mediators allowing the recipient to control the excessive inflammatory cascade induced by the infectious agent [14] . animal plasma collection should be avoided if possible as it can cause side effects collectively called "serum sickness" [15] . convalescent plasma is not a novel therapy; it is a therapy widely used in the past, both for bacterial and viral pathologies. behering and kisato were the first in 1890 to provide the basis of passive immunization, then known as serum therapy. despite limited knowledge on the structural and functional complexity of antibodies, they demonstrated that not previously immunized animals can be protected from sublethal doses of diphtheria and tetanus toxin with serum therapy. the discovery was so important that in 1901, behring earned his noble prize for it. given the early success in the 1900s, passive immunization was rapidly expanded; it was used to treat several bacterial infections including corynebacterium diphtheriae, streptococcus pneumoniae, streptococcus pyogenes, clostridium tetani, haemophilus influenzae, and neisseria meningitidis; type-specific antipneumococcal serum was used as the first-line treatment for lobar pneumonia [15] . during the first half of the 20 th century, serum therapies were successfully used to treat patients affected by many infectious diseases (anthrax, plague, scarlet fever, measles, tularemia, diphtheria, dysentery, meningococcal meningitis, rabies, and pneumococcal pneumonia) [16] . however, serum therapy for bacterial diseases suddenly stopped after the discovery of antibiotics. moreover, the tools for the correct selection of plasma were still missing; the risk of serum disease was very high in those years, as plasma was frequently prepared from the blood of hyperimmunized animals [15] . regarding viral diseases, studies about convalescent plasma use to fight pandemics of the last century are available; the reported results were positive [8] . in the early 20 th century, convalescent serum was used to fight outbreaks of viral diseases such as poliomyelitis, measles, mumps, and spanish influenza [8] . a meta-analysis by luke and colleagues reported eight studies involving 1,703 patients with 1918 influenza pneumonia from 1918 to 1925. patients were often selected among the most serious ones and received an infusion of influenza convalescent human blood products; the outcome of the treated patients was compared to that of the untreated influenza pneumonia controls. the study showed a pooled absolute reduction of 21% in the mortality rate compared to controls. unfortunately, the included studies were few and with methodologic limitations (no study was a blinded, randomized, or placebo-controlled trial; moreover, convalescent 2 biomed research international sera were developed and used in many cases without measuring antibody titers or without knowledge about viral serotypes); anyway, this treatment received consensus at the time, and it was applied in several countries [4, 8, 17] . in the modern era, the treatment of argentine hemorrhagic fever (junin virus) with convalescent immune plasma was applied as part of a nationally organized response; patients treated with immune plasma had a much lower mortality than those given normal plasma [16, 18] . convalescent plasma or immunoglobulins were administered as a last chance to reduce the mortality rate of patients with sars; several studies showed a shorter hospital stay and lower mortality in patients treated with convalescent plasma compared to those not treated with convalescent plasma. the largest study involved the treatment of 80 patients showing clinical deterioration despite treatment with methylprednisolone. earlier plasma administration was more likely to be effective: patients treated before the 14 th day had better prognosis compared to those treated later. in addition, patients who were pcr positive and seronegative for coronavirus at the time of therapy had improved prognosis. no immediate adverse reactions were observed [4, 8, 19] . positive evidence was reported for the treatment of influenza a (h5n1) too [20] . regarding the pandemic 2009 influenza a h1n1, the results from the prospective cohort study by hung and colleagues showed that plasma treatment reduced mortality (the patients involved in the study were seriously ill and required intensive care); no adverse events were observed [4, 8, 20] . a second trial by hung and colleagues was conducted on 35 patients affected by severe influenza a h1n1 during 2010 and 2011, using immunoglobulins fractionated from plasma of patients recovered from the previous 2009 influenza a h1n1. treated patients showed a lower viral load and reduced mortality rate within 5 days of symptom onset [4, 8, 21] . a meta-analysis by mair-jenkins and colleagues, including 32 studies of sars coronavirus and severe influenza, reported that convalescent plasma reduced mortality and it was safe (no relevant adverse events or complications after treatment were reported). the reduction of mortality was higher when convalescent plasma was administered earlier after symptom onset [22] . regarding ebola disease, the use of convalescent plasma was recommended by the who in 2014 as an empirical treatment during the outbreaks [8] . the first use of convalescent plasma for ebola dates back to previous times. the study of mupapa et al. involved eight patients during an outbreak in 1995; among these, seven survived [23] . the ebola-tx clinical trial tested the efficacy of convalescent plasma as a treatment for ebola in guinea: the trial confirmed convalescent plasma safety, but unfortunately, the efficacy was not proven. no association with the dose of neutralizing antibodies was apparently found, even if the levels of neutralizing antibodies were low in many plasma donations. the authors concluded that further studies were needed to assess the effectiveness of antibody doses higher than those used in their study [24] . sahr et al. used convalescent serum in sierra leone for ebola treatment: their study revealed a significantly lower fatality rate for patients treated with convalescent whole blood with respect to those receiving standard treatments [13] . a protocol for convalescent plasma in the treatment of middle east respiratory syndrome (mers) caused by a coronavirus was established in 2015. three patients with mers in south korea were treated with convalescent serum, but only two showed neutralizing activity. the authors concluded that high antibody titer (≥1 : 80) should be needed to achieve good neutralization activity [4, 8, 25] . keller and stiehm listed all the pathologies for which passive immunization has been or is currently being used. for each pathology, they specified when passive immunization is to be used for prevention versus treatment and if the efficacy has been demonstrated (they also pointed out when there is no recommendation to use the passive immunization tool, even in the case of demonstrated efficacy). more than 30 infectious pathologies were analysed. the efficacy of passive immunization in the prevention of infectious diseases has been proven for tetanus, clostridium botulinum, hepatitis a, hepatitis b, rsv (respiratory syncytial virus), cmv (cytomegalovirus), vzv (varicella zoster virus), rabies, measles, and vaccinia. in addition, passive immunization has been proven but not recommended for the treatment of respiratory infections (streptococcus, streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae) or for enterovirus infection. the efficacy of passive immunization in the treatment of infectious disease has been proven for diphtheria, tetanus, clostridium botulinum, and vaccinia and has been proven but not recommended for respiratory infections (streptococcus, streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae), parvovirus, and enterovirus [9] . as already discussed, previous studies on convalescent plasma in pandemic scenarios have shown that plasma is a safe treatment [4, 8, 13, [17] [18] [19] [20] [21] [22] [23] [24] [25] . anyway, side effects are possible for any medication; maclennan and barbara analysed the possible side effects of generic plasma administration (not only convalescent plasma) in a recipient. several factors can lead to adverse events (donor-related factors, which testing is performed on plasma, any treatment or modification to which it has been subjected, interaction between donor factors, and the patient's immune system). possible adverse reactions can be classified into three groups: immune reactions (anaphylactic/anaphylactoid reactions, mild allergic reactions, haemolysis, and transfusionrelated acute lung injury), physicochemical reactions (fluid overload, citrate toxicity, and chemicals), and infectious risks. anaphylactic reactions are uncommon, but severe and potentially life-threatening (in 2003, the incidence in the uk was approximately 0.002%). ige mediates anaphylactic reactions; the term "anaphylactoid" describes a similar reaction not mediated by ige. less severe allergic reactions are much more common and usually characterized by cutaneous symptoms, ranging from mild pruritus to urticaria and flushing. haemolysis can occur following transfusion of plasma containing high-titer anti-a or anti-b haemolysins to an a or b recipient. it could be very serious, and deaths have also been reported. to avoid this reaction, plasma should always be abo compatible; if not possible, the plasma should be tested for haemolysins and found negative for high titer of them. concomitant transfer of antibodies against other red cell antigens might occasionally cause haemolysis in the recipient. thus, donor screening procedures to detect clinically significant antibodies are essential to minimize this risk [26] . trali is an acute respiratory reaction, indistinguishable from the adult respiratory distress syndrome (ards), occurring in association with transfusion of blood components; the incidence was reported variously, ranging from 1 in 5,000 to 1 in 50,000. it is caused by the presence of antibodies against leucocyte antigens (hla antigens seem the most frequent [11] ) in donor plasma [26] . to avoid this risk, preference should be given to the use of plasma from male donors or from females who have never been pregnant, including abortions. this measure lowers the possibility to find antibodies against hla or granulocyte antigens causing trali in the donor plasma [11] . moreover, it was reported that the presence of certain antibodies may cause immune enhancement of pathogenicity, termed ade (antibody-dependent enhancement), for several viral diseases, such as dengue virus and sars [27] . physiochemical reactions can sometimes be severe, but usually not life-threatening: (i) fluid overload is one of the most common complications of transfusion and can lead to pulmonary oedema (ii) citrate toxicity depends on the action of citrate in binding calcium and therefore in reducing the availability of ionised calcium for normal neuromuscular function; it is not frequent because citrate is rapidly metabolised by the liver (iii) some units of plasma might contain chemicals (e.g., drugs) derived from the donor to which the recipient might react modern technologies allow to minimize infectious risk. firstly, bacterial transmission is not a significant risk factor as the plasma is frozen within hours of collection and processing. secondly, an accurate selection of donors and pathogen reduction processes can be applied to minimize viral infection risk (for single-unit components, methylene blue is used; for plasma pools, solvent detergent is used) [26] . specific side effects are identified for single plasma components; immunoglobulins have been associated with thrombotic events, renal toxicity, and aseptic meningitis [26] . tamburello and marando reported that treatment with human immunoglobulin during the sars-cov-2 pandemic was associated with a significantly increased risk of same-day thrombotic events (from 0.04 to 14.9%) [3] . however, the estimated risk of serious adverse events is less than 5% [26] . a recent work by joyner and colleagues explores the safeness of the use of convalescent plasma in 20,000 critically ill covid-19 patients. the cohort studied is very huge; thus, the reported results should be considered particularly reliable. serious adverse events within 4 hours of completion of covid-19 plasma transfusion were 146 (less than 1% of all transfusions). 50 events were judged surely unrelated to plasma transfusion. among the other events, there were 83 nonmortality events reported (37 reports of transfusionassociated circulatory overload, 20 reports of transfusion acute lung injury, and 26 reports of severe allergic transfusion reaction). 13 mortality events happened; they were judged only as possibly, not definitely, related to the transfusion of covid-19 convalescent plasma. notably, the vast majority of other serious adverse events, which happened within seven days of completion of the convalescent plasma transfusion, were judged to be unrelated to the plasma transfusion [28] . recently, in 2017, the who blood regulators network (brn) published a position paper, recommending the need for healthcare systems to prepare adequate infrastructures to deal with the emergence of any pandemic caused by new emerging viruses; in that paper, the brn suggested plasma from recovered patients as the first-choice treatment to be tested. based on the evidence from past experience in passive immunization, the brn explained that there was a considerable possibility that the application of whole blood (as well as plasma, serum, or immunoglobulin concentrates) from convalescent persons could be effective in the treatment/prevention of infectious disease. thus, in the absence of effective vaccines and antiviral therapies for the emerging pathogen, an organized program to collect convalescent plasma or serum from disease survivors could provide a potentially valuable empirical intervention, while data on the effectiveness and safety of its use are obtained through orderly scientific studies [16] . in fact, any blood derivative should be considered a drug, and if administered for different indications from the authorized ones, it must be tested for the specific new application [29] . epstein [31] . unfortunately, data from case reports and case series are observational, and they are not sufficient for a definitive validation of the treatment. some controlled trials are already available too; they confirm the outcomes of the first case series. a randomized control trial out of wuhan was the first to be published: 103 patients with severe or life-threatening covid-19 (52 in the convalescent plasma-treating group and 51 in the control group) were enrolled, but unfortunately, the study had an early termination due to low patient enrollment as the regional outbreak waned. contrary to expectations, the study failed to detect a statistically significant difference in the evaluated outcomes (time to clinical improvement, 28-day mortality, and time from randomization to discharge). however, convalescent plasma was demonstrated to be associated with antiviral activity in patients with covid-19 (convalescent plasma treatment was associated with higher rates of negative sars-cov-2 viral pcr results from nasopharyngeal swabs at 24, 48, and 72 hours); a statistically significant improvement was noted for the convalescent plasma treatment group compared to controls in the subgroup of patients without life-threatening covid-19 (91% improvement in the plasma group compared to 68% in the control arm). the median between the onset of symptoms and the beginning of the treatment was 30 days. this time window could have affected the study to detect a clinically important benefit of the convalescent plasma therapy, in addition to the early termination of the study and to the type of patient conditions (only severe or lifethreatening disease) [32] . the . they demonstrated that convalescent plasma is associated with reducing ventilatory requirements in patients with both severe and life-threatening diseases [33] . these results are consistent also with a recent cohort study by liu and colleagues. in this study, 39 patients were treated with convalescent plasma and were compared to 156 control patients. the authors reported a lower mortality rate among patients with severe or worse disease who received convalescent plasma and significantly better outcomes among patients transfused prior to mechanical ventilation [34] . salazar et al. enrolled 387 patients (136 transfused patients and 251 nontransfused control covid-19 patients); they found that patients transfused within 72 h of hospital admission had decreased mortality within 28 days, whereas patients transfused after 72 h of hospital admission did not. these data demonstrate that early convalescent plasma transfusion after hospital admission reduces mortality within 28 days posttransfusion [35] . two other articles deserve to be mentioned, the one of duan et al. and the other of joyner et al. duan et al. compared 10 severely ill patients treated with convalescent plasma to a historical control group of 10 severely ill patients not treated with convalescent plasma. the covid-19-transfused patients' group showed better clinical outcomes than the historical control group. all enrolled severe covid-19 patients had improvement of clinical symptoms and showed different degrees of absorption of the pulmonary lesions after convalescent plasma transfusion. the authors showed amelioration of routine laboratory criteria and pulmonary function (lymphocytopenia, an important index for prognosis in covid-19, tended to be improved after convalescent plasma transfusion). increase of neutralizing antibody titers was demonstrated [36] . joyner et al. studied the effects of convalescent plasma use in a very huge cohort of 20,000 critically ill hospitalized patients. the aim of the paper differs from the ones previously mentioned: the study was designed to demonstrate the safety of convalescent plasma. anyway, the seven-day mortality rate in this extremely high-risk cohort of patients was 8.6% only. the authors anticipated the intent to create a control comparator group using patients hospitalized with covid-19 during the same period; they will discuss potential convalescent plasma efficacy in a future publication. given the large number of observations, it is expected that the results of this study will have significant importance in evaluating the efficacy of the treatment and its reliability [28] . moreover, there are several ongoing randomized controlled trials on the role of convalescent plasma to treat covid-19 (zheng et al. estimated that the main underway trials are 48 in the world). also in this case, a positive confirmation of the results in terms of the efficacy of the treatment for covid-19 is strongly expected [37] . preparation requirements for convalescent plasma follow the standard operating procedures for plasma collection and all applicable regulations. thus, health system requirements are the same for routine plasma collection procedures via plasmapheresis. during plasma donation procedure, the blood cells and plasma are removed from the body and separated by a plasmapheresis machine; then, the blood cells are returned to the donor while plasma is collected. plasma products are stored as fresh-frozen plasma, until usage. recently, approved serological assays are necessary to detect sars-cov-2 (rt-pcr test) in serum and virologic assays [35] . regarding plasma treatment in the context of the current pandemic, the following points are worth remarking. plasma should only be collected from selected recovered individuals diagnosed with covid-19 for at least 3 weeks. at least 14 days must have elapsed since complete recovery, 5 biomed research international in order to minimize the possible risk of sars-cov-2 in the blood. the titer of anti-sars-cov-2 igg should be determined, and virus inactivation procedures should be strictly attended before using plasma [5] . actually, the recommended viral neutralization titer cut-off for covid-19 convalescent plasma is at least ≥1 : 160. this corresponds to a receptor binding domain igg titer ≥ 1 : 1350 [35] . a titer of 1 : 80 may be considered acceptable if an alternative matched unit is not available [38] . although largely experimental, the optimal dose of convalescent plasma to be administered to a covid-19 patient ranges between 200 and 500 ml [28, 31-33, 35, 36] . treatment effectiveness is expected to be better when immune plasma is collected from patients of the same city, or surrounding area, since it is assumed that these donors have defeated the same virus (virus genome can mutate); likewise, lifestyle, diet, and environment play an important role in the development of specific antibodies against the virus [39] . as a principle, convalescent plasma should be used as soon as possible in the acute stage of the disease of the recipient [4, 5] , and it is important that the titer of anti-sars-cov-2 antibodies be high. it is essential to ensure abo compatibility between donor and recipient; theoretically, transfusion of plasma from at least two donors may be better to achieve more effective immune protection from delivery of diverse antibodies. standard selection criteria for plasma donation, according to local requirements, must always be followed, as well as standard postdonation treatment of plasma [11] . unfortunately, the brn recommendations have been disregarded. at the beginning of the present pandemic, no healthcare system had already organized programs to collect convalescent plasma or serum from recovered patients to fight a potential new emerging viral pathogen. currently, some clinical studies and programs have started, but unfortunately, the procedures are very slow [7, 40, 41] . the first reported results are very encouraging and confirm the effectiveness of plasma therapy and its safety [42] [43] [44] . the classical process to approve a new drug for clinical use is long, but a terrible pandemic emergency is underway and the time to wrest the fate of many people from death is very short. it is essential that governments follow the strategy recommended by the brn in 2017. recently, the food and drug administration (fda) has also moved on this path, in consideration of the numerous evidences of efficacy and safety of plasma therapy coming from past experiences and the first scientific confirmations in the current pandemic. the administration has remarked the importance to study the safety and efficacy of covid-19 convalescent plasma enrolling patients in clinical trials. in addition, two other strategies have been authorized to allow patients to access treatment. firstly, it provides an expanded access for the use of covid-19 convalescent plasma dedicated to patients with serious or immedi-ately life-threatening covid-19 disease, who are not eligible or unable to participate in randomized clinical trials (21 cfr 312.305) . secondly, given the public health emergency, fda facilitates access to covid-19 convalescent plasma for patients with serious or immediately life-threatening covid-19 infections: the patient's physician can request a single emergency investigational new drug application to obtain expanded access for an individual patient (21 cfr 312.310) [29] . this strategy could allow to save as many lives as possible. in addition, the fda is promoting an awareness campaign to invite patients recovered from covid-19 to donate plasma [45] . expected results from ongoing trials should definitely support the researches in finding the best criteria for including/excluding convalescent plasma in covid-19 patients' treatment. as detailed in the available studies, performed analysis suggests convalescent plasma for the most serious cases and at their early stage. thus, the early recognition of the covid-19 patients who may develop critical illness is the key question for convalescent plasma treatment. they are the patients to be treated with convalescent plasma. it is known that most mild covid-19 patients can be self-recovered, and convalescent plasma may be inappropriate therapy for them. and for end-stage covid-19 patients, the convalescent plasma treatment may not be able to regress the poor outcome as demonstrated by the current studies [46] . the strategy of the fda seems the most correct, since convalescent plasma appears to be a safe and effective therapy, and a vaccine requires a long time to get ready. to date, there are no other authorized therapies against sars-cov-2. furthermore, it should not be forgotten that the other currently applied therapies (e.g., antiviral drugs and hydroxychloroquine) have remarkable side effects and are administered for compassionate use [7, 47] . another key point is that convalescent plasma should be hyperimmune and contain high antibody titers against sars-cov-2. it is still unknown how long patients have good antibody levels in their blood [48] ; therefore, at least hypothetically, the time window for convalescent plasma donation is limited to the first period after a patient's full recovery. fortunately, many patients in the world are recovering from covid-19 infection. this should be the right time to donate plasma to treat seriously ill patients. governments should be aware of this opportunity and start organizing appropriate plasma donation campaigns and adequate plasma collection programs. the author denies any conflict of interest. who pandemia who director-general's opening remarks at the media briefing on covid-19 -11 perspectives on therapeutic neutralizing antibodies against the 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significantly decreased mortality effectiveness of convalescent plasma therapy in severe covid-19 patients a scoping review of registered clinical trials of convalescent plasma for covid-19 and a framework for accelerated synthesis of trial evidence (fast evidence) recommendations for investigational covid-19 convalescent plasma, us fda could intravenous immunoglobulin collected from recovered coronavirus patients protect against covid-19 and strengthen the immune system of new patients? covid-19 convalescent plasma: phase 2 national covid-19 convalescent plasma project using therapeutic plasma exchange as a rescue therapy in covid-19 patients: a case series de donno's plasma therapy seems to work, but nobody cares convalescent plasma transfusion for the treatment of covid-19: systematic review donate covid-19 plasmau.s. food and drug administration site13 maggio effect of convalescent plasma therapy on viral shedding and survival in patients with coronavirus disease 2019 the pharmacological basis of therapeutics. goodman & gilman's, mcgraw-hill education immunity after sars-cov-2 infection key: cord-311625-d7iycdyh authors: choong, oi kuan; mehrbod, parvaneh; tejo, bimo ario; omar, abdul rahman title: in vitro antiviral activity of circular triple helix forming oligonucleotide rna towards feline infectious peritonitis virus replication date: 2014-02-20 journal: biomed res int doi: 10.1155/2014/654712 sha: doc_id: 311625 cord_uid: d7iycdyh feline infectious peritonitis (fip) is a severe fatal immune-augmented disease in cat population. it is caused by fip virus (fipv), a virulent mutant strain of feline enteric coronavirus (fecv). current treatments and prophylactics are not effective. the in vitro antiviral properties of five circular triple-helix forming oligonucleotide (tfo) rnas (tfo1 to tfo5), which target the different regions of virulent feline coronavirus (fcov) strain fipv wsu 79-1146 genome, were tested in fipv-infected crandell-rees feline kidney (crfk) cells. rt-qpcr results showed that the circular tfo rnas, except tfo2, inhibit fipv replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular tfo rnas-transfected cells. furthermore, the binding of the circular tfo rna with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. the strength of binding kinetics between the tfo rnas and their target regions was demonstrated by nanoitc assay. in conclusion, the circular tfos have the potential to be further developed as antiviral agents against fipv infection. feline infectious peritonitis virus (fipv) is an enveloped virus with a nonsegmented, positive sense, single-stranded rna genome. fipv is grouped as feline coronavirus (fcov), under the family coronaviridae. fcov is divided into two biotypes, namely, feline enteric coronavirus (fecv), a ubiquitous enteric biotype of fcov, and fipv, a virulent biotype of fcov [1] . the relationship between these two biotypes still remains unclear. two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. internal mutation theory stated that the development of fip is due to the exposure of cat to variants of fcov which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] . study has shown that about 40-80% of cats are detected with fecv shedding in their faeces [4] . about 12% of these fecv-positive cats have developed immune-mediated fatal fip disease [4] . the prevalence of fip among felines is due to continual cycles of infection and reinfection of fecv and indiscernible clinical symptoms of infected cats with fecv at an early stage before the progressive development of fipv. vaccination against fipv with an attenuated, temperature-sensitive strain of type ii fipv induces low antibody titre in kittens that have not been exposed to fcov. however, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . in addition, antibodies against fipv do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as antibody-dependent enhancement [1] . besides vaccines, several antiviral drugs such as ribavirin, 2 biomed research international interferons, and immunosuppressive drugs have been used as treatments for fipv-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . however, those treatments were ineffective. hence, there is still significant unmet medical need to develop effective treatments and prophylactics for fipv infection. triple helix forming oligonucleotide (tfo) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex dna [9] . furthermore, double helical rna or dna-rna hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . hence, tfo has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . the main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular tfo rnas against fipv replication. serotype ii strain wsu 79-1146 (atcc no. vr-1777) was grown in crfk cells. a serial 10-fold dilution of fipv was prepared from the working stock. confluent 96-well plate was inoculated with 100 l of each virus dilution/well. the plate was incubated in a humidified incubator at 37 ∘ c, 5% co 2 . cytopathic effects (cpe) development was observed. the results were recorded after 72 hours and the virus tissue culture infective dose 50 (tcid 50 ) was calculated using reed and muench's method [17] . oligonucleotide rna. the triple helix forming oligonucleotides (tfos) were designed based on the genome sequence of fipv serotype ii strain wsu 79-1146 (accession no: ay994055) [18] . tfos, which specifically target the different regions of the fipv genome, and one unrelated tfo were constructed ( table 1 ). the specificity of the tfos was identified using blast search in the ncbi database. the designed linear tfos were synthesized by dharmacon research (usa), whereby the 5 and 3 ends of the linear tfos were modified with phosphate (po 4 ) group and hydroxide (oh) group, respectively. these modifications were necessary for the circularization of linear tfo. the process of circularization, using the t4 rna ligase 1 (ssrna ligase) (new england biolabs inc., england), was carried out according to the manufacturer's protocol. after ligation, the circular tfo rnas were recovered by ethanol precipitation and the purity of the circular tfo rnas was measured using spectrophotometer. denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. then, the gel was prerun at 20 to 40 v for 45 minutes. five l of tfo rna mixed with 5 l of urea loading buffer was heated at 92 ∘ c for 2 minutes and immediately chilled on ice. it was run on the gel at 200 v for 45 minutes. finally, the gel was stained with ethidium bromide (sigma, usa) and viewed with a bio-rad gel doc xr system (ca, usa). (emsa) . the target regions of the fipv genome were synthesized by dharmacon research (usa) ( table 1) . each tfo rna was mixed with the target region in 1x binding buffer containing 25 mm tris-hcl, 6 mm mgcl 2 , and 10 mmnacl in a final volume of 10 l and subsequently incubated at 37 ∘ c for 2 hours. the sample was run on 15% native polyacrylamide gel at 80 v, in cool condition. the stained gel was viewed by a bio-rad gel doc xr system. regions. the binding strength was measured using a nano isothermal titration calorimeter (itc) (ta instruments, newcastle, uk). the rna sample mixtures, consisting of circular tfos (0.0002 mm), were incubated with their respective synthetic target regions (0.015 mm) using 1x binding buffer as the diluent. the experiment was run at 37 ∘ c with 2 l/injection, for a total of 25 injections. data was collected every 250 seconds and analyzed using the nanoanalyze software v2.3.6 provided by the manufacturer. this experiment was conducted in crfk cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. one hundred nm of tfo rnas was separately transfected into the crfk cells using a hiperfect transfection reagent (qiagen, germany), as per the manufacturer's protocol. the plate was incubated at 37 ∘ c with 5% co 2 for 6 hours. then, the cultures were infected with 100tcid 50 of fipv serotype ii strain wsu 79-1146 for 1 hour at 37 ∘ c (100 l/well). finally, the viral inoculum was replaced by fresh maintenance media (mem containing 1% fbs and 1% pen/strep). virus-infected and uninfected cells were maintained as positive and negative controls, respectively. the morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ c prior to rna extraction. inhibition. different concentrations of circular tfo1 rna (25 nm, 50 nm, 100 nm, and 500 nm) were transfected into crfk cells. the plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ c with 5% co2. the cells were processed as described above. madin-darby canine kidney (mdck) cell (atcc no. ccl-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. transfection was performed the same as before. one hundred nm of circular tfo rna was transfected into mdck cells. following 6 hours orf1a/1b and 530-541 orf1a/1b and 7399-7411 orf1a/1b and 14048-14061 * highlighted in bold indicated the binding region. * * unrelated circular tfo. [20, 21] , respectively. the reverse transcriptase quantitative real-time pcr (rt-qpcr) was performed using a bio-rad cfx96 real-time system (biorad, usa). the reaction was amplified in a final volume of 25 l using a sensimix sybr no-rox one-step kit (bioline, uk), which consisted of 12.5 l 2x sensimix sybr no-rox onestep reaction buffer, 10 m forward and reverse primers, 10 units ribosafe rnase inhibitor, and 5 l template rna. absolute quantification approach was used to quantify qpcr results where a standard curve of a serial dilution of virus was plotted before the quantification. amount of the virus in the samples was quantified based on this standard curve. analysis. data statistical analysis was performed using spss 18.0. data were represented as mean ± se of three independent tests. one-way anova, tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. the target regions were 5 untranslated region (5 utr), open reading frames (orfs) 1a and 1b, and 3 untranslated region (3 utr) ( table 1 ). the tfos were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. both ends of the duplex tfos were ligated with a linker sequence or clamps (c-c) to construct circular tfo rna. denaturing page assay was carried out after the ligation process to determine the formation of the circular tfo. as shown in figure 1 , the circular tfo rnas migrated faster than the linear tfo rnas, when subjected to 20% denaturing page. target region. the binding ability was determined using electrophoretic mobility shift assay (emsa) [23] . the appearance of the slow mobility band indicates the successful hybridization of circular tfo rna with its target region. the binding ability of different tfo rnas (tfo1 to tfo5) against their target regions was determined by emsa (figure 2) . tfo1, tfo3, tfo4, and tfo5 showed slow mobility band, while tfo2 showed the lack of an upward shifted band. this indicates the possession of triplex binding ability for all circular tfo rnas, except tfo2. tfo rna. study on the interaction and hybridization of tfo towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. as shown in supplementary figure 1 (table 3) . the antiviral effect of circular tfo rnas was investigated by rt-qpcr assay at 72 hours after transfection. the results showed viral rna genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with tfo1, tfo2, tfo3, tfo4, tfo5, and tfo7, respectively. the data analyzed by one-way anova, tukey post hoc test showed significant high viral rna genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular tfo1, tfo3, tfo4, and tfo5 treatments ( ≤ 0.05). the viral rna copies of circular tfo2, linear tfo3 and tfo4, and unrelated circular tfo7 rnas transfected cells also showed high viral rna copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( figure 3 ). the morphological changes of the cells were also captured 72 hours after transfection. the cells transfected with circular tfo1, tfo3, tfo4, and tfo5 appeared to be in good condition following virus inoculation, while the cells transfected with circular tfo2 and linear tfo3 and tfo4 showed visible cytopathic effect (cpe), the same as virus inoculated cells (supplementary figure 2) . furthermore, cells transfected with tfo only remain viable indicating that tfo treatment is generally not toxic to the cells. hence, these results illustrated the capacity of circular tfo rnas (except tfo2) to inhibit fipv replication. concentrations on fipv replication. circular tfo1 was used to examine the dose-response relationship as a representative to other tfos. the experimental conditions were identical to that of the previous experiment, except for tfo1 concentrations of 25 nm, 50 nm, 100 nm, and 500 nm. there was no significant reduction in viral rna genome copies using the concentration of 25 nm tfo1. the other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. however, no significant difference was detected in copy numbers from all of these concentrations ( figure 4 ). the specificity of the tfo towards fipv was tested, using tfo1 and tfo5, as the proper representatives of tfos, on influenza a virus h1n1 new jersey 8/76. the analyzed data using one-way anova, tukey post hoc test did not show significant reductions in the copies of viral rna for both tfos compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary figure 3 ). complex structure g4/cir4 figure 2 : emsa analysis. emsa analysis illustrated the binding of circular tfo 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. binding of each circular tfo except circular tfo2 to its respective target forms a complex that migrates slower than unbound tfo. g1 to g5 represent the target region for circular tfo1 to tfo5 and cir1 to cir5 represent the circular tfo1 to tfo5, respectively. in the replication process [24] . meanwhile, the orf1a/1b of fipv are translated into polyproteins that are cleaved into nonstructural proteins which assemble into replicationtranscription complexes together with other viral proteins [24] . hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit fipv replication. development of antiviral therapies against fipv using sirna [25] and viral protease inhibitors [26] figure 4 : tfo1 dose-response study for inhibiting fipv replication. the concentrations of 50 nm and higher showed significant antiviral effects. 50 nm of circular tfo1 rna was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nm showed 4-fold reduction. data are averages of 3 independent tests (mean ± se). * significantly different from fipv-infected group. as potential new treatments against fipv infection. in this study, circular triple helix forming oligonucleotide (tfo) rnas, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. tfo1 and tfo2 targeted the 5 and 3 utrs of the viral genome, respectively. tfo3 to tfo5 targeted different regions of the orf1a/1b on fipv genome. prior to in vitro antiviral study, the ligated circular tfos were evaluated using page analysis. all of the circularised tfo showed faster migration pattern compared to the linear tfo; however, only slight variation was detected for some of the tfo (figure 1 ). the reason for this is not clear but probably due to the differences in length and the tertiary structures of the tfos leading to differences in the migration rate. emsa was used to show the binding capability of each circular tfo towards the target region in the fipv genome except for tfo2 which showed lack of formation of complex structure upon hybridization ( figure 2) . the emsa result also concurred with the antiviral study, where all circular tfos (except tfo2) were able to demonstrate a significant reduction in the viral rna genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in tfo-transfected cells (figure 3 ). however, no antiviral properties were detected from the linear tfos and unrelated circular tfo7 rna, confirming that the antiviral activity is associated with specific binding of circular tfos towards targeted regions. furthermore, the binding of the circular tfo to the target region was confirmed by nanoitc analysis; where the low value and high stability allowed tfos to compete effectively with the target regions for inhibiting transcription in cell-free systems. since, tfo1 shows the lowest value (table 3) , the antiviral properties of this tfo were evaluated in doseresponse study. as shown in figure 4 , 50 and 100 nm of tfo1 showed similar antiviral effects indicating the potential therapeutic application of tfo1 on fipv replication. however, increasing the concentration of tfo1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the tfo into the cells. in addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of fipv in crfk cells showed that by 2 hours approximately 67% of fipv 79-1146 were internalized by crfk cells by endocytosis increasing to more than 70% at 3 hours [27, 28] . the above finding probably also explained the reason why no antiviral effect was detected when the transfection of the tfo was performed on virus-infected cells (data not shown). the antiviral properties, as demonstrated by the circular tfos, were probably associated with the binding of the tfo to the target region, based on both the watson-crick and hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . therefore, the triplex formation is tightly bonded and not easy to detach. furthermore, the circular tfos were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . however, there are various factors that may limit the activity of tfos in cells like intracellular degradation of the tfo and limited accessibility of the tfo to the target sites which can prevent triplex formation [31] . these findings may also explain the inability of the designed tfo1 to inhibit further virus replication in dose-response study (figure 4) . various molecular-based therapies against infectious diseases and cancer have been developed and tested. however, only the sirna-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . recently, mcdonagh et al. [25] developed sirna with antiviral activity against the fipv 79-1146, where the designed sirna was able to reduce the copy number of viral genome compared with virus-infected cells. the potential therapeutic application of tfos, such as linear tfo conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and tfo to inhibit the transcription of 1(i) collagen in rat fibroblasts [14] , has also been reported. in addition, short tfo conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . these studies indicated the flexibility of using tfo-based oligonucleotides as a potential molecular-based therapy. in this study, we demonstrated short circular tfo rnas between 28 and 34 mers (table 1) , which are able to inhibit fipv replication by binding to specific target regions of the fipv genome. all designed circular tfos (except tfo2) showed significant inhibitory effects against fipv replication. the tfos that formed triplex structures showed antiviral effects towards fipv replication. the reason why tfo2 failed to show any interaction with the target region or antiviral activity is probably due to the length of tfo2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (figure 2 ), be effective enough to suppress viral rna transcription, and eventually inhibit virus replication. nevertheless, the inability of tfo2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. in vitro antiviral study which showed no antiviral property for unrelated tfo (tfo7) and also inability of circular tfo1 and tfo5 to inhibit influenza a virus h1n1 infected cells confirms the specificity of the tfos' activity. in conclusion, the circular tfo rna has the potential to be developed as a therapy against fipv in cats. however, further studies on tfo specificity, actual mechanism of circular tfo rna in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future. antibody-dependent enhancement occurs upon re-infection with the identical serotype virus in feline infectious peritonitis virus infection feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses intrahost diversity of feline coronavirus: a consensus between the circulating virulent/avirulent strains and the internal mutation hypotheses? the european advisory board on cat diseases inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro feline infectious peritonitis treatment of cats with feline infectious peritonitis use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis specific inhibition of transcription by triple helix-forming oligonucleotides sequence-specific recognition of double helical rna and rna⋅dna by triple helix formation triplex forming oligonucleotides against type 1(i) collagen attenuates liver fibrosis induced by bile duct ligation targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides accessibility of nuclear dna to triplex-forming oligonucleotides: the integrated hiv-1 pro virus as a target antiparallel polypurine phosphorothioate oligonucleotides form stable triplexes with the rat 1(i) collagen gene promoter and inhibit transcription in cultured rat fibroblasts design of a novel triple helix-forming oligodeoxyribonucleotide directed to the major promoter of the c-myc gene inhibition of human prostate cancer xenograft growth by 125i labeled triple-helin forming oligonucleotide directed against androgen receptor a simple method of estimating fifty per cent endpoints polyacrylamide gel electrophoresis of rna detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr attenuation of influenza virus infectivity with herbal-marine compound (hesa-a): an in vitro study in mdck cells targeting pyrimidine single strands by tripler formation: structural optimization of binding effect of cations on purine⋅purine⋅pyrimidine triple helix formation in mixed-valence salt solutions a contemporary view of coronavirus transcription in vitro inhibition of feline coronavirus replication by small interfering rnas a biodegradable polymersome containing bcl-xl sirna and doxorubicin as a dual delivery vehicle for a synergistic anticancer effect attachment and internalization of feline infectious peritonitis virus in feline blood monocytes and crandell feline kidney cells feline infectious peritonitis virus-infected monocytes internalize viral membrane-bound proteins upon antibody addition targeting of single-stranded dna and rna containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides triplex dna: fundamentals, advances, and potential applications for gene therapy dna binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the p2 promoter of the human c-myc gene structural diversity repertoire of gene silencing small interfering rnas the rna silencing endonuclease argonaute 2 mediates specific antiviral immunity in drosophila melanogaster this work was supported by grant no. ergs/1-2012/5527123 from the ministry of higher education, government of malaysia. the funder had no role in the study design, data collection and analysis, or preparation of the paper. the authors have declared that no conflict of interests exists. oi kuan choong, abdul rahman omar, and bimo ario tejo codefined the research theme. oi kuan choong designed biomed research international 7 and carried out the laboratory experiments, analyzed and interpreted the data, and drafted the paper. oi kuan choong, abdul rahman omar, and parvaneh mehrbod revised the paper thoroughly. all authors have read and approved the final paper. key: cord-299552-rgrm8dil authors: bianchi, martina; benvenuto, domenico; giovanetti, marta; angeletti, silvia; ciccozzi, massimo; pascarella, stefano title: sars-cov-2 envelope and membrane proteins: structural differences linked to virus characteristics? date: 2020-05-30 journal: biomed res int doi: 10.1155/2020/4389089 sha: doc_id: 299552 cord_uid: rgrm8dil the coronavirus disease 2019 (covid-19) is a new viral infection caused by the severe acute respiratory coronavirus 2 (sars-cov-2). genomic analyses have revealed that sars-cov-2 is related to pangolin and bat coronaviruses. in this report, a structural comparison between the sars-cov-2 envelope and membrane proteins from different human isolates with homologous proteins from closely related viruses is described. the analyses here reported show the high structural similarity of envelope and membrane proteins to the counterparts from pangolin and bat coronavirus isolates. however, the comparisons have also highlighted structural differences specific of sars-cov-2 proteins which may be correlated to the cross-species transmission and/or to the properties of the virus. structural modelling has been applied to map the variant sites onto the predicted three-dimensional structure of the envelope and membrane proteins. covid-19 has become a planetary emergency which is seriously threatening human health [1, 2] . development of effective therapeutic and prevention strategies is significantly hampered also by the lack of detailed structural information on virus proteins, although several crystallographic structures of sars-cov-2 proteins are now available [3] [4] [5] . in this report, a structural comparison between the sars-cov-2 surface proteins from different isolates with homologous proteins from closely related viruses such as those from bat and pangolin is described. this work has been focussed onto the envelope (e) and membrane (m) proteins that, along with the spike, form the virus protein interface to the external environment. the spike glycoprotein has been already extensively studied, and a few crystallographic structures are available in the protein data bank [3] [4] [5] [6] ; consequently, this protein has not been specifically addressed within this note. identification of local structural differences, even minimal, to the closest virus proteins may indicate the mutations that enabled sars-cov-2 to cross species and/or to acquire its peculiar pathogenic properties [7, 8] . indeed, a number of examples have been reported in the scientific literature suggesting how even single point mutations in virus proteins can significantly alter their biology and pathogenesis [9, 10] . therefore, comparative studies may shed light on the molecular mechanisms through which an epidemic of epizootic origin can emerge and may also suggest molecular targets for therapeutics or reverse vaccinology experiments. nucleotide and protein sequences have been taken from genbank [11] data repository. blast suite [12] has been used for databank searches; jalview [13] and mafft [14] have been used for multiple sequence display and alignment, respectively. transmembrane helix prediction has been obtained by tmhmm [15] , memsat [16] , and protter [17] . cd-hit program [18] has been used for sequence clustering. homology modelling relied on swiss-model [19] , modeller [20] , or hhpred [21] and structure display and analysis on open-source pymol [22] . when necessary, i-tasser [23] has been used as an alternative source of ab initio homology models. modelling. from the genbank repository, 797 complete genomes of sars-cov-2 have been collected (the full list is reported in supplementary data). the tblastn program has been used to extract the sequences of e and m proteins from each genome. to remove redundancy within each e and m protein set, cd-hit clustering has been applied at 100% sequence identity level: identical sequences have been assigned to the same group for which only one representative has been considered for further analysis. the sars-cov-2 e and m protein sets have been grouped into three and seven clusters, respectively. this finding suggests that within the 797 genomes three and seven variants of the e and m proteins can be observed, respectively. e and m homologous proteins from closely related virus have been retrieved from the genbank using the tblastn tool. protein. the e protein is conserved across β-coronaviruses. only three variants have been found in the sars-cov-2 e set collected. sequence comparisons show that the sars-cov-2 e protein from the reference genome (refseq code yp_009724392) is identical to the sequences from pangolin cov mp798 and bat cov covzxc21, covzc45, and ratg13 isolates. the multiple sequence alignment reported in figure 1 demonstrates that a distinguishing feature of sars-2-cov e variants is the presence of arg at position 69 that substitutes glu, gln, asp in other homologous sars-cov e proteins. this site is followed by a deletion in position 70 corresponding to gly or cys in the other proteins. sars-cov-2 e sequences differ from the homologous proteins also at positions 55-56, where the dyad ser-phe replaces thr-val (except in bat coronavirus isolate btky72, accession code ky352407). variants of the sars-cov-2 e protein differ at positions 37 and 72 where his substitutes a leu and leu replaces a conserved pro, respectively. the size of each envelope variant cluster is reported in table 1 along with accession codes and definitions of the isolates. a homology model of the e protein has been built with modeller using as a template the pentameric ion channel structure of sars-cov protein identified by the pdb code 5x29. this sequence shares 91% identity to sars-cov-2 e protein and covers the segment encompassed by positions 8-65. figure 2 displays the structure of the homology model of the sars-cov-2 e protein assembled as a pentameric viroporin-like protein. figure 2 displays also the position of the variant sites onto the three-dimensional model. prediction of the transmembrane helices is difficult in a short protein. therefore, transmembrane topology cannot be assigned reliably. likewise, experiments have not clarified definitively which portions of the e protein are exposed to the external or internal side of the virus membrane [24] . the m glycoprotein is conserved across the β-coronaviruses. however, seven variants of sars-cov-2 m protein were identified in the collected set, while only three variants were observed for the e protein ( figure 3) . the multiple sequence alignment shows a remarkable similarity (98% identity) among the sars-cov-2 m variants and the sequences from bat and pangolin isolates. however, a difference at the n-terminal position (figure 3 ) can be observed: the insertion of a ser residue at position 4 table 1 along with accession codes and definitions of the isolates. noteworthy, the protein from the sars-cov-2 nihe isolate (accession code mt127115) possesses an arg instead of a conserved gly at position 89 ( figure 3 ). the mutation occurs within a predicted transmembrane helix and, if confirmed, may have a significant impact on the protein properties ( figure 3) . the three-dimensional model of the m protein has been taken from the i-tasser server (code qhd43419) as other methods failed to find any suitable template. however, it should be mentioned that hhpred found a weak local affinity, albeit below the statistical significance level, to 4n31, a peptidase-like protein from streptococcus pyogenes essential for pilus polymerisation. biomed research international be considered with great caution and should be treated as a low-resolution approximation of the real structure. according to the prediction of the transmembrane helix topology, the n-and c-terminal portions of the m protein are exposed outside and inside the virus particle, respectively ( figure 4 ). previous studies pointed out that e and m proteins could be important for viral entry, replication, and particle assembly within the human cells [24, 25] . according to the accepted theories, the current covid-19 pandemic has been caused by the cross-species transmission of a β-coronavirus normally hosted by bats and, perhaps, pangolin to humans [3, 26] . in this paper, e and m proteins from 797 sars-cov-2 genomes have been compared to the counterparts taken from the most closely related virus also to evaluate the potential role of amino acid mutations in the epizootic origin of covid-19. e protein is a minor component of the virus membrane though it is deemed to be important for many stages of virus infection and replication [24, 25] . sequence comparison has shown that this protein is identical to the counterparts of specific bat and pangolin coronavirus isolates, even though the sars-cov-2 sequence seems to possess specific modifications and characteristics with respect to other sars covs. in particular, arg69, a positively charged amino acid, replaces glu or gln residues, negatively charged and neutral, respectively, in the homologous cov proteins. moreover, a deletion specific to sars-cov-2 proteins flanks this position. unfortunately, it is not possible to predict reliably whether the sites of these modifications are exposed to the internal or external side of the membrane. in any case, the substitution and the deletion appear a rather drastic change and may have a significant impact on conformational properties and possibly on protein-protein interactions. further structural studies are needed. however, it may be hypothesized that these changes can also affect the olig-omerization process necessary to form a transmembrane ion channel. it has been demonstrated that m protein is more prevalent within the virus membrane, and it is deemed to be important for the budding process of coronaviruses. indeed, during the process of virus particle assembly, this protein interacts with the nucleocapsid, envelope, spike, and membrane glycoprotein itself [25] . moreover, in alphacoronaviruses, it has been demonstrated that this protein cooperates with the spike during the cell attachment and entry [27] . therefore, mutations occurring at the n-terminus region, which is exposed to the virus surface, could play a key role in the host cell interaction. in conclusion, the analyses here reported show the structural similarity of e and m proteins to the counterparts from pangolin and bat coronavirus isolates. at the same time, comparisons have highlighted structural differences specific of sars-cov-2 proteins which may be correlated to the cross-species transmission and/or to the properties of the virus. although further studies are needed, it is clear that these amino acid variations have been important for the virus evolutionary history, and the results may hint at how similar mutations within the coronavirus family can lead in the next years to other epizootic epidemic events similar to the one that we are experiencing these days. all sequence data are available in the genbank repository. the complete list is available in the supplementary materials. the authors declare that there is no conflict of interest regarding the publication of this paper. the 2019-new coronavirus epidemic: evidence for virus evolution severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges the global spread of 2019-ncov: a molecular evolutionary analysis structure, function, and antigenicity of the sars-cov-2 spike glycoprotein cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of sars-cov-2 by full-length human ace2 covid-2019: the role of the nsp2 and nsp3 in its pathogenesis cross-species transmission of the newly identified coronavirus 2019-ncov distinct mutation in the feline coronavirus spike protein cleavage activation site in a cat with feline infectious peritonitis-associated meningoencephalomyelitis two-amino acids change in the nsp4 of sars coronavirus abolishes viral replication genbank gapped blast and psi-blast: a new generation of protein database search programs jalview version 2-a multiple sequence alignment editor and analysis workbench mafft multiple sequence alignment software version 7: improvements in performance and usability secreted protein prediction system combining the psipred protein structure prediction server protter: interactive protein feature visualization and integration with experimental proteomic data cd-hit: accelerated for clustering the next-generation sequencing data swiss-model: homology modelling of protein structures and complexes protein structure modeling with mod-eller a completely reimplemented mpi bioinformatics toolkit with a new hhpred server at its core the axpymol molecular graphics system, version 2.0, schrödinger, llc the i-tasser suite: protein structure and function prediction coronavirus envelope protein: current knowledge membrane binding proteins of coronaviruses genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding membrane protein of human coronavirus nl63 is responsible for interaction with the adhesion receptor this work has been in part funded by a grant to sp from sapienza university of rome (rp11916b74b27c4d). the supplementary data consist of an excel file containing the list of the 797 sars-cov-2 genomes retrieved from genbank and analysed in the article. for each genome, the sequence header is displayed as found in the genbank. the header reports the accession code in the first field of each line after the ">" character. (supplementary materials) key: cord-026595-imn2jxcu authors: qamar, mariam khan; shaikh, babar tasneem; afzal, aamir title: what do the dental students know about infection control? a cross-sectional study in a teaching hospital, rawalpindi, pakistan date: 2020-06-01 journal: biomed res int doi: 10.1155/2020/3413087 sha: doc_id: 26595 cord_uid: imn2jxcu background: dental students are exposed to various infections and infective sources during their training, and on this aspect, their level of knowledge is suboptimal and practices are risky. therefore, improving their knowledge and practices would contribute significantly to infection control. objective: to ascertain the level of understanding of senior dental students regarding the infection control in the dental practice. methods: a cross-sectional study was conducted among dental students (3rd year and 4th year) of the foundation university dental college, pakistan. the sample consisted of 100 third year dental students and 88 fourth year students. a self-administrated questionnaire was used for data collection which consisted of fourteen close-ended items. frequencies of knowledge, attitudes, and practice were calculated separately by using spss 21.0 software. results: almost half of the students would not use any antiseptic for sterilizing their hands, and only two-third would ask their patient to use an oral mouth rinse before starting the treatment. many students did not the optimal temperature of the autoclave for sterilization of the instruments. only one-third would wear the personal protective equipment during a procedure. around one-third of the study participants reported that ineffective sterilization during clinical practice can transmit infection from one patient to another. conclusion: knowledge on infection control among the dental students is though weak, practices are not as per standards but attitudes are positive and encouraging for taking steps and complying with measures on infection control. dental students are one of the dental health care professionals who are at a high risk of exposure to infections because of their direct contact with the patients, infected instruments, and hospital environment. cross infection is a major concern to all the dental health care professionals. it is defined as the transmission of infection between the staff and the patient within the hospital environment [1] . among these, the most serious oral infections are caused by bacteria that colonize in the oral cavity including mycobacterium tuberculosis, influenza virus, and streptococci. students are equally vulnerable to cross infections that are caused by the hepatitis b virus (hbv), hepatitis c virus (hcv), and other viruses [2] . moreover, they are also at a risk of percutaneous occupational injuries and eye exposure while treating the patients [3] . only through strict safety precautions and implementation of guidelines for infection control, we can prevent these mishaps from happening. the center for disease control and prevention (cdc) in the united states has updated its guidelines on infection control in dental settings. the aim of these guidelines is to ensure a safe working environment to prevent the transmission of nosocomial and occupational infections among the dental health professionals [4, 5] . dental colleges are responsible for providing proper training to their students to ensure safety of the patients, implementing infection control measures, and establishing a safe working environment [6] . several studies have been conducted to assess the practices and knowledge of dental students and have demonstrated poor compliance of the students to infection control measures. a study conducted in india to assess the infection control practices among dental students showed that only one-tenth of the respondents adhere to the infection control measures [1] . similar studies have been conducted worldwide to investigate the knowledge and practices of dental students on infection control [6] [7] [8] [9] [10] [11] [12] [13] [14] , and a general consensus is that students need awareness and must be protected in the unsafe environment. fauji foundation hospital in rawalpindi city houses a huge number of medical and dental students for educational and training purposes. this hospital is a busy tertiary health facility catering to mostly ex-army servicemen and their families. it has a fully equipped dental outdoor clinic where the dental undergraduate students come on daily basis for observations, education, hands-on training, or apprenticeship. nevertheless, there is paucity of data with regard to infection control practices among dental students within the hospital environment in pakistan. for this purpose, it is imperative to ascertain the understanding of the senior dental students who have attended the module of infection prevention and control during their study course. this study will help better understand the gaps and deficiencies in the dental college curriculum and will sensitize and educate the future dental surgeons in adopting the necessary infection prevention practices. a cross-sectional study was conducted among dental students (3 rd year and 4 th year) attending the foundation university dental college, pakistan. the duration of study was two months from 14 th july 2019 to 14 th september 2019. the sample size (n = 188) was calculated by using the who sample size calculator with the following parameters: confidence level = 95%, anticipated population proportion = 95:5% [6] , and absolute precision required = 3%. a total of 228 dental students studying in the college included 121 third year and 107 fourth year students. out of these approaches, 82.6% (n = 100) students from the third year and 82.2% (n = 88) students from the fourth year gave a complete response and therefore were included in the study. as per the exclusion criteria, the students of first and second year were excluded from the study because training in infection control is provided in the dentistry curriculum of third year and fourth year. written informed consent was taken from all the participants of the study. the institutional review board of the hospital granted the requisite permission to conduct the study. a total of 188 dental students completed the questionnaire comprising of 14 items. there were 6 questions related to "knowledge," 5 questions related to practice, and 3 questions were based on "attitude." a self-administered questionnaire developed by singh et al. [6] was distributed to the dental students. the consistency of the questionnaire was assessed using cronbach's alpha (α = 381). each student was given fifteen minutes to fill the questionnaire in silence without discussing with each other. they were not asked to put their names on the forms, hence keeping the anonymity intact. data was entered and analyzed on spss v.21. descriptive statistics was done in terms of mean and standard deviation. frequency and percentage were calculated for qualitative variables. independent sample t-test was used to compare the mean score of knowledge, attitude, and practice among both groups. p value ≤ 0.05 was taken as a level of significance. written verbal consent was obtained from all the respondents. study received approval from the ethics and research committee of the foundation university, rawalpindi, dated january 2, 2019. a total of 188 dental students responded to our questionnaire completely and hence included in the study results. hence, the study population comprised of 28 male and 168 female students. table 1 shows the analysis of cross tabs between the independent (gender and class of study) and dependent variables which yielded no significant statistical association (i.e., p > 0:05). table 2 shows that although a majority (94%) reported washing their hands before and after examination of the patient, yet only half of them (49%) would use an antiseptic solution for washing their hands. there were 64% participants who preferred patients to have an oral mouth rinse before commencement of any treatment procedure. the majority (98%) of the students considered isolation as an important measure for the infection control. with regard to vaccination, 85% got vaccinated for hepatitis b, 45% for tetanus, and only 15% of the students were vaccinated for tuberculosis, but surprisingly, 18% have not received any vaccine at all. on the subject of sterilization of instruments, 95% reported use of autoclave for the purpose and only 68% reported using the same for 15 minutes (optimal time) and only 60% thought that 120 degrees is the required temperature on which the autoclave should be used. around 80% study participants considered that hepatitis b has the highest rate of transmission via saliva. in case of direct blood contact with a hiv patient, 62% would opt for anti-hiv immunoglobulins. during the work at a dental surgery, the use of face mask and gloves as an infection control measure was practiced by 38% while 32% would wear an eye protector, and only one-third of them (29%) would wear all of them. after using a pair of gloves on the patient, 57% would dispose them off but 43% think that they can reuse them after washing. only 37% reported that ineffective sterilization during clinical practice can transmit infection from one patient to another, although a large majority (98%) believe that besides instruments, disinfecting the dental chair, clinic, and doctor's office is necessary. in table 3 , it has been observed that there is a significant positive correlation between the mean score of knowledge and attitude r = 0:781, p ≤ 0:05; however, there was no differences hands are the foremost significant reservoir for many pathogens. handwashing is therefore considered as an effective method of prevention of infection [15] . in the current study, the majority of the students described that hands should be washed and the students described plain soap and antiseptic solution before and after the examination as a preferred method. similar results were seen in a study conducted in germany where most of the trainee dentists washed their hands before and after the procedure [8] . nonetheless, there are studies that show poor compliance of the students with handwashing [9] . a study conducted in yemen reported only 43% of the students adhere to handwashing [9] . in the present study, most of the dental students were vaccinated with hepatitis b vaccine which was in concurrence with the other studies [12] [13] [14] . this rate is much higher than the study conducted in india showing that only 38% of the students were vaccinated against hepatitis b [6] . the dental students had good knowledge of autoclaving and sterilization similar to other studies [6, 9] . wearing of face mask and eye protection was observed in only one-third of the students. similar results were recorded in other studies [9] . however, a good majority of the dental students in our study thought that hepatitis b infection and not tuberculosis is transmitted through saliva, which was quite surprising. another surprising finding in our study was that two-thirds of the students considered that ineffective sterilization during clinical practice cannot transmit infection from one patient to another. this highlights the lack of awareness of the concept of cross infection. the majority of the students did believe that disinfection of dental chairs and the office of the dentist is also required. this concurs with the findings of other studies included in our literature review [2, 3] . importance of continuous-based infection control lectures and training could help in raising the level of knowledge regarding the subject [16] . we recommend that every hospital needs to set up infection control measures in accordance with the guidelines. for both the students and faculty of a hospital to improve their practice and knowledge of infection control, hospitals need to address the need for quality assurance and implementation of infection control measures. it is highly recommended that reinforcement training of the students through periodic workshops on infection precaution and control should be planned to highlight the significance of infection control [17] . another recommendation is that of vaccination, especially hepatitis b, which should be mandatory for all medical and dental students prior to their admission. the strength of this study is that it was conducted with a senior group of dental students (3 rd and 4 th year) who have studied the module on infection prevention and control. the use of a self-administered questionnaire itself is one of the limitations of this study; therefore, objectivity in responding to certain questions might have been compromised. results could be generalized with caution because similar group of students in a private hospital setting with better infection control and prevention standard operating procedures might exhibit different behaviors and practices. we concluded that there was lack of correct knowledge on infection control among the dental students, yet they showed a positive attitude towards infection control measures. however, a greater compliance and supervision would be needed. further studies in similar settings and utilizing the mix methods with qualitative enquiry would be beneficial to understand certain careless attitudes and behaviors among dental students. available with first author. the authors declare that they are no conflict of interests. compliance with infection control programs in private dental clinics in jordan attitudes and practices of infection control among senior dental students at college of dentistry, university of sharjah in the united arab emirates infection control: knowledge and compliance among saudi undergraduate dental students updated cdc recommendations for the management of hepatitis b virus-infected health-care providers and students guidelines for infection control in dental health-care settings-2003 knowledge, attitudes, and practice regarding infection control measures among dental students in central india cross-infection control in malaysian dental practice rate of compliance with hand hygiene by dental healthcare personnel (dhcp) within a dentistry healthcare first aid facility knowledge, attitudes, and practice of infection control among dental students at sana'a university knowledge, attitude and practices about hepatitis b and infection control measures among dental students in patiala infection control measures in private dental clinics in lebanon knowledge, attitude, and practice of needle stick and sharps injuries among dental professionals of bangalore, india awareness of droplet and airborne isolation precautions among dental health professionals during the outbreak of corona virus infection in riyadh city, saudi arabia knowledge, attitude and practice of infection control measures among dental practitioners in public setup of karachi, pakistan: cross-sectional survey compliance with infection control practices in a university hospital dental clinic knowledge, attitude and compliance of infection control guidelines among dental faculty members and students in ksu evaluation of final-year turkish dental students' knowledge, attitude, and self-perceived competency towards preventive dentistry appreciation is hereby extended to the students of foundation medical and dental university for participating in our study. mkq, bts, and aa were all responsible for defining the initial research question and developing the protocol and study design. mkq and aa were involved in the collection of data and took lead in writing the manuscript. bts contributed to the completion of the manuscript and has critically revised it. all authors read and approved the final manuscript. key: cord-354730-hfau2odb authors: wang, rong; zhang, yan-jin title: antagonizing interferon-mediated immune response by porcine reproductive and respiratory syndrome virus date: 2014-07-03 journal: biomed res int doi: 10.1155/2014/315470 sha: doc_id: 354730 cord_uid: hfau2odb interferons (ifns) are important components in innate immunity involved in the first line of defense to protect host against viral infection. porcine reproductive and respiratory syndrome virus (prrsv) leads to severe economic losses for swine industry since being first identified in early 1990s. prrsv interplays with host ifn production and ifn-activated signaling, which may contribute to the delayed onset and low level of neutralizing antibodies, as well as weak cell-mediated immune response in infected pigs. prrsv encodes several proteins that act as antagonists for the ifn signaling. in this review, we summarized the various strategies used by prrsv to antagonize ifn production and thwart ifn-activated antiviral signaling, as well as the variable interference with ifn-mediated immune response by different prrsv strains. thorough understanding of the interaction between prrsv and host innate immune response will facilitate elucidation of prrsv pathogenesis and development of a better strategy to control prrs. porcine reproductive and respiratory syndrome (prrs) is an important infectious disease, causing huge economic losses to the swine industry worldwide [1, 2] . the prrs clinical signs include respiratory disorders, abortion in pregnant sows, and variable mortality in piglets. prrs was first identified in the usa in 1987 and subsequently in europe. the causative agent of the disease is the prrs virus (prrsv), a positive-sense single-stranded rna virus, belonging to the arteriviridae family in the order nidovirales [3] . according to the genetic differences, prrsv is grouped into two genotypes: european (type 1) and north american (type 2), represented by lelystad virus (lv) and vr-2332 strains, respectively. the genome of prrsv is about 15 kb in length with 10 open reading frames (orfs) [3] . orf1a and orf1b comprise 80% of the viral genome and encode viral enzymes involved in virus replication. in addition, polypeptides from the two orfs are processed into 14 nonstructural proteins (nsps), including nsp1 , nsp1 , nsp2, nsp2tf, and nsp3∼12 [3, 4] . orf2a, orf2b, orf3 through orf7, and orf5a code for eight structural proteins: gp2, envelop protein (e), gp3∼ 5, membrane protein (m), nucleocapsid protein (n), and orf5a protein [3, 4] . swine are the only known host of prrsv. prrsvinfected pigs develop a delayed onset of neutralizing antibodies and a weak cell-mediated immune response [5, 6] . the main target cells for prrsv infection in vivo are porcine pulmonary alveolar macrophages (pams), which play a crucial role in host immune response [7] . in order to successfully invade host, prrsv has evolved various strategies to interfere with host innate immunity. some of the prrsv proteins take part in the modulation of ifn-mediated immune response. host innate immune responses play a key role against early viral infection. interferons are major components of inmate immunity and have diverse biological functions including antiviral activity, antiproliferative activity, stimulation of t cell cytotoxic activity, and modulation of immune response [8] . there are three types of interferons. in human, type i interferons include ifn-, ifn-, ifn-, ifn-, and ifn[9, 10] . in addition, ifn-, ifn-, and ifn-(or limitin) have been identified as type i ifns in swine, ruminant, and mice, respectively [11] . almost all cell types are capable of producing ifn-/ ; however, plasmacytoid dendritic cells (pdc) are considered to be the major source of ifn-production during viral infection [12, 13] . type ii ifn contains only ifn-, whose production is restricted to activated t cells, natural killer cells, and macrophages [14] . type iii ifns comprise ifn-1, ifn-2, and ifn-3 (also known as interleukin-(il-) 29, il-28a, and il-28b, resp.), which are mainly generated by dendritic cells [11] . considering the major roles in antiviral response by type i ifns, we focus on this type of ifns and discuss the prrsvmediated interference with their production and signaling. this review summarizes the recent advances in the research of prrsv interference with ifn-mediated innate immunity, the viral proteins involved, and their molecular mechanisms, as well as diverse effects by different strains and in different cell types. a few relevant reviews on prrsv interplay with innate immunity were published previously [15] [16] [17] . readers are encouraged to read them if interested as these reviews were written in different angles to address the issue with diverse scopes, though there is some overlap in certain topics. this review is arranged into sections of ifn induction, ifn-activated signaling, ifn-stimulated genes, and perspective. host pattern recognition receptors (prrs) for rna viruses include toll-like receptors (tlrs) and rig-(retinoic acid inducible gene-) i-like receptors (rlrs). tlrs that can detect viral rna are tlr3, tlr7, and tlr8 [18] . the rlr family of prrs comprises rig-i and melanoma differentiationassociated gene 5 (mda-5) [19] . both rig-i and mda-5 signal through adaptor ips-1 (also known as mavs, cardif, and visa) on the outer membrane of the mitochondria [20] . tlr3 and rlr can recognize double-stranded rna (dsrna) of viral genome or replication intermediate of rna viruses. activation of tlr and rlr signaling pathways leads to activation of interferon regulatory factor 3 (irf3), irf7, and nf-b. these transcription activation factors translocate into the nucleus and result in induction of type i ifns and expression of inflammatory cytokines, which not only lead to an antiviral state of the neighboring uninfected cells, but also serve as key regulators to evoke adaptive immune response. at least 39 functional type i ifn genes have been identified in porcine chromosomes 1 and 10 [21] . these ifn genes include 17 ifn-subtypes, 1 ifn-, 11 ifn-, 7 ifn-, 1 ifn-, 1 ifn-, and 1 ifn-. prrsv is sensitive to type i ifns and the sensitivity is confirmed in vitro and in vivo. pretreatment of pams with porcine ifn-resulted in significant reduction of prrsv yield [22] . pretreatment of marc-145 cells and porcine pulmonary alveolar macrophages (pams) with porcine ifninhibited prrsv replication [23] . pigs that were inoculated with recombinant adenovirus for ifn-expression and challenged one day later with prrsv had lower febrile responses, reduced lung lesion, and delayed viremia and antibody response compared to controls [24] . therefore, for invading host immune clearance, prrsv has evolved multiple strategies to antagonize the host ifn induction. cells. prrsv appears to inhibit synthesis of type i ifns in pigs infected with type 1 strains, while swine transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) induced high level of ifn[22, 25] . ifncould not be detected in the lungs of pigs in which prrsv actively replicated. it was estimated that the ifn-inducing capacity of prrsv is at least 159-fold lower than that of prcv [22] . prrsv infection of pams leads to no ifn-production and when the cells were superinfected with tgev, no ifnwas detected either [25] . the prrsv suppression of ifn induction correlates with the virus replication. plasmacytoid dendritic cells (pdcs) are thought to be the major source of ifnin vivo. prrsv fails to induce porcine pdcs to produce ifn-, while pseudorabies virus (prv), swine influenza virus (siv), and tgev stimulated the pdcs to synthesize ifn[26, 27] . moreover, presence of prrsv markedly reduced the typical ifn-response of pdcs to tgev or toll-like receptor 9 agonist. loving et al. showed that prrsv replicated in monocyte-derived dcs but not lung dcs and the response of both cell types to prrsv was only limited to ifn-transcription [28] . additionally, for marc-145 cells prrsv replication also significantly inhibited the dsrna-induced type i ifn expression [29] [30] [31] . these data suggest that prrsv infection directly interferes with type i ifn induction in vivo and in vitro. the prrsv proteins that are found to be antagonists of ifn induction include nsp1, nsp2, nsp11, and n ( figure 1 and table 1 ) [30] [31] [32] [33] [34] [35] [36] . nsp4, a 3c-like serine protease that is responsible for most of the nonstructural protein processing [37] , was found to inhibit ifn-promoter activation in a reporter assay [31] , but no further characterization was reported. further study is needed to elucidate the mechanism. also nsp1 has been studied in more detail than others. nsp1 is self-cleaved into nsp1 and nsp1 subunits, both of which mainly localize in the cell nucleus and dramatically inhibit ifn-expression by affecting the irf3 signaling pathway [32] . ifn-promoter reporter assay was performed in hek293t cells in the study. the result showed that nsp1 and its two cleavage products, nsp1 and nsp1 , inhibited the activation of ifn-promoter (p125-luc) and an artificial promoter containing three irf3 binding sites (p55-cib-luc) after sv40 stimulation. prrsv nsp1 and nsp1 blocked the induction of ifn-at downstream of irf3 activation but had no effect on the phosphorylation and translocation of irf3. it suggested that nsp1 and nsp1 may have a direct effect on the formation of the transcription enhanceosome on the ifnpromoter inside the nucleus. kim et al. showed that nsp1 inhibited irf3 association with creb-binding protein (cbp) in the nucleus but had no effect on irf3 phosphorylation figure 1 : interference of type i ifn production by prrsv proteins. activation of rlr pathway and signaling by viral dsrna is shown. viral dsrna is generated during prrsv replication. "p" besides irf3 and irf7 indicates phosphorylation. red-colored blocks indicate prrsv proteins known to inhibit the signaling molecules indicated. prrsv nsp1 inhibits irf3 association with cbp, enhances cbp degradation, and interferes with i b degradation. and nsp1 inhibits irf3 phosphorylation and nuclear translocation. also nsp2 inhibits irf3 phosphorylation and nuclear translocation, interferes with i b polyubiquitination, and prevents its degradation. and nsp11 inhibits irf3 phosphorylation and nuclear translocation via degrading ips-1 mrna. ifn signaling blocking stat1/stat2 nuclear translocation [55] and nuclear translocation [30] . immunoprecipitation with anti-irf3 antibody found that interaction between cbp and irf3 in prrsv-infected marc-145 cells or nsp1-transfected hela cells was weaker than in cells treated with polyi:c alone [30] . the expression of nsp1 also enhanced cbp degradation, which can be rescued by mg132 treatment, a proteasome inhibitor. no interaction between nsp1 and cbp was found. the process is independent of the pcp activity of nsp1 [30] . beura et al. showed that nsp1 interfered with irf3 signaling pathway by inhibiting dsrna-induced irf3 phosphorylation and nuclear translocation [31] . the discrepancy is possibly because an nsp1 that is 27-residue longer than its authentic form was used in beura's study. another possible reason is that different prrsv strains were used as a couple of other studies showed that prrsv replication significantly blocked dsrna-induced irf3 activation in marc-145 cells [38, 39] . and nsp1 was also found to downregulate irf3 protein level and inhibit its phosphorylation [39] . in our laboratory, we observed that prrsv infection of marc-145 cells led to reduction of irf3 protein level (unpublished data). luo et al. [38] showed prrsv blocked ifn-production and irf3 nuclear translocation via significantly inhibiting activation of ips-1 in rig-i signaling pathway. the structure-function studies of nsp1 and nsp1 identified critical motifs of the proteins in inhibition of ifn induction. the zinc-finger (zf) domain in the c-terminus of nsp1 is critical for this protein to antagonize both ifninduction and nf-b activation, especially the 14 amino acids at c-terminal of the nsp1 [40] . shi et al. [41] screened a series of nsp1 c-terminal truncated mutants and revealed that the amino acid residue f176 of nsp1 is essential for the inhibition of ifn-induction. the residue f176 played a role in both tlr3 signaling and rig-i signaling pathways [41] . double mutations k130a/r134a (type 1 prrsv) or k124a/r128a (type 2 prrsv) in a highly conserved motif of nsp1 , gkylqrrlq, dampened the nsp1 inhibition of ifn induction [42] . moreover, recovered recombinant viruses with the nsp1 mutations by reverse genetics induced higher level gene expression of type i ifns than that of wild type viruses. also nsp2 inhibits ifn induction by blocking irf3 phosphorylation and nuclear translocation. the cysteine protease domain (pl2) of nsp2 was necessary for antagonizing activation of irf3 pathway [33] . the cysteine protease domain (pl2) at the n-terminus of nsp2, which belongs to the ovarian tumor (otu) protease family, was shown to inhibit type i ifn induction by interfering with the nf-b signaling pathway [35] . the otu domain possesses deubiquitinase activity, which interferes with the polyubiquitination of i b and subsequently prevents its degradation. recovered recombinant viruses with mutations in the otu domain of nsp2 by reverse genetics were found to be unable to inhibit nf-b activation as effectively as the wild type virus. nsp11 is an endonuclease [43] and ifn antagonist [31] . nsp11 suppressed the activation of ifn-promoter and the expression of irf3-mediated genes. the endoribonuclease activity of nsp11 was essential for nsp11 to inhibit dsrnainduced ifn-induction [36] . the amino acid residue h129 of nsp11, a presumed catalytic histidine, was involved in the inhibition of irf3 phosphorylation. it seems that the inhibition of irf3 activation is due to the nsp11 endoribonuclease activity, which can cleave mrna of ips-1 [15] , the adaptor molecule for rig-i and mda-5. the ifn antagonizing activity is not restricted to nonstructural proteins of prrsv. structural proteins, such as the n protein, were found to downregulate ifn-mrna level in polyi:c-treated immortalized pam cells [34] . n protein interferes with dsrna-induced phosphorylation and nuclear translocation of irf3. the multiple components of prrsv are involved in the interference with ifn induction. the nsps are early proteins and n is a late one, which may play roles at different stages of viral replication. induction. the effect of prrsv replication on ifn induction appears to be variable among different strains and different cell types. prrsv field isolates have variable suppressive effect on ifn-induction in pam cultures and the suppression was found at posttranscriptional stage [44] . this is not unexpected as prrsv strains are divergent in genomic sequences. prrsv infection of monocyte-derived dendritic cells (mo-dc) induced the transcription of ifn-/-but no detectable ifn-in culture supernatant, suggesting a blockage at posttranscriptional stage [45] . prrsv activated the transcription of ifn-in a pi3k-dependent manner in mo-dc cells. prrsv infection of marc-145 cells inhibits ifn gene expression [29] by interfering with the ips-1 activation in the rig-i signaling pathway [38] . a variety of type 1 and type 2 prrsv strains were found to stimulate ifnsecretion by pdc via tlr7 pathway and the effect did not require live virus [46] . the suppressive effect on pdc may be strain dependent. a novel isolate, a2mc2, induced ifns in both marc-145 and pam cells and virus replication was needed for ifn induction [47] . ifn-2 and elevation of isgs were detected in a2mc2-infected cells. sequencing analysis indicated that a2mc2 was closely related to vr-2332 and ingelvac prrs mlv with an identity of 99.8% at the nucleotide level [47] . there were a total of 28 nucleotide (nt) variations when compared to vr-2332, resulting in 14 amino acid changes scattered from nt 4681 to the end of the genome. compared to both vr-2332 and mlv, a2mc2 has 15 unique nucleotides. yet the mechanism of this strain inducing ifns is not known and it is intriguing to note that the first 4.6 kb of its genome is identical to vr-2332. its nsp1 and nsp1 proteins should be able to inhibit ifn induction. we hypothesize that the unique nucleotides in a2mc2 genome resulting in special rna structures or unique dsrna formation during early viral replication could evoke ifn production. it is worth to note that the induction of ifns is dose dependent. the virus is able to replicate when the inoculum is at less than 0.1 moi, which induces limited ifns that cannot suppress the virus replication [47] . a2mc2 infection of pigs resulted in earlier onset and higher level neutralizing antibody against homologous and heterologous strain than mlv vaccine strain that is highly homologous in sequence [48] . type i ifns are critical to innate immunity against viral infections and play an important role in the stimulation of adaptive immune response [49, 50] . the activation of ifn signaling leads to the induction of antiviral responses. the signaling of type i ifns is initiated after they bind to their receptors on the cell surface [51] [52] [53] . this receptor binding activates janus kinases (jak), which phosphorylates both the signal transducers and activators of transcription 1 (stat1) and stat2. the phosphorylated stat1 and stat2 form heterodimers, followed by interaction with interferon regulatory factor 9 (irf9) and subsequently formation of heterotrimers, also known as interferon-stimulated gene factor 3 (isgf3). translocation of isgf3 into the nucleus followed by binding to consensus dna sequences leads to the expression of ifnstimulated genes (isgs), which then take part in antiviral responses. [54] [55] [56] . prrsv replication in marc-145 cells suppresses jak/stat signaling stimulated by addition of ifn-to the culture [54] . transcripts of isg15, isg56, and protein stat2 were significantly reduced compared to mockinfected cells. the phosphorylation of both stat1 and stat2 was unaffected. immunoprecipitation with stat1 or stat2 antibody for marc-145 cell lysates was performed and the result showed no significant difference for stat1/stat2 heterodimer formation between prrsv-infected and mockinfected cells. further study showed that the nuclear translocation of stat1/stat2 heterodimer was blocked. prrsv infection of pam cells also blocks jak/stat signaling shown by reduction of isg expression after stimulation with external ifn-, while a vaccine strain ingelvac prrs mlv had little effect, possible due to its less efficient replication in the primary cells [54] . signaling. prrsv proteins that interfere with ifn-activated signaling include nsp1 , nsp7, nsp12, gp3, and n ( figure 2 and table 1 ) [54] [55] [56] . prrsv nsp1 was found to block the nuclear translocation of stat1 and significantly inhibit the expression of isgs [54] . by observing stat1-gfp distribution under fluorescence microscopy, chen et al. [32] noticed that stat1-gfp accumulated in cytoplasm in hek293t cells with nsp1 expression. further studies on nsp1 revealed that it induced the degradation of karyopherin-alpha1 (kpna1, also called importin-alpha5), which is known to mediate the nuclear import of isgf3 [56] . the n-terminal domain of nsp1 was involved in the ubiquitin-proteasomal degradation of kpna1. residue 19 of nsp1 was found to be essential in inducing kpna1 degradation and inhibiting ifn-mediated signaling as the residue change from valine to isoleucine diminished the suppressive effect. notably, prrsv infection of marc-145 cells by vr-2332 and vr-2385 also reduces kpna1 expression, whereas infection by ingelvac prrs mlv does not. mlv nsp1 has no effect on kpna1 expression or ifn signaling but gains the suppressive function when residue 19 is changed to valine [56] . other prrsv proteins including nsp7, nsp12, gp3, and n were also found to be able to inhibit ifn-activated signaling [55] . n protein inhibits ifn-activated stat1 nuclear translocation, albeit less effective than nsp1 [55] . signaling. variable effect on ifn signaling among prrsv strains was found [55] . among six prrsv strains (vr-2385, ingelvac prrs mlv, vr-2332, nvsl97-7895, mn184, and lelystad) tested, all but mn184 inhibited ifn signaling in marc-145 cells, and all but mlv and nvsl97-7895 blocked the ifn activation in pams. the result also demonstrated that the interference with ifn signaling by prrsv was variable in different infected-cell types. nsp1 from the six strains was cloned and all but mlv nsp1 inhibited ifn signaling when overexpressed [55] . the ifninducing a2mc2 strain has no effect on jak/stat signaling activated by ifn[47] . type i ifns (e.g., ifn-and ifn-) drive the expression of more than 300 genes that encode proteins with antiviral, antiproliferative, proapoptotic, and proinflammatory functions [57] . among the antiviral isgs, the best studied ones are 2 ,5 -oligoadenylate synthetases (oass), ribonuclease l rnasel, the dsrna-activated protein kinase (pkr), p56 (isg56, interferon-induced protein with tetratricopeptide repeats 1 (ifit1)), mx1 (myxovirus (influenza virus) resistance 1), and isg15. prrsv nsp2 was shown to inhibit the antiviral function of ifn-stimulated gene 15 (isg15) by the deubiquitinase activity of the otu domain of nsp2 ( figure 2 and table 1 ) [58] . isg15 is an ubiquitin-like antiviral protein [59, 60] . isg15 conjugation (isgylation) to substrate proteins follows a process similar to that of ubiquitin conjugation. isgylation of many important immune-related molecules leads to the activation of host innate immune response. as mentioned above, the cysteine protease domain (pl2) at the n-terminus of nsp2 belongs to otu-containing superfamily of proteases (dubs), which possesses deubiquitinating activity [58] . sun et al. revealed that nsp2 was an antagonist for the antiviral activity of isg15 by reducing isg15 production and conjugation. the n-terminal pl2 domain of nsp2 was crucial for the antagonizing function. pkr mediates translational control by phosphorylating the protein translation initiation factor eif2 , resulting in inhibition of protein synthesis and viral replication [61] . addition of 2-aminopurine (2-ap), an inhibitor of pkr, restored prrsv replication in ifn--treated cells [62] . addition of 2-ap to recombinant swine ifn--primed marc-145 cells restored prrsv replication but did not rescue the virus in ifn--primed pam cells [63] . these results showed the important role of pkr in the ifn-activated antiviral figure 2 : interference with type i ifn-activated jak/stat signaling pathway and antiviral isgs. ifn-/-binds to their receptors ifnar-1 and ifnar-2 on cell membrane, which activates jak/stat pathway. "p" besides stat1 and stat2 indicates phosphorylation. prrsv nsp1 inhibits isgf3 nuclear translocation via inducing degradation of kpna1, which is essential for mediating the nuclear import of isgf3. n protein also inhibits isgf3 nuclear translocation. and nsp2 reduces isg15 production and conjugation via its deubiquitination activity. signaling. we found that prrsv is able to inhibit the polyi:cinduced activation of pkr, as well as its downstream effector eif2 (unpublished observation). it is not known if prrsv interferes with other isgs. considering the important roles of the isgs in deterring invading pathogens, one can imagine that prrsv must have evolved strategies to evade them during its replication. further study on the interplay of prrsv and isgs will provide insights into such strategies. prrsv infection in pigs leads to delayed production and low titer of neutralizing antibodies [5] , as well as weak cellmediated immune response [6] . partly the reason is possibly because of prrsv interference with ifn-mediated innate immunity. prrsv infection appears to inhibit synthesis of type i ifns in vivo and in vitro with the exception of some atypical strains that induce ifn production, such as a2mc2. the mechanism for the interference is at multiple steps from inhibition of irf3 activation, cbp interaction with irf3, and posttranscriptional suppression. prrsv also block ifn-activated signaling, which results in suppression of the expression of antiviral isgs. the mechanism is prrsvmediated blocking of isgf3 nuclear translocation. the prrsv interference with the innate immunity is at multiple levels, from ifn induction and ifn-activated signaling to activity of isgs. given the divergence of prrsv strains in sequences, variation of these activities in innate immunity is not a surprise, whereas the multifold interplay between the virus and host may determine the consequences. in addition, type i ifns are proinflammatory cytokines. the protective effect of ifns in vivo may be context dependent. the ifns in proper amount at right site and time should be protective. otherwise, its elevation might be a consequence of inflammation during prrsv infection. a typical example 7 is that high-pathogenic prrsv induces high level ifn-but causes high mortality in pigs [64] . further study is needed to elucidate the contribution of prrsv effect on innate immunity to its pathogenesis and the modulation of adaptive immune responses. assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states family arteriviridae arterivirus molecular biology and pathogenesis effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs the level of virus-specific t-cell and macrophage recruitment in porcine 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relationship within the tlr7, 8 and 9 subfamily innate immune recognition of viral infection ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction differential expression and activity of the porcine type i interferon family in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) recombinant swine beta interferon protects swine alveolar macrophages and marc-145 cells from infection with porcine reproductive and respiratory syndrome virus adenovirus-mediated expression of interferon-delays viral replication and reduces disease signs in swine challenged with porcine reproductive and respiratory syndrome virus interferon-response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists north american porcine reproductive and respiratory syndrome viruses inhibit type i interferon production by plasmacytoid dendritic cells differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc-145 cells modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein 1 in marc-145 and hela cells porcine reproductive and respiratory syndrome virus nonstructural protein 1 modulates host innate immune response by antagonizing irf3 activation identification of two autocleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 antagonizes interferon regulatory factor 3 activation porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferonproduction by inhibiting irf3 activation in immortalized porcine alveolar macrophages the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp11 was essential for nsp11 to inhibit ifn-induction structure and cleavage specificity of the chymotrypsin-like serine protease (3clsp/nsp4) of porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferonproduction by interfering with the rig-i signaling pathway porcine reproductive and respiratory syndrome virus (prrsv) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in marc-145 cells nonstructural protein 1-alpha subunit-based inhibition of nf-b activation and suppression of interferon-production by porcine reproductive and respiratory syndrome virus amino acid at position 176 was essential for porcine reproductive and respiratory syndrome virus (prrsv) non-structural protein 1 (nsp1 ) as an inhibitor to the induction of ifn targeted mutations in a highly conserved motif of the nsp1 protein impair the interferon antagonizing activity of porcine reproductive and respiratory syndrome virus biochemical characterization of arterivirus nonstructural protein 11 reveals the nidovirus-wide conservation of a replicative endoribonuclease porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes porcine reproductive and respiratory syndrome virus activates the transcription of interferon alpha/beta (ifn-/ ) in monocyte-derived dendritic cells (mo-dc) impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-responses by plasmacytoid dendritic cells induction of type i interferons by a novel porcine reproductive and respiratory syndrome virus isolate enhancing neutralizing antibody production by an interferon-inducing porcine reproductive and respiratory syndrome virus strain interferon signalling network in innate defence immunomodulatory functions of type i interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins transcriptional responses to polypeptide ligands: the jak-stat pathway how cells respond to interferons porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat1/stat2 nuclear translocation variable interference with interferon signal transduction by different strains of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus nsp1 inhibits interferon-activated signal transduction by inducing karyopherin-1 degradation interferons at age 50: past, current and future impact on biomedicine nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15 a human 15-kda ifn-induced protein induces the secretion of ifn ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses the double-stranded rnadependent protein kinase pkr: structure and function inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine marked differences between marc-145 cells and swine alveolar macrophages in ifn -induced activation of antiviral state against prrsv experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord-335620-xqokfg3l authors: wang, anqi; sun, lipei; wang, mingshu; jia, renyong; zhu, dekang; liu, mafeng; sun, kunfeng; yang, qiao; wu, ying; chen, xiaoyue; cheng, anchun; chen, shun title: identification of ifitm1 and ifitm3 in goose: gene structure, expression patterns, and immune reponses against tembusu virus infection date: 2017-03-13 journal: biomed res int doi: 10.1155/2017/5149062 sha: doc_id: 335620 cord_uid: xqokfg3l as interferon-stimulated genes (isgs), interferon-inducible transmembrane proteins 1 and 3 (ifitm1 and ifitm3) can effectively inhibit the replication of multiple viruses. here, goose ifitm1 and ifitm3 were cloned and identified for the first time. the two proteins share the same topological structure and several important sites critical for the antiviral functions in other species are conserved in the goose. goose ifitm1 and ifitm3 are most closely related to their respective orthologs in ducks; these proteins exhibited high mrna transcript levels in immune-related tissues, including the thymus, bursa of fabricius, and harderian gland, compared to other tissues. moreover, goose ifitm1 was highly constitutively expressed in gastrointestinal tract tissues, while goose ifitm3 was expressed in respiratory organs. furthermore, goose ifitm3 was activated in goose peripheral blood mononuclear cells (pbmcs) infected with tembusu virus (tmuv) or treated with toll-like receptors (tlrs) agonists, while only the r848 and poly (i:c) agonists induced significant upregulation of goose ifitm1. furthermore, goose ifitm1 and ifitm3 were upregulated in the sampled tissues, to some extent, after tmuv infection. notably, significant upregulation of goose ifitm1 and ifitm3 was detected in the cecum and cecal tonsil, where tmuv was primarily distributed. these data provide new insights into the immune effectors in geese and promote our understanding of the role of ifitm1 and ifitm3 in the defense against tmuv. types i and ii interferons (ifns) are critical for establishing an antiviral state, which is mediated by downstream ifnstimulated genes (isgs) [1] . viruses have evolved diverse strategies to escape immune defenses [2] . however, virus evasion of the interferon-inducible transmembrane proteins (ifitms) restriction is not apparent. recently, ifitms have become a popular research topic with the discovery that the immune-related ifitms (ifitm1, ifitm2, and ifitm3) inhibit the early replication of multiple viruses, including influenza a virus (iav), dengue virus (denv), west nile virus (wnv), severe acute respiratory syndrome coronavirus (sars cov), hepatitis c virus (hcv), vesicular stomatitis virus (vsv), ebola virus (ebov), and human immunodeficiency virus type 1 (hiv-1) [3] [4] [5] [6] [7] [8] . ifitms are a subfamily of a larger transmembrane protein family called dispanins [9] , which are generally considered to be comprised of ifitm1, ifitm2, ifitm3, ifitm5, and ifitm10 [10] . ifitm4p, a pseudogene, is located on mouse chromosome as ifitm6 and ifitm7 [11] . moreover, ifitm1, ifitm2, and ifitm3, as viral restriction factors, are known as immune-related ifitms, the antiviral activity of which is conserved from prokaryotes to vertebrates. in addition, the immune-related ifitms exhibit high constitutive expression in target cells and are strongly induced by types i and ii ifns [12] . ifitms have common domains that consist of n-and c-termini, two transmembrane domains, and a conserved cytoplasmic domain and share common properties: (1) the proteins contain a cd225 domain composed of transmembrane domain 1 (tm1) and a cytoplasmic domain and (2) two exons encode transmembrane polypeptides [13] . among these domains, the n-terminal region plays an important role in the correct cellular localization of ifitm3. removing the n-terminal 21 amino acids of ifitm3 results in a loss of association with the endosomal compartments and relocation to the cell surface, thereby abrogating its antiviral function [14] . ifitm1 possesses a shorter n-terminal region and predominately localizes at the cell periphery and in early endosomes [4] . the different cellular localization of ifitm1 and ifitm3 likely underlies their diverse antiviral efficiency against various viruses [15] . ifitm1 exerts more resistance to viruses that fuse at the plasma membrane or early endosome, such as hiv-1 [8] . however, ifitm3 is more proficient than ifitm1 at preventing infection by the late endosomal-or lysosomal-entering viruses, including iav and denv [3] . at present, ifitms have been widely identified in vertebrates, and homologs of ifitms are even found in bacteria [16] . however, little attention has been paid to ifitms in birds. only a small number of bird ifitm sequences have been deposited in public databases, including gallus gallus ifitm1-like (genbank: xm_001233949), anas platyrhynchos ifitm1 (genbank: kf584226), gallus gallus ifitm3-like (genbank: xm_420925), anas platyrhynchos ifitm3 (genbank: kf584228), serinus canaria ifitm3 (genbank: xm_009102512), and nipponia nippon ifitm3 (genbank: xm_009463652). in addition, it was reported that chicken ifitm3 [17] and duck ifitm3 [18] could restrict influenza viruses, but information regarding goose ifitms was unavailable. tembusu virus (tmuv) is a newly emerging pathogenic virus that, together with denv, wnv, japanese encephalitis virus (jev), and yellow fever virus (yfv), belongs to the genus flavivirus [19] . the tmuv genome is a single-stranded rna with an open reading frame that encodes three structural proteins, namely, the core (c), membrane (prm), and envelope (e) proteins, and seven nonstructural proteins [20] . in china, the virus was initially isolated from ducks and can cause severe egg drop syndrome in laying ducks, resulting in huge economic losses. however, recent studies showed that chickens, geese, and house sparrows are also susceptible to this virus [21] [22] [23] . most importantly, serum samples from workers in the duck industry contained tmuv antibodies, which may indicate potential zoonotic transmission [24] . in view of the significant antiviral activity of ifitms in the ifn-mediated innate antiviral defense and the lack of studies focusing on avian ifitms, goose ifitm1 and ifitm3 were cloned for the first time. in addition, the amino acid sequences and topological structures of these proteins were predicted. to understand the evolutionary relationship of goose ifitm1 and ifitm3, a phylogenetic tree was constructed. moreover, the tissue distribution of goose ifitm1 and ifitm3 in healthy geese was identified. it has been demonstrated that ifitms play an important role in blocking the entry of pathogenic flaviviruses such as denv and wnv. as tmuv is a novel member of flavivirus, the interaction between tmuv and ifitm-mediated immune responses is unknown. interestingly, we observed that goose ifitm1 and ifitm3, to different degrees, positively responded to tmuv infection both in vivo and in vitro. collectively, our results promote the research of avian ifitms and may provide the molecular foundation for further research regarding the contribution of goose ifitm1 and ifitm3 to the innate immune responses to tmuv infection. 2.1. animals, viruses, and agonists. all geese were purchased from the waterfowl breeding center of sichuan agriculture university and provided with sufficient water, fodder, and vegetables prior to and during experiments. all experiments were approved by the institutional animal care and use committee of sichuan agriculture university (number xf2014-18), sichuan, china. tmuv was a kind gift of the avian diseases research center of sichuan agricultural university. the agonists r848 (invivogen, usa), poly (i:c) (sigma, usa), odn2006 (invivogen, usa), and lps (invivogen, usa) were used to mimic treatment with single-stranded rna, double-stranded rna, synthetic oligonucleotides, and lipopolysaccharides, respectively. the animals were euthanized, and then the various tissues were rapidly removed and frozen in liquid nitrogen. total rna was extracted from tissues using trizol (invitrogen, carlsbad, ca, usa) following the manufacturer's protocol and then reverse transcribed using 5x all-in-one rt mastermix (abm, canada), which could remove the genomic dna. finally, the synthetic cdna was stored at −80 ∘ c until needed for cloning and quantitative real-time pcr (qrt-pcr). coding sequences (cds). the primers ifitm1-f, ifitm1-r, ifitm3-f, and ifitm3-r (all sequences of the optimal primers used in this study are listed in table 1 ) were designed based on the conserved regions of their respective counterparts in chicken and duck to amplify the partial sequences of goose ifitm1 and ifitm3. subsequently, the full-length cdna of ifitm3 and the 5 -untranslated region (utr) of ifitm1 were obtained by rapid amplification of cdna ends (race). briefly, the gene-specific primers (gsps), including 5 gsp0, 5 gsp1, 5 gsp2, 3 gsp1, and 3 gsp2, were designed based on the obtained partial sequences. for 5 -race, a homopolymeric tail was added to the 5 -end of the synthetic cdna using terminal deoxynucleotidyl transferase and dctp (takara, japan), and then the first-strand cdna was synthesized using the primer 5 gsp0 and a hiscript 1st strand cdna synthesis kit (vazyme, usa). the 5 -end of the target gene was obtained following two rounds of nested pcr using the primer 5 gsp1 with the abridged anchor primer (aap) and 5 gsp2 with the abridged universal amplification primer (auap). for 3 -race, the adapter primer (ap) was used to amplify the first-strand cdna. the 3 -end of ifitm3 was also amplified by nested pcr with the primer pairs 3 gsp1/ap1 and 3 gsp2/ap2. the primer ifitm3-3 r, which was used for amplifying the 3 -utr of ifitm3, was designed according to the predicted ifitm3 sequence from goose transcriptome data [25] . finally, the complete cds of ifitm1 and ifitm3 were amplified using primer star max dna polymerase (takara, japan) and were cloned into the pmd19-t vector (takara, japan) for sequencing. healthy geese. to evaluate the tissue expression profiles of ifitm1 and ifitm3, various tissues, including the brain, bursa of fabricius, cecum, cecal tonsil, gizzard, heart, harderian gland, kidney, liver, lung, muscle, pancreas, proventriculus, small intestine, skin, spleen, thymus, and trachea, were collected from two-week-old gosling and adult goose. the mrna expression levels of ifitm1 and ifitm3 in the various tissues were detected by the bio-rad cfx96 real-time pcr detection system (bio-rad, usa) and evagreen 2x qpcr mastermix-no dye kit (abm, canada) according to the manufacturer's protocol. the relative expression of target genes was calculated using the livak and schmittgen 2 −δδct method [26] and was normalized to goose gapdh. pbmcs were separated from goose blood using a goose peripheral blood lymphocyte separation kit (tbd sciences, china) according to the manufacturer's protocol. then, pbmcs were cultured and maintained in rpmi 1640 medium (gibco, usa) at 37 ∘ c in 5% co 2 overnight. subsequently, the cells were treated with different toll-like receptors (tlrs) agonists, including r848 (5 g/ml), poly (i:c) (30 g/ml), odn2006 (50 g/ml), and lps (25 g/ml), or challenged with 50 l tmuv. cells treated with pbs were used as an experimental control, and the treated cells were challenged with each agonist or virus in quadruplicate and incubated for 6 h. finally, the cells from each treatment group were harvested for qrt-pcr analysis of the expression levels of goose ifitm1 and ifitm3. ifitms in vivo. ten three-day-old goslings were divided into two groups with five goslings in each group. one group of goslings was infected with 500 l of tmuv (6.3 × 10 −6 tcid 50 /ml), and the second group was inoculated with an equivalent amount of normal saline. five days after infection (dpi), three goslings from each group were euthanized, and eight tissues (bursa of fabricius, cecum, cecal tonsil, harderian gland, lung, small intestine, spleen, and thymus) were collected immediately for rna isolation and cdna synthesis. the relative expression levels of ifitm1 and ifitm3 in each sample were assessed by qrt-pcr as described above. goslings. the full-length e gene from tmuv was amplified using primer star max dna polymerase (takara, japan) and subcloned into the pet-32a(+) vector. the temperature and primers were optimized with two negative controls (depc water) and two positive controls (gpv and h9n2 templates). three separate dilution series (10 −1 to 10 −8 ) of recombinant plasmid were prepared for the establishment of standard curves. the viral copy numbers (log 10 ) were normalized per 1 g of total rna [27] . the viral copy number in tmuv-infected gosling tissues was quantified by qrt-pcr. the potential open reading frames (orfs) were predicted by orf finder (https://www .ncbi.nlm.nih.gov/gorf/gorf.html), and the corresponding amino acid sequences of cdna were translated with dna-man software. the molecular weight was calculated using expasy (http://www.expasy.org/tools/). the transmembrane helices prediction was performed by tmhmm 2.0 (http:// www.cbs.dtu.dk/services/tmhmm/). the nucleotide sequences of ifitm1 and ifitm3 from numerous species were compared by pairwise identity analysis using the species demarcation tool [28] . the multiple alignment analysis of amino acid sequences was performed using clustalw [29] . the phylogenetic evolutionary tree was constructed by mega6 using the neighbor-joining method [30] . the qrt-pcr data were analyzed by bio-rad cfx manager software and graphpad prism 5 with an unpaired two-tailed -test. ifitm3. the full-length cdna of goose ifitm1 (genbank: kx594328) is 649 bp, with a 55-bp 5 -utr, a 390-bp orf, and a 204-bp 3 -utr. the identified goose ifitm3 (genbank: kx594327) contains a 426-bp cd and 19-bp utrs ( figure 1 ). the cds of goose ifitm1 and ifitm3 encode 129 and 141 amino acids (aa) with molecular weights of 14.0 kda and 15.33 kda, respectively. the nucleotide sequences from fourteen species were compared by pairwise sequence alignment analysis ( figure 2 ). the 2d color-coded matrix indicates that goose ifitm1 shares the highest identity with its counterpart in anas platyrhynchos (genbank: kf584228.1) (93.3%), followed by 70.6% shared identity with the gallus gallus gene (genbank: xm_420925), while it shared less than 60% identity with the sequences of primate genes. in addition, the nucleotide sequence of goose ifitm3 shared 94.5%, 83.4%, and 74.7% identity with the anas platyrhynchos, gallus gallus, and serinus canaria counterparts, respectively. however, only 64.4% identity was shared between goose ifitm1 and ifitm3 (tables s1 and s2 in supplementary material available online at https://doi.org/10.1155/2017/5149062). the pairwise identity scores of ifitm1 and ifitm3 are shown in tables s1 and s2, respectively. proteins. since these two molecules are transmembrane proteins, the topological structures were predicted by the tmhmm server. two transmembrane helices were found in both proteins, 1 t a a c t g a g t g g a c c c a a g c a c c g g t g c t c c a a c t c c c c g c t g c t g c c a g g c t g c c t c c t t a a c c c c c c c t g c c c c a a a c c c c a t c a c c c t t c c g a c c c c c t c c g t c c a g c t g a g g g g g t g c a c c c g c c g a g g t g a c c c c g t t g g g a g t g a g c c t c t g g g g t g a g c t c a a g a c g a a t a a a g c a t t c c t c a c t c c c t t t c a a a a a a a a a a a a a a a a a a a figure 2 : the 2d color-coded matrix of ifitm1 and ifitm3. the nucleotide sequences of ifitm1 (a) and ifitm3 (b) from various species were compared by pairwise alignment using the species demarcation tool. the color spectrum scheme was assigned according to the pairwise identity scores. goose ifitm1 and ifitm3 shared the highest identity with their respective counterparts in duck. tm1 cil tm2 ctd ifitm1 ifitm1 ifitm1 ifitm1 ifitm1 ifitm3 ifitm3 ifitm3 ifitm3 ifitm3 ifitm1 ifitm1 ifitm1 ifitm1 ifitm1 ifitm3 ifitm3 ifitm3 ifitm3 ifitm3 53 52 51 82 70 74 74 predicted to be oriented toward the extracellular space. multiple amino acid sequence alignments show several conserved residues that are critical for the antiviral function and localization of ifitms. a highly conserved yxxφ motif appears in the ntd of ifitm3, which promotes the internalization of ifitm3. however, ifitm1, with a comparatively short ntd, lacks this yxxφ motif. in addition, three other yxxφ motifs are conserved in ifitm3 of chickens, ducks, and geese. two cysteines (c60 and c61) in tm1 are conserved in ifitm3 and undergo palmitoylation [31] . in addition, three lysines (k72, k77 and k93) that are the sites of ubiquitination are also present in ifitm3 [32] (figure 3 ). to explore the relationship of ifitm1 and ifitm3, as well as the evolutionary dynamics of goose ifitm1 and ifitm3, a phylogenetic tree was constructed using the amino acid sequences of ifitm1 and ifitm3 from diverse species. the phylogenetic tree indicated that ifitm1 and ifitm3 of diverse species were separated into two clusters. the avian ifitm1 and ifitm3 were clustered into two groups, respectively. predictably, goose ifitm1 and ifitm3 were closely related to their homologs in ducks (figure 4) . the mrna expression levels of ifitm1 and ifitm3 in eighteen tissues collected from healthy geese were assessed by qrt-pcr. in gosling, goose ifitm1 was highly expressed in the cecum, moderately expressed in the lung, bursa of fabricius, trachea and cecal tonsil, and minimally expressed in the rest of the tissues. however, goose ifitm3 was widely expressed in all collected tissues. high relative expression levels of goose ifitm3 were detected in the harderian gland and lung compared to the other tissues ( figure 5 ). meanwhile, muscle tissue exhibited the lowest transcription level of goose ifitm3. in adult goose, comparatively high expression levels of goose ifitm1 were observed in the thymus and small intestine. in addition, the production of goose ifitm3 was easily detected in the thymus followed by the lung, skin, and bursa of fabricius but was only faintly exhibited in the muscle tissue of adult goose ( figure 5 ). challenged pbmcs. ifitms have been reported to be broadspectrum antiviral molecules [15, 33] . once host cells are infected, the invasive pathogens are recognized by pattern-recognition receptors (prrs) [34] , which drive the pan troglodytes ifitm3 capra hircus ifitm3 xenopus laevis ifitm3 gallus gallus ifitm3 anser cygnoides ifitm3 anser cygnoides ifitm1 mus musculus ifitm1 bos taurus ifitm1 homo sapiens ifitm1 production of ifns. subsequently, the interaction of ifns and ifn receptors triggers the activation of isgs such as ifitms [1] . here, whether goose ifitm1 and ifitm3 were activated in response to exogenous stimulation was monitored by qrt-pcr. as expected, goose ifitm1 and ifitm3 actively responded to treatment with either tmuv or tlrs agonists. tmuv and all the agonists led to extremely significant upregulation of goose ifitm3 in comparison with the pbs group ( figure 6(b) ). however, only poly(i:c) and r848 effectively caused significant upregulation of goose ifitm1 (figure 6(a) ). to investigate the impact of tmuv infection on the transcription levels of goose ifitm1 and ifitm3, the relative expression levels of immune-related and epithelial tissues from goslings treated with normal saline or infected with tmuv were examined. as shown in figure 7 , goose ifitm1 and ifitm3 were universally upregulated in response to tmuv infection. compared to the mock-infected group, goose ifitm1 in the cecum, cecal tonsil, harderian gland, and spleen was significantly upregulated in the tmuvinfected group. in addition, goose ifitm1 in the thymus was extremely significantly upregulated after tmuv infection (figure 7(a) ). moreover, extremely significant upregulation of goose ifitm3 was observed in the cecum at 5 dpi with tmuv. meanwhile, the transcription levels of goose ifitm3 were obviously upregulated in the cecal tonsils and lungs from tmuv-infected animals (figure 7 (b)). furthermore, relatively high viral copy numbers were observed in these corresponding tissues, although the viral copy numbers in lung tissues were lower than those of other tissues (figure 8 ). since ifitms, as cell-intrinsic restriction factors, were first identified to have a role in the restriction of influenza and flavivirus infection in 2009 [3] , the antiviral function of ifitms has been widely studied in various species, including human [35, 36] , pig [37, 38] , and mouse [39, 40] . however, in avian species, ifitms were identified in only a small number of birds. the goose ifitm1 and ifitm3 identified here will facilitate a better understanding of the ifitm family in avian species. as always, the nomenclature of ifitm1 and ifitm3 in chickens and ducks was confusing and controversial. according to the gene synteny with mammals, the predicted chicken figure 7 were analyzed for the corresponding viral copy numbers by an absolute quantification method in triplicate. the viral copy numbers (log 10 ) were normalized to 1 g of total rna. ifitm3-like (genbank: xm_420925) and chicken ifitm1like (genbank: xm_001233949) were renamed chicken ifitm1 and ifitm3, respectively. in addition, the names of the previously identified duck ifitm3 (genbank: kf584228) and duck ifitm1 (genbank: kf584226) were reversed. the pairwise sequence alignment analysis of nucleotide sequences showed that goose ifitm1 shares a much higher identity with its counterparts in duck (genbank: kf584228) (93.3%) and chicken (genbank: xm_420925) (70.6%) than with duck ifitm3 (genbank: kf584226) (63.5%) and chicken iftm3 (genbank: xm_001233949) (59.4%). a similar result was observed in the nucleotide sequences alignment of goose ifitm3 with chicken ifitm1 and ifitm3, as well as duck ifitm1 and ifitm3 (table s2 ). the phylogenetic analysis indicated that the ifitm1 and ifitm3 clusters were divergent subgroups. goose ifitm1 and the renamed chicken ifitm1 and duck ifitm1, together with the mammalian ifitm1, were classified into the same subgroup. meanwhile, goose ifitm3 and the renamed duck ifitm3 were located in the same monophyletic group, which was closely related to the renamed chicken ifitm3. in summary, the results revealed that it was necessary to rename the ifitm1 and ifitm3 in chicken and duck and that goose ifitm1 and ifitm3 have been conserved during evolution. the minor ifitm3 allele (snp rs12252-c), which is equivalent to the n-terminal 21-amino acid-truncated ifitm3, was strikingly enriched in patients who were severely infected with the 2009 pandemic h1n1 virus [35, 41] . in line with the above findings, vsv-g envelope proteinmediated virus entry could not be inhibited by the ifitm3 mutant (the n-terminal 21-amino acid deletion) [14] . these results highlight the important role of the n-terminal region of ifitm3 in antiviral functions [14] . in addition, the 20-yxxφ-23 sorting signal in the n-terminal region is critical for the internalization of ifitm3 from the cell periphery to endosomal compartments where it acts to impede virus entry [42] . the n-terminal yxxφ motif was observed in the n-terminal region of goose ifitm3. we predicted that the conserved motif in goose ifitm3 is also involved in the internalization and antiviral actions. moreover, three other yxxφ motifs that might be required for the endosomal localization and antiviral functions of duck ifitm3 [18] are also conserved in ifitm3 of chickens and geese. however, whether these yxxφ motifs in goose ifitm3 are critical for correct cellular localization and inhibition of virus entry has yet to be confirmed. analysis of the tissue distribution of goose ifitm1 and ifitm3 in geese showed that both goose ifitm1 and ifitm3 were readily detected in some immune-related tissues, including the thymus, bursa of fabricius, and harderian gland. in addition, the expression of goose ifitm1 was restricted and primarily confined to the gastrointestinal tract tissues (cecum, small intestine, and gizzard), which was observed previously in both chickens [17] and ducks [18] . in contrast, goose ifitm3 was constitutively and ubiquitously expressed in various tissues. notably, high expression levels of goose ifitm3 were observed in respiratory tract tissues (lung and trachea), the target tissues of infection with influenza a viruses, compared to the other tissues, which might contribute to the inhibition of influenza a virus replication [43] . it is notable that goose ifitm1 and ifitm3 both exhibit the lowest levels of expression in muscle tissues, similar to observations in chickens [17] . in vitro, we explored the immunological characteristics of goose ifitm1 and ifitm3. the mrna level of ifitm3 in pbmcs sharply increased with tmuv infection and with r848, poly (i:c), odn 2006, or lps treatment, although goose ifitm1 was prominently expressed only in the r848-and poly (i:c)-stimulated groups. r848, poly (i:c), odn 2006, and lps are known as tlr7, tlr3, tlr21, and tlr4 agonists, respectively. all four agonists and the tmuv genome can be recognized by prrs, which then activate downstream signal transduction pathways triggering the production of cytokines, such as ifns, that induce the transcription of isgs, including ifitms [1, [44] [45] [46] . previous studies demonstrated that immune-related ifitms were significantly upregulated in highly pathogenic avian influenza-infected ducks [18, 47] . however, the available information on whether ifitms positively respond to tmuv infection is scant. obviously, the expression levels of goose ifitm1 and ifitm3 displayed an upward trend in all the tested tissues, especially the cecum (figure 7) , where tmuv was primarily distributed (figure 8 ). high viral copy numbers were also observed in the intestinal tract tissues of tmuv-infected ducks [48] . in addition, goose ifitm1 was intensively transcribed in the tmuv-infected spleen and thymus (figure 7) , where the significant upregulation of the ifns was previously observed [48] . after tmuv infection, the virus was intensively located in the collected tissues, which reasonably explains why higher expression levels of goose ifitm1 and ifitm3 appeared in the tmuv-infected gosling tissues than those of the mock-infected group. in addition, the lowest viral copy numbers were seen in lung tissues, which might be attributed to the high expression level of ifitm3 in the tmuv-infected goose lungs. in light of the positive response of goose ifitm1 and ifitm3 to tmuv infection, we speculate that goose ifitm1 and ifitm3 might facilitate ifn-mediated defenses against tmuv. goose ifitm1 and ifitm3 were characterized for the first time. in addition, high mrna levels of goose ifitm1 and ifitm3 were presented in some immune-related tissues. meanwhile, goose ifitm1 and ifitm3 were primarily expressed in intestinal tract tissues and respiratory organs, respectively. furthermore, goose ifitm1 and ifitm3 were activated in response to tmuv infection in vitro and in vivo. these results indicated that goose ifitm1 and ifitm3 might be potential antiviral effectors that restrict tmuv infection. a diverse range of gene products are effectors of the type i interferon antiviral response inverse interference: how viruses fight the interferon system the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm1 is a tight junction protein that inhibits hepatitis c virus entry interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms interferon-induced transmembrane protein-mediated inhibition of host cell entry of ebolaviruses the ifitm proteins inhibit hiv-1 infection the dispanins: a novel gene family of ancient origin that contains 14 human members evolution of vertebrate interferon inducible transmembrane proteins the fragilis interferon-inducible gene family of transmembrane proteins is associated with germ cell specification in mice ifitm-family proteins: the cell's first line of antiviral defense the small interferoninduced transmembrane genes and proteins the n-terminal region of ifitm3 modulates its antiviral activity by regulating ifitm3 cellular localization ifitms restrict the replication of multiple pathogenic viruses ifitms from mycobacteria confer resistance to influenza virus when expressed in human cells chicken interferoninducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro duck interferon-inducible transmembrane protein 3 mediates restriction of influenza viruses flaviviruses analysis of the complete genome of tembusu virus, a flavivirus isolated from ducks in china adapted tembusu-like virus in chickens and geese in china identification and molecular characterization of a novel flavivirus isolated from geese in china characterization of a tembusu virus isolated from naturally infected house sparrows (passer domesticus) in northern china tembusu virus in human, china immune-related gene expression patterns in gpv-or h9n2-infected goose spleens analysis of relative gene expression data using real-time quantitative pcr and the 2-δδct method tembusu virus infection in cherry valley ducks: the effect of age at infection sdt: a virus classification tool based on pairwise sequence alignment and identity calculation multiple sequence alignment with the clustal series of programs mega6: molecular evolutionary genetics analysis version 6.0 palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 s-palmitoylation and ubiquitination differentially regulate interferoninduced transmembrane protein 3 (ifitm3)-mediated resistance to influenza virus the broad-spectrum antiviral functions of ifit and ifitm proteins pathogen recognition and innate immunity ifitm3 restricts the morbidity and mortality associated with influenza the cterminal sequence of ifitm1 regulates its anti-hiv-1 activity bat and pig ifn-induced transmembrane protein 3 restrict cell entry by influenza virus and lyssaviruses evolutionary characterization of pig interferoninducible transmembrane gene family and member expression dynamics in tracheobronchial lymph nodes of pigs infected with swine respiratory disease viruses expression profile and histological distribution of ifitm1 and ifitm3 during h9n2 avian influenza virus infection in balb/c mice human, pig, and mouse interferon-induced transmembrane proteins partially restrict pseudotyped lentiviral vectors interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals identification of an endocytic signal essential for the antiviral action of ifitm3 ifitm3 inhibits influenza a virus infection by preventing cytosolic entry the history of toll-like receptors-redefining innate immunity toll-like receptor downstream signaling interferon response by toll-like receptor signaling a comparative analysis of host responses to avian influenza infection in ducks and chickens highlights a role for the interferon-induced transmembrane proteins in viral resistance the sequential tissue distribution of duck tembusu virus in adult ducks the authors disclose that there are no financial and personal relationships with other people or organizations that could inappropriately influence this work. anqi wang and shun chen contributed equally as co-first authors of this work. key: cord-347200-dtwhd6zy authors: ivanova, daria; krempels, ryan; ryfe, jennyfer; weitzman, kaitlyn; stephenson, david; gigley, jason p. title: nk cells in mucosal defense against infection date: 2014-08-14 journal: biomed res int doi: 10.1155/2014/413982 sha: doc_id: 347200 cord_uid: dtwhd6zy conventional natural killer cells (nk cells) provide continual surveillance for cancer and rapid responses to infection. they develop in the bone marrow, emerge as either nk precursor cells, immature, or mature cells, and disperse throughout the body. in the periphery nk cells provide critical defense against pathogens and cancer and are noted to develop features of adaptive immune responses. in the tightly regulated and dynamic mucosal tissues, they set up residency via unknown mechanisms and from sources that are yet to be defined. once resident, they appear to have the ability to functionally mature dependent on the mucosal tissue microenvironment. mucosal nk cells play a pivotal role in early protection through their cytolytic function and ifnγ production against bacteria, fungi, viruses, and parasitic infections. this review presents what is known about nk cell development and phenotypes of mucosal tissue resident conventional nk cells. the question of how they come to reside in their tissues and published data on their function against pathogens during mucosal infection are discussed. dissecting major questions highlighted in this review will be important to the further understanding of nk cell homing and functional diversity and improve rational design of nk cell based therapies against mucosal infection. natural killer cells (nk cells) are a first line of defense against invading pathogens and cancer. recent studies focused on development and functional diversity of innate immune cells have led to the reclassification of these cell types into a large group known as innate lymphoid cells (ilcs) [1] . this is due to their origin from the common lymphoid progenitor (clp) but unlike their t cell and b cell counterparts, they do not activate the recombination activation genes (rga1/2) and do not undergo antigen receptor rearrangement. there are three main groups, group 1, of which conventional nk cells are members, group 2, and group 3. each grouping is based on the functionality and transcriptional regulation of cell type development. nk cells are members of group 1 ilcs due to their ability to produce ifn and be cytolytic. their activation and function rely on recognition of pathogeninfected cells through activating receptors (kirs in humans and ly49 in mice) and proinflammatory cytokines. nk cells can also regulate immunity. during systemic infections they produce il-10 and with high viremia can target dcs and t cells, thus modifying immunological memory [2] [3] [4] [5] . as such, nk cells have many roles, in protection, in helping to maintain immune homeostasis, and in long term immunity. nk cells are found in many tissues. this includes bone marrow (bm), blood, liver, thymus, and spleen. mucosal sites that harbor nk cells include the lung, the small and large intestine and colon of the gastrointestinal tract (gi), and the uterus, cervix, ectocervix, and vagina of the female reproductive tract (frt). much of how they gain access to these sites and provide function (protection, immunoregulation) is just beginning to be understood. the review focuses on recent work and the current understanding of the regulation of mucosal tissue residency of nk cells and nk cell functional importance at mucosal sites relevant to both mouse and human systems. we will not address ilc2 and ilc3 populations as those have been reviewed elsewhere [6, 7] . in humans and mice, nk cells develop from the common lymphoid progenitor (clp) in the bone marrow [8] . clps in the mouse bm differentiate into a pre-nk precursor 2 biomed research international (pre-nkp) with a phenotype of lin − cd117 − cd127 + and express some nk cell specific receptors including nkg2d and 2b4 (cd244) and negative for classical nk cell markers nk1.1 and cd49b. pre-nkp then express the -chain receptor for il-15 (cd122) and cd11b and are now defined to be nk precursors (nkp). il-15 is a cytokine required for their development as shown by il-15 ko mice, which predominantly lack nk cells [9] . interestingly, infection of il-15 ko mice with the protozoan parasite toxoplasma gondii or il-15 ko, il-15r ko, and rag2/il-2r ko mice with mcmv infection results in rapid expansion of nk cells [10, 11] . these studies support il-15 as an important cytokine for promoting nk cell development in the absence of infection. however, they demonstrate that other non--chain cytokines such as il-12 during infection can stimulate nk expansion and activity independent of il-15. once cd122 is expressed, nkps now become responsive to il-15 and develop further into immature nk cells (ink) marked by cd27 lo cd11b lo and the progressive expression of activating and inhibitory receptors. murine nk cells have been further defined based on the expression of cd27 and cd11b into 4 stages of maturation that correspond with their production of inflammatory cytokines and cytolytic activity [12, 13] . after the ink stage they progress in phenotype from the cd27 lo cd11b lo to cd27 hi cd11b lo then from cd27 hi cd11b hi to cd27 lo cd11b hi . human nk cells develop from hematopoietic stem cells (hscs) through a clp as in mice with differentiation points designated as development stages (stages 1-5) [8] . stages 2 and 3 cells are cd56 − and cd94 − and become responsive to il-15. stages 4 and 5 cells acquire the expression of the human classical nk cell marker cd56 and are now considered to have differentiated into the nk cell phenotype. stage 4 cells are cd56 bright fc riii − (cd16 − ), produce high levels of cytokines, have low cytolytic capabilities, and are considered immature. stage 5 cells are cd56 dim cd16 + , are both cytolytic and capable of producing high amounts of ifn , and are considered mature. nk cells are present systemically in bone marrow, secondary lymphoid structures, peripheral nonlymphoid organs, and blood. in lymph nodes, the majority of nk cells are considered immature and are cd56 bright cd16 − in humans and cd27 hi cd11b lo-med are in mice [8, 14] . in nonlymphoid organs, such as gut and lung, resident conventional nk cells have varying phenotypes based on maturation and function. the factors governing the phenotype and function of resident nk cells at these sites are unknown. however, nk cell diversity at these different sites is likely dependent upon the integration of environmental signals to promote their needed activity. additionally, the ultimate source (blood, bm, or lymph node (ln)) of resident nk cells in these organs is not well described. while human and murine nk cells undergo the above-mentioned developmental stages, once they exit the bm could migrate to either the blood stream or into secondary lymphoid structures. whether immature nk cells from ln or more mature cells from the blood seed peripheral tissues during the steady state is unclear. recently, liver nk cells were demonstrated to be distinct in development from thymic and splenic nk cells [15] . therefore mucosal tissue specific nk cells could also differentiate in situ rather than be seeded by ln or peripheral blood precursors. regardless, there are several necessary steps for this post-bone-marrow phase of nk cell development and function at mucosal sites. these steps include migration, changes in phenotype, education, and maturation. in addition to what controls homing of nk cells to mucosal tissues, the mechanisms behind how mucosal nk cells adjust to their resident environments are unclear and will be important to dissect. the current model of nk cell development and migration suggests that nk cells likely emerge from bm as a mix of mature and immature cells. immature cells mature and acquire organ specific phenotypes in the extramedullary tissues including secondary lymphoid tissues and liver [14] [15] [16] [17] [18] . mature nk cells circulate to different tissues and are then modified by tissue microenvironments via cytokine milieu, growth factors, or chronic inflammation [7, 19] . migration from bm to a specific tissue is a complex and critical first step to establishing residency. this process is likely different for each nonlymphoid tissue and has not been well described. a good example of the complexity of this process is how specific cd8 t cell populations are recruited to the gut to become intraepithelial lymphocytes (iels). intestinal mucosa homing of iels requires a priming event near the tissue itself or in the mesenteric lymph node (mln). priming is dependent upon an interaction with mucosal ccr7 + cd103 + dcs and acquisition of 4 7 integrin and ccr9 expression [20] [21] [22] [23] . given the intense research in this area, the gut iel model for tissue homing may be a useful starting point to base mechanistic studies on nk cell recruitment to mucosal nonlymphoid tissues. 3.1. resident nk cells of the lung mucosa. 10% of total lung resident lymphocytes are nk cells. this is a higher nk cell number than other nonlymphoid organs highlighting their importance in this tissue [24] . they are primarily found along with other lymphocytes in the lung epithelium and vascular tissues [24, 25] . in humans, lung resident nk cells are cd56 dim cd16 + and mostly nkp46+ similar to nk cells in the blood suggesting a mature phenotype capable of being cytotoxic and producing cytokines [26] . in mice the more mature nk cell phenotype is found in the lung epithelium, which is cd27 lo cd11b hi , very similar in function to the human subset. in the naïve state, these two phenotypes of nk cells in both human and mouse compose 80-90% of all nk cells present in the lung tissue [13, 25] . the maturation status of most lung nk cells resembles those from blood. however a recent study identified a population of nk cells in the lung capable of being further differentiated [27] . this study demonstrated that, unlike bone marrow precursors, the lung precursor cells when cultured in vitro expressed more ly49 receptors. these results suggest that both mature and immature nk cells are present in the lung and that the murine lung microenvironment could condition nk cells separately from the bone marrow. in humans, other than classical nk cell marker phenotype (cd56 and cd16) approximately 30% of lung resident nk cells express kirs including kir2dl2, kir2dl3, kir3dl1 and kir2ds2. nearly 80% of the cd56 bright population expressed cd94 similar to the phenotypes found in peripheral blood [26, 28] . murine and human lung microenvironments may differ in their ability to modify resident nk cells. in humans support for tissue specific microenvironment conditioning of nk cells comes from studies of pulmonary sarcoidosis and nonsmall-cell lung cancer [26, 28] . nk cells express less kir in bronchiolar lavage fluid (balf) in these situations than in the naïve state. interestingly, peripheral blood nk cells also had lower kir expression. whether or not the change observed in balf was from migrating cells is not known. the observations in balf support a role for tissue specific microenvironment conditioning of nk cells. however, the more mature phenotype of resident nk cells in the lung is suggestive of a blood origin ( figure 1 ). currently, much is still not known about the mechanisms behind these changes and whether in the healthy state tissue resident human nk cell phenotypes can be modified by the lung. of the three innate lymphoid cell populations found in the gut, conventional nk cells of the gut are classified to belong to the ilc1 subset [1] . nk cells are present in all gut tissues (small intestine, large intestine, and colon) as cells of the iel and lp compartments. they can also be found in smaller numbers in peyer's patches (pp) and mlns. unlike the lung, gut mucosal nk cells in humans are predominantly cd56 bright with few that express cd16 indicating that they may be similar to immature nk cells found in secondary lymphoid structures [29] [30] [31] . nk cells in the murine intestinal mucosa also appear immature. iel and lp nk cells of naïve mice are cd27 hi cd11b med and cd27 lo cd11b lo , respectively [32] [33] [34] . further support for an immature phenotype for both murine and human gut nk cells is evident from functionality. resident human cd56 bright and mouse cd27 hi nk cells produce large amounts of the proinflammatory cytokine ifn and exhibit low cytolytic activity when tested in vitro [19, 31] . after infection, significant changes occur in nk cell frequencies in the small intestine and lamina propria. whether peripheral nk cell infiltration occurs or whether there are phenotypic changes in resident nk cell populations has not been investigated. infiltration of peripheral blood nk cells probably occurs albeit to a lesser extent and the nk cell dependent response may rely more on an nk cell interaction with activated gut mucosal dcs in the mln and/or expansion of activated lp nk cells at the site of infection ( figure 2 ). however, given the diversity of gut mucosal ilc populations with wide ranging function, determining the level of infiltration of new cells may be very difficult. against pathogens, but also successful vascular remodeling of the uterus during pregnancy and fetal implantation [35, 36] . in humans, frt nk cells resemble an immature phenotype of nk cells similar to that found in the digestive tract and are cd56 bright cd16 − [37] . nk cells found in the uterus are phenotypically distinct from others in the frt being cd9 + and chemokine receptor like −1 + (cmklr1). cd9 is a tetraspanin family member important for cell adhesion and migration and cmklr1 is a receptor for chemerin, which promotes migration to the decidua and vascular remodeling during pregnancy [38, 39] . they are also positive for kir molecules (kir2dl4) and cd94/nkg2a. uterine nk cells exhibit functional characteristics of immature nk cells having high cytokine production and low cytotoxic potential. murine uterine nk cells resemble their human counterparts as having a more immature phenotype being cd27 med cd11b hi [36] . they also express ly49 receptors including ly49d, h, c, i, g, and a. they are different from human uterine nk cells in that they exhibit lower cytolytic activity and are more important in contributing to vascular remodeling for proper fetal implantation [40] . vaginal resident nk cells in humans are different from their uterine counterparts in that they are cd16 + and cd94 − [39] . they also had a high potential of producing inflammatory cytokines including ifn and lower cytotoxic potential. they express cd56 to a similar level as immature [41] . given their importance, a more thorough analysis of their phenotype would be interesting and informative to pursue. homing to mucosal sites. the mechanisms behind establishment of resident nk cells at mucosal sites are still not well described. chemokines most likely play a very important role in this process and mucosal associated nk cells express a vast array of chemokine receptors including, cxcr1, ccr2, ccr7, ccr5, and cx3cr1 [42] . additional factors are important in homing of nk cells to these sites including chemerin for female reproductive tract resident cells and sphingosine-1 phosphate family member s1p5 [43] . given the diversity of expression of these receptors on nk cells, each tissue could regulate which cells migrate to which organ. an intriguing model behind how nk cells set up residency in different tissues has been recently proposed [8, 43, 44] . this model suggests that, unlike secondary lymphoid tissues and blood, nk precursor cells migrate from the bm into the blood then migrate further to different sites in the body including mucosal tissues. once they have migrated, organ specific environments influence their development. likely contributors include estrus cycling hormones, inflammatory milieu such as il-15 by somatic cells, tgf , il-10, and the resident microbiomes. factors from these sources could induce nk precursor differentiation to attain different levels of maturation and education. new data on tissue specific difference in nk cell differentiation and studies investigating whether nk cells can be differentiated from tissue specific precursors may support the hypothesis that nk precursor cells seed peripheral organs and that they differentiate independent of the bone marrow [27, 45] . this would allow these tissue resident nk cells to be "educated" to have very specific functions important at sites to which they migrate [8] . an alternative hypothesis is possible. in certain mucosal tissues, especially those with established microbiomes, such as gut, frt, and recently described in the lung [46] , nk cells may require a priming event from draining apc populations. these apcs program ln nk cells in mucosal associated lymphoid tissue (malt) to migrate to a specific site (figure 2 ). malt includes lymph nodes such as the mesenteric lymph node (mln). this hypothesis is supported by the conserved phenotypic characteristics of gut and female reproductive tract nk cells. they more closely resemble immature cells by being cd56 med-bright cd16 + and cd27 + cd11b + with higher cytokine production in humans and mice, respectively. as mentioned above, lung nk cells resemble a more mature phenotype and could come directly from the peripheral blood ( figure 1 ). despite the lung nk cell phenotype, the presence and the effect of the lung microbiome on the development of asthma and chronic inflammation suggest that nk cells could also be modified in situ. regardless, support for the malt apc priming hypothesis could be taken from the process of t cell (iel) recruitment into the intestinal epithelium. interactions between gut mucosal t cells and cd103 + dcs via crtam1 in both the steady state and during infection are required for recruitment [47] . iels during pathogenic situations require ifn producing mucosal dcs to be primed to home to the gut [20] . interestingly, a recent report demonstrates that small intestine iels emerge from the thymus as recent thymic emigrants which express differing levels of the gut homing integrin 4 7 [22] . 4 7 is required for iel trafficking to the gut [21] . since nk precursors and other ilc populations in secondary lymphoid tissues express varying levels of this integrin, it may be possible that an nk-dc interaction is a requirement for immature nk cells to be signaled to home to mucosal sites. altogether, this is a possible mechanism important for homing that would be interesting to explore. during mucosal infections of humans and mice, nk cells are recruited to sites of infection and play an important role in immune defense [6, 48] . soon after infection and ensuing inflammation (type i ifn and il-12), resident nk cells respond, produce ifn and tnf , and become cytotoxic [49] . additional nk cells are recruited from the periphery within a few days of these events and contribute to these responses. as mentioned before, although the cytokine il-15 is required for development and the expansion of developing nk cells, other cytokines and signals can cause nk cells to expand and respond to infection [11] . investigation into how nk cells in il-15 ko or il-15r mice increase in number with mcmv infection demonstrated that il-12 promotes their expansion and activation. additionally, stimulation of nk cells through the activating receptor ly49h via m157 of mcmv also contributed to this response. therefore the cytokine milieu present in the different mucosal tissues in addition to activating signals stimulated that by diverse pathogens help nk cells respond to infection. peripheral nk cell migration occurs in all mucosal tissues during bacterial, fungal, viral, and parasitic infection [6, 37, 43, 44] . recruitment to inflamed tissues is largely dependent upon chemokines and the ligation of their cognate receptors on nk cells. once activated conventional nk cells at these sites are vital for initial containment of pathogens and preventing their systemic spread. the lung is a site of entry for viral, bacterial, and fungal infection. of all nonlymphoid tissues, the lung contains the largest number of conventional nk cells. humans and mice with genetic deficiencies resulting in loss of nk cells are more susceptible to pulmonary infections [50] [51] [52] . many viruses including influenza, coronavirus (severe acute respiratory syndrome (sars), middle eastern respiratory syndrome (mers)), herpes, and respiratory syncytial virus (rsv) infect via the airways. nk cells were demonstrated early to respond to influenza infection in the lungs of humans through their production of ifn [53] . cytolytic activity of these cells is also important for control of influenza infection including pandemic h1n1 influenza [54] . nk cell ctl activity in response to influenza is mediated through antibodies and adcc or via the recognition of viral haemagglutinin by the natural cytotoxicity receptors nkp46 [55] . in mice infected with influenza a (pr8), infiltrating nk cells are cd27 low cd11b hi and appear to be of a mature phenotype [56] . in line with this, they also have greater cytolytic activity than ifn production suggesting that they could be nk cells migrating in from the blood or replicating in situ [57] . in addition to il-12, il-15 has been shown to play a role in nk cell responses in the lung [52, 58, 59] . il-15 in complex with il-15r can help in recruitment and activation of nk cells during rhinovirus infection and il-15 when blocked with an antibody appears to prevent nk cell recruitment into the bal during influenza infection in mice [58, 59] . nk cell protective function in the lung against extracellular staphylococcus aureus also appears to require il-15 [52] . il-15 likely contributes to the expansion and synergizes with il-12 and ifn for proper activation of nk cells in the lung. overall, it appears that, in the mouse model, nk cells are required for survival against influenza infection [60] . nk cells functioning to control influenza can also cause severe pathology in the lungs [56] . in mice given a high dose of influenza, depletion of nk cells resulted in better outcome against infection. a reason behind these differences could be attributed to the proinflammatory cytokines produced during initial viral encounter that help nk cells respond. il-12 and type i ifns stimulate nk cells to produce ifn [61] . however, high production of these cytokines in response to pulmonary infection can lead to overproduction of ifn resulting in tissue destruction. nk cells play a role in early immunity and control of lung fungal and bacterial infections. nk cells help in early control of cryptococcus neoformans and aspergillosis through their cytolytic activity and ifn production [62] [63] [64] [65] . by producing ifn these cells play a critical role in control of several bacterial infections including staphylococcus aureus, klebsiella pneumonia, and legionella infections [52] . in the case of klebsiella, nk cell production of il-22 may also be important in establishing immunity. in response to mycobacterium tuberculosis (mtb) infection, ifn and ifn are critical for control of the bacterium, the former via restriction of macrophage infiltration into the lung and the latter through inhibition of microbial growth [66] . as with many infections, nk cells respond to both type i ifn and il-12 during mtb infection resulting in their production of ifn and are critical for early survival in the mouse model as demonstrated in rag2−/− common--chain−/− and il-12p40−/− mice [67] . however, depletion of nk cells has shown that, like some fungal models, nk cells can contribute to early control of mtb but are not required for long term survival against infection [68] . overall, nk cells are very important for early control of pathogens in the lung. the majority of this protective response is governed by the production of ifn by these cells and can be regulated via il-12 and ifn / provided by myeloid populations. activation is also aided by the integration of activation signals through activating receptors on the cells. nk cell can also be pathogenic in the lung during infection from overproduction of ifn . questions still remaining that would be interesting to address are how to boost nk cell responses to this infection without causing overt tissue pathology and also what is the advantage of having nk cells present in the lung to promote this early control. are nk cells more important for immune stimulation or regulation or do they have bifunctional roles as a plastic cell able to respond to the environment? further investigation of the biology of these cells in the lung is needed to answer these questions. infections. as mentioned above, ilc populations in the gi tract are very diverse and have many roles from innate immune control of pathogens to regulation of autoimmunity [7, 8, 44] . ifn producing conventional nk cells helps to control many infectious pathogens in the gut. although nk cells appear to be dispensable for listeria monocytogenes infection in mice [69] , in a rat model of infection, they were essential in they were essential for early control of the bacterium [70] . investigation of salmonella infection in mice demonstrated that il-15 and nk cells were required [71] . il-15 ko mice had greater bacterial burdens than wt animals. depletion of nk cells also resulted in the great colonization of the murine gut with salmonella. despite previous studies where il-15 ko mice are still able to mount a robust nk cell response during viral and parasitic infection due to il-12 production this did not appear to occur during bacterial infection. this difference may highlight a difference in gut nk cells or variation in gut nk cell responses to different pathogens [10, 11] . recently, lp nkp46+ nk1.1+ ilcs were shown to contribute to gut pathology and ileitis during toxoplasma gondii infection [72] . their function relied on il-15 and resulted in ccl3 dependent recruitment of inflammatory monocytes into the lamina propria. gut nk cells therefore may have several functions, not only in controlling infections directly, but also in the coordination and regulation of immune responses in the intestinal mucosa. nk cell ifn is also important in control of gut citrobacter rodentium and yersinia enterolytica infections [73] [74] [75] . ifn production from macrophages was critical for this response. interestingly, trif signaling downstream of tlr4 was required for this macrophage induced nk cell activity. nk cells in the gi tract are involved in control of parasitic infection. nk cells production of il-17 in response to toxoplasma gondii infection in the gut was dependent upon il-6 [76] . in another model, nk cell dependent and independent ifn was required to control cryptosporidium parvum infection in mice [77, 78] . nk cells in humans are also important for innate control of gut mucosal infections. in hiv patients, individuals who are known as spontaneous controllers have greater numbers of iel associated nk cells than those who are classified as nonresponders [79] . control of hiv in humans and also siv in macaques could be due to nk cell derived il-17 and il-22 production in iels and dependent upon an interaction with gut mucosal cd103+ dendritic cells [80] . nk cells may contribute to hiv pathogenesis early in infection as tested with an siv model. this was shown when 4 7 was blocked by antibody treatment in macaques [81] . trafficking of nk cells blocked with this treatment reduced siv dissemination and viral burdens. nk cell activation itself may also contribute through recruitment of cd4 t cells providing the virus with more replicative niche to survive. however, later nk cells could play a substitutive role in protection. since 4 7 was required for nk cell recruitment in this model, these results give support to the hypothesis that, in the gut, nk cell homing depends upon an interaction with mucosal dendritic cells (figure 2, top) . overall nk cells in gut mucosa play an important role in protection against orally acquired infections and further dissection is needed to understand how to take advantage of this cell type to optimize protection. there is a wide distribution of nk cells in mice and humans throughout the female reproductive tract, from the uterus, endometrium, and cervix to the ectocervix and vagina [39, 82] . the resident cells in these tissues are all capable of producing ifn and cytolytic activity through adcc. the importance of ifn was noted early in response to vaginal infection of mice with hsv-2 where it was shown to help in resolving this infection in mice [83] . subsequent studies in mice revealed that a major source in the vaginal mucosa for this cytokine was nk cells [84] . this was demonstrated using rag2−/− common-chain−/− mice, which lack t, b and nk cells and rag2−/− mice, which lack only t and b cells. rag2−/− common-chain−/− mice were 100 fold more susceptible to infection than rag2−/− mice. although it was shown later on that il-15 could stimulate an anti-hsv-2 response independent of nk cells [85] , as in gut mucosa infections, il-15 was important in stimulating nk cell protective responses. recruitment of nk cells was also important for the ability of nk cells to protect against vaginal hsv-2 infection. ccr5−/− mice were more susceptible and had higher viral loads in the cns after vaginal infection most likely due to impaired nk cell trafficking to the site of infection [86] . using a rag2−/− common--chain−/− humanized mouse model, human nk cells were observed to be recruited into the genital tract of female mice infected with hsv-2 suggesting that nk cell innate protection could be important in humans as well [87] . further evidence of a potential role for nk cells in human reproductive tract comes from siv-macaque models where treatment of siv infected animals with ifn and il-12 resulted in enhanced activation and adcc function of cd56 + cd16 + cells in the vaginal mucosa [88] . much investigation is still needed to understand nk cell responses in the vaginal mucosa as they do appear to be important for protection, but they can also contribute to disease pathology as seen in early hiv infection. uterine nk cells play two important roles in that they are required for tissue reorganization, vascularization, implantation of the fetus, and tolerance. at the same time they play a role in providing fetal protection during infection (figure 2 , bottom) [89] . what regulates the balance between tolerance and protection is not clear. infection with listeria monocytogenes results in placental colonization [90] . nk cells tested for their ability to prevent this colonization in mice were shown to not have an impact. although il-15 was required for uterine nk cell development and their recruitment to this site in the naïve state, a lack of il-15 did not prevent the production of ifn early during listeria infection. therefore, il-15 in the uterus appears to play a more important role in nk cell recruitment but less of a role in early nk cell control of infection [91] . other factors may be required to regulate their responsiveness to infection in this tissue. human uterine nk cells did not respond to direct tlr triggering but required an interaction with tlr stimulated accessory cells from the uterus to produce ifn [92] . therefore, pathogen stimulation could cause an imbalance towards inflammation rather than tolerance at the maternalfetal interface. this is supported by a study investigating toxoplasma gondii infection induced abortion in mice [93] . t. gondii induces a potent th1 response including nk cell production of ifn . parasite infection is also known to cause spontaneous abortion in the first trimester of pregnancy. infection of pregnant wild type mice in this study resulted in fetal resorption while infection of pregnant ifn r−/− mice had 50% reduction in effect. surprisingly, this was shown to be independent of nk cells. however, recent studies using decidual nk and trophoblast cells from humans have demonstrated that activity of these nk cells is enhanced upon t. gondii infection [94] . this increase in activity was due to the increased expression of nkg2d by decidual nk cells after in vitro stimulation with toxoplasma [95] . nk cells may sense their environment and be tuned commensurate with what functions are needed. this possibility is supported by the theory of nk cell reeducation as what occurs when nk cells develop a memory phenotype [96] . since uterine nk cells recruited in the steady state are less mature, as noted by their surface marker phenotype, the cytokine/hormone milieu changes the functional phenotype needed for successful pregnancy or immune protection develops in situ. another possibility is that new nk cells from the periphery could be recruited and contribute to pathology. pathogen stimulated that apcs migrating to the draining lymph node activate newly recruited nk cells that are then driven to protect. much needs to be addressed in regard to the mechanisms behind frt nk cell function as there is relatively little known. conventional nk cells are found in all mucosal tissues and play an important role in first line of defense against bacterial, fungal, viral, and parasitic infections. significant questions still remain in regard to (1) how they gain entry into each of the specific tissues, (2) what sources of nk cell precursors provide resident nk cells in different tissues, and (3) how their diverse functional responses are regulated in different mucosal tissues. for mucosal tissue resident nk cells in the naïve state, lung nk cells may represent a more mature phenotype and could come from the blood stream. gut mucosal and frt nk cells may be derived from nk precursor or immature nk cells after receiving initial address directions and programming from apcs in secondary lymphoid structures. during infection, resident mucosal tissue nk cells respond primarily through ifn production, which contributes directly to early control of pathogens. inflammation (il-12, ifn / , ifn , and chemokines) at the site of infection then results in expansion of resident cells and recruitment of peripheral nk cells which provide additional protection. resident nk cells can change their functional phenotype based on the intensity of the inflammatory signals resulting in pathology in different tissues. further studies will be important to elucidate mechanisms behind all of these processes and lead to the development of nk cell dependent therapies as well as vaccine approaches that could be useful in not only infectious disease, but also autoimmunity and cancer. innate lymphoid cells-a proposal for uniform nomenclature systemic but not local infections elicit immunosuppressive il-10 production by natural killer cells the aryl hydrocarbon receptor promotes il-10 production by nk cells adaptive immune features of natural killer cells adaptive immune 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vitro and in vivo the transcription factor e4bp4 is not required for extramedullary pathways of nk cell development lamina propria ckit + immune precursors reside in human adult intestine and differentiate into natural killer cells imbalance of nkp4 4+ nkp46 − and nkp44 − nkp46 + natural killer cells in the intestinal mucosa of patients with crohn's disease ifn--producing dendritic cells are important for priming of gut intraepithelial lymphocyte response against intracellular parasitic infection the role of beta7 integrins in cd8 t cell trafficking during an antiviral immune response origin, trafficking, and intraepithelial fate of gut-tropic t cells the light and dark sides of intestinal intraepithelial lymphocytes organ-specific features of natural killer cells natural killer cell distribution and trafficking in human tissues natural killer cells infiltrating human nonsmall-cell lung cancer are enriched in cd56 ℎ cd16 − cells and display an impaired capability to kill tumor cells nk cell development from a novel progenitor found in the murine lung characterisation of natural killer cells and cd56+ t-cells in sarcoidosis patients intestinal intraepithelial lymphocytes contain a cd3 − cd7 + subset expressing natural killer markers and a singular pattern of adhesion molecules human small-iintestinal epithelium contains functional natural killer lymphocytes cd8-natural killer cells are greatly enriched in the human gastrointestinal tract and have the capacity to respond to bacteria microbial flora drives interleukin 22 production in intestinal nkp46+ cells that provide innate mucosal immune defense influence of the transcription factor rorgammat on the development of nkp46+ cell populations in gut and skin ror t and commensal microflora are required for the differentiation of mucosal interleukin 22-producing nkp46 + cells functions of uterine natural killer cells are mediated by interferon gamma production during murine pregnancy mhcdependent inhibition of uterine nk cells impedes fetal growth and decidual vascular remodelling organ-specific phenotypic and functional features of nk cells in humans chemerin regulates nk cell accumulation and endothelial cell morphogenesis in the decidua during early pregnancy unique characteristics of nk cells throughout the human female reproductive tract ly49 receptors: innate and adaptive immune paradigms cytokine profile of mouse vaginal and uterus lymphocytes at estrus and diestrus unique subpopulations of cd56 + nk and nk-t peripheral blood lymphocytes identified by chemokine receptor expression repertoire natural killer cell distribution and trafficking in human tissues organ-specific features of natural killer cells a human cd34(+) subset resides in lymph nodes and differentiates into cd56bright natural killer cells the microbiome of the lung crtam controls residency of gut cd4+cd8+ t cells in the steady state and maintenance of gut cd4+ th17 during parasitic infection the role of natural killer cells in pulmonary immunosurveillance natural killer cells in infection and inflammation of the lung human natural killer cell deficiencies natural killer cell deficiency nk cells play a critical protective role in host defense against acute extracellular staphylococcus aureus bacterial infection in the lung interferon induction and increased natural killer-cell activity in influenza infections in man antibody-dependent cellular cytotoxicity is associated with control of pandemic h1n1 influenza virus infection of macaques recognition of haemagglutinins on virus-infected cells by nkp46 activates lysis by human nk cells nk cells exacerbate the pathology of influenza virus infection in mice immune dysregulation in severe influenza il-15 complexes induce nk-and t-cell responses independent of type i ifn signaling during rhinovirus infection il-15 participates in the respiratory innate immune response to influenza virus infection in vivo treatment of mice and hamsters with antibodies to asialo gm1 increases morbidity and mortality to pulmonary influenza infection natural killer cell responses during viral infections: flexibility and conditioning of innate immunity by experience role of natural killer cells in resistance to cryptococcus neoformans infections in mice an acidic microenvironment increases nk cell killing of cryptococcus neoformans and cryptococcus gattii by enhancing perforin degranulation il-18 contributes to host resistance against infection with cryptococcus neoformans in mice with defective il-12 synthesis through induction of ifn-gamma production by nk cells chemokine-mediated recruitment of nk cells is a critical host defense mechanism in invasive aspergillosis dynamic roles of type i and type ii ifns in early infection with mycobacterium tuberculosis nk cellderived ifn-differentially regulates innate resistance and neutrophil response in t cell-deficient hosts infected with mycobacterium tuberculosis nk cells respond to pulmonary infection with mycobacterium tuberculosis, but play a minimal role in protection conventional alpha beta t cells are sufficient for innate and adaptive immunity against enteric listeria monocytogenes the role of natural killer cells in the defense against listeria monocytogenes lessons from a rat model interleukin-15 and nk1.1 + cells provide innate protection against acute salmonella enterica serovar typhimurium infection in the gut and in systemic tissues interleukin-15-dependent nkp46 + innate lymphoid cells control intestinal inflammation by recruiting inflammatory monocytes cd3 − nk1.1 + cells aid in the early induction of a th1 response to an attaching and effacing enteric pathogen natural killer cells protect against mucosal and systemic infection with the enteric pathogen citrobacter rodentium host innate recognition of an intestinal bacterial pathogen induces trifdependent protective immunity il-6 promotes nk cell production of il-17 during toxoplasmosis innate immune responses against cryptosporidium parvum infection roles for nk cells and an nk cell-independent source of intestinal gamma interferon for innate immunity to cryptosporidium parvum infection altered distribution of mucosal nk cells during hiv infection loss of mucosal cd103 dcs and il-17 and il-22 lymphocytes is associated with mucosal damage in siv infection blocking of alpha4beta7 gut-homing integrin during acute infection leads to decreased plasma and gastrointestinal tissue viral loads in simian immunodeficiency virus-infected rhesus macaques innate and adaptive immunity in female genital tract: cellular responses and interactions interferon-enhances resolution of herpes simplex virus type 2 infection of the murine genital tract interleukin-15 and natural killer and nkt cells play a critical role in innate protection against genital herpes simplex virus type 2 infection nk and nkt cellindependent contribution of interleukin-15 to innate protection against mucosal viral infection susceptibility of ccr5-deficient mice to genital herpes simplex virus type 2 is linked to nk cell mobilization mucosal innate and adaptive immune responses against herpes simplex virus type 2 in a humanized mouse model enhancement of natural killer cell activation and antibody-dependent cellular cytotoxicity by interferon-and interleukin-12 in vaginal mucosae sivmac251-infected macaca fascicularis origin, phenotype and function of human natural killer cells in pregnancy the uterine nk cell population requires il-15 but these cells are not required for pregnancy nor the resolution of a listeria monocytogenes infection central role of interleukin-15 in postovulatory recruitment of peripheral blood cd16(-) natural killer cells into human endometrium tlrs mediate ifn-gamma production by human uterine nk cells in endometrium toxoplasma gondii-induced foetal resorption in mice involves interferon--induced apoptosis and spiral artery dilation at the maternofoetal interface changes of human decidual natural killer cells cocultured with yfp-toxoplasma gondii: implications for abnormal pregnancy toxoplasma gondii infection regulates the balance of activating and inhibitory receptors on decidual natural killer cells re-educating natural killer cells the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord-316181-ccauw70y authors: yang, fude; dong, xiaoxv; yin, xingbin; wang, wenping; you, longtai; ni, jian title: radix bupleuri: a review of traditional uses, botany, phytochemistry, pharmacology, and toxicology date: 2017-05-16 journal: biomed res int doi: 10.1155/2017/7597596 sha: doc_id: 316181 cord_uid: ccauw70y radix bupleuri (chaihu) has been used as a traditional medicine for more than 2000 years in china, japan, korea, and other asian countries. phytochemical studies demonstrated that this plant contains essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols. crude extracts and pure compounds isolated from radix bupleuri exhibited various biological activities, such as anti-inflammatory, anticancer, antipyretic, antimicrobial, antiviral, hepatoprotective, neuroprotective, and immunomodulatory effects. however, radix bupleuri could also lead to hepatotoxicity, particularly in high doses and with long-term use. pharmacokinetic studies have demonstrated that the major bioactive compounds (saikosaponins a, b(2), c, and d) were absorbed rapidly in rats after oral administration of the extract of radix bupleuri. this review aims to comprehensively summarize the traditional uses, botany, phytochemistry, pharmacology, toxicology, and pharmacokinetics of radix bupleuri reported to date with an emphasis on its biological properties and mechanisms of action. radix bupleuri, also called "chaihu" in chinese, is derived from the dried roots of bupleurum chinense dc. and bupleurum scorzonerifolium willd. [1] . as a traditional herbal medicine, radix bupleuri has been used widely for the treatments of influenza, fever, inflammation, malaria, menstrual disorders, and hepatitis in china, japan, korea, and other asian countries [2, 3] . according to ancient chinese medical literatures, radix bupleuri is capable of regulating the exterior and interior metabolisms, dispersing evil heat from the superficies, soothing the liver, and promoting yang and qi (representing "life energy" or "life force" in tcm theories). in recent decades, investigations of radix bupleuri have focused on its biological activities, including its anti-inflammatory [4, 5] , anticancer [6, 7] , antipyretic [8] , antimicrobial [9] , antiviral [10] , hepatoprotective [11] , and immunomodulatory effects [12] . in addition, radix bupleuri also exhibited significant effects on membrane fluidity [13] . these studies have resulted in the isolation of essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols from this plant [14] . among them, triterpenoid saponins are known to be the major bioactive compounds [15, 16] . saikosaponins a and d are commonly used as chemical standards for quality evaluation of radix bupleuri in the current chinese pharmacopoeia and recent publications. however, an increasing number of recently published studies have reported adverse effects of radix bupleuri. the purpose of this review is to provide updated, comprehensive information on the traditional uses, botany, phytochemistry, pharmacology, toxicology, and pharmacokinetics of radix bupleuri based on scientific literatures in the past few decades. this study will facilitate exploring the therapeutic potential of this plant and evaluate future research opportunities. radix bupleuri, which is characterized by a wide spectrum of biological and pharmacological effects, has been used as a famous traditional chinese medicinal herb with a history of medical use in china. according to tcm theory, radix bupleuri is thought to regulate the exterior and interior curing rhinitis and nasosinusitis bioactive compounds owing to their antifungal and antiinflammatory activities [37, 38] . in one study, the essential oils in radix bupleuri were extracted by steam distillation and solvent extraction and then analyzed by gc/ms; 78 peaks were identified. among these peaks, the major volatile compounds were 3-methylbutanal (7.24%), pentanal (5.74%), hexanal (20.11%), furan-2-carbaldehyde (25.23%), and heptanal (12.07%) [39] . however, in another study, the results showed that e-2-heptanal, furan, 2-pentyl, and e-2-nonenal were some of the main compounds of the oil [40] . triterpenoid saponins are the main active components of radix bupleuri, which exhibit a broad spectrum of biological and pharmacological effects, including analgesic, immunomodulatory, hepatoprotective, immunomodulatory, anti-inflammatory, antitumor, and antiviral activities [3, [41] [42] [43] . currently, approximately 35 saponins have been isolated from radix bupleuri ( figure 2 ) [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] . among them, saikosaponins a, c, and d are the major bioactive constituents found in radix bupleuri; however a variety of minor saikosaponins have also been isolated [16] . the cytotoxic and antiproliferative effects of saikosaponins a and d have been attracting much interest in recent years [55] . additionally, more information about the intimate relationship between the structural characterization of saikosaponins and their cytotoxic evaluations is very necessary. radix bupleuri have been identified, including (2z,8z,10e)pentadecatriene-4,6-diyne-1-ol ( ), (2z,8e,10e)-pentadecatriene-4,6-diyne-1-ol ( ), (2z,8z,10e)-heptadecatriene-4,6-diyne-1-ol ( ), and bupleurynol ( ) ( figure 3 ) [56, 57] . radix bupleuri exerts a great variety of pharmacological activities due to its complexbioactive compounds. an overview of the pharmacological studies on radix bupleuriis presented in detail in the following sections. widely used for the treatment of several types of chronic inflammatory diseases. the crude polysaccharides (80 mg/kg) isolated from the roots of bupleurum chinense dc. significantly attenuated lung injury by inhibiting the level of myeloperoxidase (mpo), tumor necrosis factor-(tnf-), and serum nitric oxide (no) [66] . chun et al. reported that saikosaponins from radix bupleuri exhibited anti-inflammatory activity on inflammatory processes including inhibition of inflammatory exudation, capillary permeability, inflammatory mediators release, migration of white cells, connective tissue hyperplasia, and a variety of allergic inflammation [67] . ma et al. was the first to show that saikosaponins exerted anti-inflammatory activity on paw edema mainly via regulating the nicotinate and nicotinamide metabolism and arachidonic acid metabolism [5] . zhu et al. found that saikosaponin a (ssa) exhibited an inhibitory effect on proinflammatory cytokines in lps-stimulated macrophages. the mechanism of these actions involved the regulation of mapk and nf-b signals pathways [68] . in another study, ssa dose-dependently inhibited the production of ros, tnf-, il-8, cox-2, and inos in lps-stimulated human umbilical endothelial cells (huvecs) by upregulating of the lxr -abca1 signaling pathway [69] . moreover, lee et al. showed that saikosaponin c (ssc) was also shown to inhibited lps-induced apoptosis in huvecs via inhibition of caspase-3 activation and caspase-3-mediated-fak degradation [70] . zhao et al. showed that ssa also suppressed tnf-and il-6 concentrations in the intestines of septic rats through the inhibition of the nucleotide-binding oligomerization domain 2 (nod2)/nf-b signaling pathway [71] . saikosaponin d (ssd) has been reported to inhibit pge 2 production and intracellular free ca 2+ concentration ([ca 2+ ]i) in a concentration-dependent manner with an ic 50 value of 3 m in c6 rat glioma cells [72] . in addition, several studies showed that a wide range of radix bupleuri preparations also exhibited antiinflammatory effects in in vitro and in vivo model. li et al. showed that saireito and its active components (ssd) could suppress the proliferation of mesangial cells and expansion of the mesangial matrix in the rat glomerulonephritis model [73] . in experimental chronic pancreatitis rats model, "chai-hu-shu-gan powder" exerted anti-inflammatory and antifibrotic effects by inhibiting the expression of nuclear factor-b (nf-b) and tnf-mrna in the pancreas [74] . furthermore, it also reduced the abnormally high plasma level of cholecystokinin, improved the gastric movement, and avoided nausea and flatulence [75] . in another experiment, a chinese herbal formula called "rcm-101" (containing flos magnoliae, radix bupleuri, radix glycyrrhizae, radix angelicae sinensis, etc.) inhibited the no production and inos protein expression in lps-stimulated rat aorta and raw 264.7 macrophages [76] . radix bupleuri also possessed anticancer/antitumor effect. the acetone extract of bupleurum scorzonerifolium could inhibit the proliferation of a549 human lung cancer cells in a dose-dependent manner via causing cell cycle arrest in the g2/m phase, increasing microtubule stabilization, suppressing telomerase activity, activating erk 1/2 and caspase-3/9 in a549 cells [77] [78] [79] . saponins isolated from radix bupleuri also exhibited significantly anti-proliferative activity in human non-small cell lung cancer a549 cells through fas-dependent apoptotic pathway [80] . su et al. found that the water extracts of radix bupleuri could enhance 5-fluorouracil-induced cytotoxicity in hepg2 hepatoma cells through cell arrest at the late g1/early s phase, while protecting normal blood lymphocytes [6] . ssd showed very potent activity against the hepg2 cell line with an ic 50 value of 12.5 mg/ml. the mechanism of cytotoxicity was attributed to the induction of apoptosis through activation of caspase-3 and caspase-7, which subsequently resulted in poly-adp-ribose polymerase (parp) cleavage [81] . sarcoplasmic/endoplasmic reticulum ca 2+ atpase (serca), leading to the increase of intracellular calcium ion levels and activating the ca 2+ /calmodulin-dependent kinase kinase--(camkk -) amp-activated protein kinase-(ampk-) mammalian target of rapamycin (mtor) signaling cascade, endoplasmic reticulum (er) stress, and unfolded protein responses (upr) [43] . several chinese medicine preparations containing radix bupleuri also have been traditionally used in the treatment of tumors and cancer. the water extracts of "long dan xie gan wan" exerted a significant growth inhibitory effect in hl60 and ht29 cancer cell lines, indicating that this formulation may possess some chemotherapeutic potential [83] . treatment with "xiao-chai-hu decoction" exhibited a significantly lower incidence of hepatocellular carcinoma and reductions in cancer pain and tumor size. the underlying mechanism of the antitumor activities is based on stimulation of the reticuloendothelial system (res) and is closely related of tnf production [84] [85] [86] . wen et al. reported that the acetone extract of radix bupleuri possessed a significant antivirus effect on acute respiratory tract infections with h1n1 virus infection and suppressed influenza a virus-induced rantes secretion in h1n1-infected a549 cells at a concentration of 100 and 200 g/ml, suggesting that radix bupleuri might be beneficial for the treatment of chronic inflammatory conditions followed by viral infection [10] . ssc has been reported to show effective anti-hbv activity through inhibiting dna expression of hbsag, hbeag, and hbv [81] . treatment with "xiao-chai-hu decoction" (20 g/ml, 3 days; 20 g/ml, 6 days) could inhibit the production of hbv ( < 0.0001) and the expression of hbeag. moreover, crude saponins of bupleurum chinense dc. could inhibit the replication of hbv ( < 0.0001) [87] . similarly, in another study, yin et al. showed that ssd isolated from the meoh extract of bupleurum chinense dc. exhibited significant bioactivity in inhibiting dna replication of hbv [88] . the antiviral activity of saikosaponins (a, b 2 , c, and d) and their mode of actin were examined. the results showed that all saikosaponins exerted antiviral activity on human coronavirus-229e at concentrations of 0.25-25 m, and the strongest activity was observed for saikosaponin b 2 with an ic 50 of 1.7 m. this mechanism might involve interference in the early stage of viral replication, such as absorption and penetration of the virus [89] . the water extract of radix bupleuri was reported to exert its antipyretic effect on dry yeastinduced high fever rats. the mechanism is related to the adjustment of synthesis and exudation of cyclic adenosine monophosphate (camp) and arginine vasopressin (avp) [90] . a novel in situ gel system for nasal delivery of the essential oil from radix bupleuri was prepared. the results suggested that radix bupleuri in situ gel can be more effective than the solution in the treatment of fever [91] . a similar study showed that the essential oil extracted from the herb exhibited dose-dependent antipyretic capacity on both fevered rabbits and rats [92] . the ethanol extract of bupleurum chinense dc. exerted a remarkable bacteriostatic effect on gram-negative microorganism helicobacter pylori. the bioactive minimum inhibitory concentration (mic) value was 60 mm [93] . saikosaponins isolated from radix bupleuri have been reported to exhibit antibacterial activity, particularly against pseudomonas aeruginosa and listeria monocytogenes. the protective effect was attributed to the immunomodulatory action on macrophages [94] . "chaihu injection" has also been tested for possible antimicrobial activity in vitro. the results demonstrated that mild inhibition of staphylococcus aureus was observed but no effects were observed against staphylococcus albus, neisseria gonorrhoeae, diplococcus pneumoniae, haemolytic streptococcus, or pseudomonas aeruginosa [95] . the liver protective effects against ccl 4 induced liver injury were investigated after treatment of mice with raw and vinegar-baked radix bupleuri (5 g/kg/day) for 14 days. the results showed that both raw and processed radix bupleuri showed liver protective effects against ccl 4 induced liver injury, and the vinegar-baked radix bupleuri exerted better effects than that of raw radix bupleuri [96] . pretreated with saikosaponins, especially ssa or ssd, showed remarkable inhibition of d-galactosamineinduced hepatic injury through decreasing the activity of glucose-6-phosphatase and nadph-cytochrome c reductase and increasing 5 -nucleotidase activity [97] . similarly, bupleurosides iii, vi, ix, and xiii and saikosaponin b 3 isolated from bupleurum scorzonerifolium willd. were also found to exhibit protective effect on the d-galactosamineinduced cytotoxicity in primary cultured rat hepatocytes [98] . further studies also demonstrated that the protective effects of saikosaponins isolated from bupleurum chinense dc. could prevent hepatocyte injury through regulating intracellular calcium levels [99] . in a rat model with ccl 4 induced acute hepatic injury, the hepatic enzyme levels (got, gpt, and alp) and the lipid peroxidation in the liver were significantly reduced by the administration of ssd [100] . additionally, ssd significantly reduced collagen i deposition and alanine aminotransferase level on liver fibrosis rats and decreased the concentration of transforming growth factor 1 (tgf-1). moreover, ssd was able to alleviate hepatocyte injury from oxidative stress. the effect of ssd on liver fibrosis may be related to its ability to reduce lipid peroxidation [101] . the effects of radix bupleuri on spontaneous lymphatic vessel activity. the results indicated that radix bupleuri significantly increased the amplitude of spontaneous activity of lymphatic vessels in a concentration-dependent manner, and the mechanisms of this effect seem to be independent of endothelial function [102] . eugenin ( ) and saikochrome a ( ) isolated from the meoh extracts from bupleurum scorzonerifolium possessed immunosuppressive effect on human peripheral blood t cells via inhibiting cd28-costimulated activation [62] . ssd (10 mg, intraperitoneally) significantly activated peritoneal macrophages in terms of enhancement of phagocytic activity, increased level of cellular lysosomal enzyme, and suppressed the response of plaque-forming cells to heterologous erythrocytes by stimulating t and b cells in a dose-dependent manner [103] . moreover, ssd modulated lymphocyte activity through suppressing the t cell response and increasing the b cell response to different mitogens and the interleukin-(il-) 2/il-4 production through a receptor-bypassed pathway [41, 104, 105] . in another experiment, wong et al. found that ssd was shown to inhibit okt3/cd28-costimulated human t cell proliferation and pma, pma/ionomycin, and con a-induced mouse t cell activation in vitro. the underlying mechanisms involved downregulation of nf-kb signaling by suppression of ikk and akt activities [106] . autophagy is a complex process in cells, which occurs through the formation of doublemembrane vesicles (autophagosomes), which are engulfed by cytoplasmic molecules. then, the autophagosome fuses with the lysosomes, leading to degradation of long-lived proteins, aggregated proteins, and damaged organelles [107] [108] [109] . moreover, autophagy might be triggered by hypoxia, nutritional deprivation, radiation, chemical drugs, and other stimulants [110] . autophagy contributes to the pathogenesis of diverse diseases, such as neuronal degeneration, inflammatory bowel disease, aging, and cancer [111, 112] . in the previous study, law et al. demonstrated that the protective pharmacological effects of radix bupleuri might be attributed to its autophagy induction. the autophagic effect of radix bupleuri played an important role in relieving liver disease-related symptoms through anti-inflammatory, organ-protective, and aggregate removal functions. furthermore, the anticancer effects of radix bupleuri could be attributed to its autophagy induction. radix bupleuri has been found to be an effective treatment against depression by regulating metabolite, hormone, and neurotransmitter levels via autophagy-mediated lipid metabolism [113] . the effect of the ethanol extract from radix bupleuri on cytochrome 450 isoform activities using a six-drug cocktail approach was evaluated; the results demonstrated that radix bupleuri had strong induction activity on the cyp2e1, cyp2d6, and cyp3a4, which may lead to potential plant drug-drug interactions [114] . radix bupleuri was shown to be the inhibitor ofglucuronidase. the inhibition rate of radix bupleuri extracts rb1 (high molecular weight polysaccharides), rb2 (ethanol soluble/water insoluble component), rb3 (extracted by nbutanol, soluble in water), and rb4 (low molecular weight water soluble parts) on the activity of -glucuronidase was found to be 45.15%, 33.94%, 24.94%, and 34.54%, respectively [115] . in pentylenetetrazol (ptz) induced epilepsy rats model, ssa isolated from radix bupleuri significantly reduced seizure severity and duration while it markedly elevated seizure latency and downregulated the cytokines expression of p-mtor, p-70s6k, l-1 , and tnf-through inhibiting mtor signaling pathway [116] . he et al. demonstrated that ssa obviously reduced lipoprotein uptake to block foam cell formation and the expression of lox-1 and cd36, boosted cholesterol efflux, and the expression of abca1 and ppar through inhibiting pi3k/akt/nf-b/nlrp3 signaling pathway [117] . in another experiment, ssc exerted a potent effect on inducing human umbilical vein endothelial cells (huvecs) viability and growth. furthermore, ssc also induced endothelial cells migration and capillary tube formation. the underlying mechanisms might be related to the gene expression or activation of matrix metalloproteinase-2 (mmp-2), vascular endothelial growth factor (vegf), and the p42/p44 mitogenactivated protein kinase (mapk, erk) [118] . in addition, ssc was shown to exhibit inhibitory activities against alzheimer's disease (ad) via suppressing the secretion of a peptides and abnormal tau hyperphosphorylation-mediated microtubule depolymerization. moreover, ssc suppressed a peptideinduced brain endothelial apoptosis, indicating that ssc might be a novel therapeutic tool for treating human ad and other neurodegenerative diseases [119] . it was shown by liu et al. for the first time that four polyacetylenes ( -) from radix bupleuri potently exhibited an antidepressant activity by inhibiting the reuptake of serotonin, norepinephrine, and dopamine. the mechanism might be mediated by increasing the level of monoamines, particularly 5-ht and ne [56] . zhu et al. suggested that ssa and ssd exhibited the anthelmintic activity against dactylogyrus spp. infecting goldfish. the effective concentration (ec 50 ) values for ssa and ssd were 1.46 and 0.74 mg â��1 , respectively [120] . bupleuri possesses a wide spectrum of pharmacological effects, including anti-inflammatory effect, anticancer effect, antiviral effect, antipyretic effect, antibacterial effect, hepatoprotective effect, and immunomodulatory effect (table 3 ). based on these pharmacological effects, we can conclude that the extracts and the compounds from this plant can prevent or treat certain diseases, such as cancer, fever, malaria, hepatitis, and ad. however, there is not enough systemic data of these chemical compounds and their pharmacological effects. thus, in the future, the pharmacological effects and the possible molecular mechanisms of the pharmacological activities of radix bupleuri must be urgently explored on our modern understanding of these diseases' pathophysiologies. radix bupleuri has been used for thousands of years as an important traditional herb in china. however, the toxic effects of radix bupleuri in clinical applications have been gradually reported. several studies have found that the liver is the main organ affected by toxicity, particularly in longterm use. major symptoms of liver injury induced by radix bupleuri included transaminase lifts, hepatitis, and jaundice. however, liver functions can return to normal levels after a specific period [121] . radix bupleuri has been reported to exhibit acute hepatitis and acute hepatic necrosis. the mean total daily dose was 18.0 â± 33.5 g, which was more than the chinese pharmacopoeia recommended range of 3 to 10 g [122] . moreover, radix bupleuri had been implicated in multiple cases of acute hepatitis both as an ingredient alone and within a particular formulation "xiao-chai-hu-tang" (also known as syo-saiko-to in japanese) [123] . lee et al. demonstrated that two chinese herbal products containing radix bupleuri might increase their risks of liver injury in hbv-infected patients. however, further mechanistic research on the hepatotoxicity of radix bupleuri in the presence of hbv infection is warranted [124] . in addition, the essential oil of radix bupleuri induced acute hepatotoxicity with asynchronous state, higher heart rate, and fast breathing [125] . the total saponins isolated from radix bupleuri could also cause evidently liver damage in dose-dependent manner manifested as hepatocyte organic lesion and liver function changes, as well as hepatocyte death [126] . a selective and sensitive lc-ms/ms method was developed and validated for simultaneous determination of ssa, b 2 , c, and d in rat plasma afteroral administration of the ethanolwater (50 : 50, v/v) extract of radix bupleuri for the first time. the results demonstrated that ssa, c, and d were absorbed rapidly with max less than 30 min [127] . in another pharmacokinetics experiment of rats, liu et al. was the first to develop an uplc-pda-ms method to determine the pharmacokinetics of four polyacetylenes after i.g. administration of 95% ethanol extract of radix bupleuri. the results showed that compounds and were not detected in rat serum, whereas compounds and exerted a fast distribution phase followed by a relatively slow elimination phase ( 1/2 , 4-7 h) [56] . in traditional chinese medicine, radix bupleuri has long been used regulate the exterior and interior metabolisms, disperse evil heat from superficies, sooth the liver, and promote yang and qi. it has been widely used to treat various diseases in china, japan, korea, and other asian countries for many centuries. a total of 74 compounds including essential oils, triterpenoid saponins, polyacetylenes, flavonoids, lignans, fatty acids, and sterols have been isolated and identified from radix bupleuri . pharmacological studies have revealed that radix bupleuri possesses a variety of biological effects, including anti-inflammatory, anticancer, antiviral, antipyretic, antibacterial, antiobesity, immunomodulatory, hepatoprotective, neuroprotective, and autophagic effects . however, there are some aspects that need to be further investigated. radix bupleuri is an ingredient of many patent medicines or prescriptions. although modern experiments have confirmed that this drug alone exhibits multiple pharmacological activities, it is important to investigate the molecular mechanisms of radix bupleuri combined with other herbs based on traditional uses. furthermore, the pharmacological effects of only a few of the ingredients, such as saikosaponins, flavonoids, and the essential oils, have been investigated. some polyacetylenes, lignans, and sterols have not been sufficiently researched in terms of their pharmacological effects. radix bupleuri shows both hepatoprotection and in vitro [89] antipyretic effect dry yeast-induced high fever rats adjusts the synthesis and exudation of camp and avp in vivo [90] turpentine-induced fever rabbits decreases body temperature the essential oil in vivo [91] turpentine-induced fever rabbits and rats the essential oil in vivo [92] antibacterial effect in vivo [113] hepatotoxicity, which appears to be contradictory. this phenomenon is similar to that of polygonum multiflorum thunb. [128] . based on the literature, the main reasons are likely the administration dosage and delivery time. high doses and long-term drug delivery are more likely to result in liver toxicity, whereas low doses and short-term drug delivery might result in liver protection. therefore, this issue needs further study. in conclusion, this review summarized the traditional uses, botany, phytochemistry, pharmacology, and toxicology of radix bupleuri. moreover, it has provided a new foundation for further research on its mechanism of action and the development of better therapeutic agents employing radix bupleuri in the future. it is anticipated that the comprehensive and detailed research on toxicity, pharmacodynamics, pharmacokinetics, and molecular mechanism are necessary to be explored to develop its bioactive compounds as effective drugs. the authors have declared that there are no conflicts of interest regarding the publication of this paper. fude yang and xiaoxv dong contributed equally 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with chinese herbal products containing radix bupleuri in 639, 779 patients with hepatitis b virus infection acute toxicity of volatile oil from bupleurum chinense in rats and mice dose-time-toxicity" relationship study on hepatotoxicity caused by multiple dose of total bupleurum saponin crude extracts to rats analysis of saikosaponins in rat plasma by anionic adducts-based liquid chromatography tandem mass spectrometry method traditional usages, botany, phytochemistry, pharmacology and toxicology of polygonum multiflorum thunb.: a review this work was financially supported by the collaborative innovation construction plan of beijing university of chinese medicine (no. 2013-xtcx-03). key: cord-031416-ytbs95wi authors: sabzpoushan, s. h. title: a system biology-based approach for designing combination therapy in cancer precision medicine date: 2020-08-26 journal: biomed res int doi: 10.1155/2020/5072697 sha: doc_id: 31416 cord_uid: ytbs95wi in this paper, we have used an agent-based stochastic tumor growth model and presented a mathematical and theoretical perspective to cancer therapy. this perspective can be used to theoretical study of precision medicine and combination therapy in individuals. we have conducted a series of in silico combination therapy experiments. based on cancer drugs and new findings of cancer biology, we hypothesize relationships between model parameters which in some cases represent individual genome characteristics and cancer drugs, i.e., in our approach, therapy players are delegated by biologically reasonable parameters. in silico experiments showed that combined therapies are more effective when players affect tumor via different mechanisms and have different physical dimensions. this research presents for the first time an algorithm as a theoretical viewpoint for the prediction of effectiveness and classification of therapy sets. study of cancer as the second leading cause of human mortality is essential. early diagnosis and appropriate therapies can be a significant help to the improvement of cancer survivals. although surgery in the case of solid tumors, antitumor drugs, radiation, and immunotherapy have been the treatment of choice in some instances, but ineffectiveness of treatments, drug resistance, side effects of therapies, and tailoring treatment to the individual characteristics of each patient are still major clinical problems. where precision medicine will allow researchers to predict more accurately which therapies will work better in which groups of people, combination therapy is a keystone of cancer therapy and potentially reduces drug resistance, while simultaneously providing therapeutic anticancer benefits, such as reducing tumor growth and metastatic potential, arresting mitotically active cells, reducing cancer stem cell populations, and inducing apoptosis. a high percent of oncology drugs and therapies fails in clinical trials [1] . this imposes extra expenses to patients and causes the loss of time in cancer therapies. mathematical models, in silico experiments, and simulations can be a great help for evaluation of different therapies and examining diverse strategies of drug therapies. ten major characteristics of cancer, known as cancer hallmarks, have been universally recognized as (1) unlimited multiplication, (2) evasion from growth suppressors, (3) promoting invasion and metastasis, (4) resisting apoptosis, (5) stimulating angiogenesis, (6) maintaining proliferative signaling, (7) elimination of cell energy limitation, (8) evading immune destruction, (9) genome instability and mutation, and (10) tumor-enhanced inflammation [2] . regarding above hallmarks, it is plausible that we attribute drug therapy efficiencies to individuals' genome, i.e., individual's heterogeneity should be taken into account in cancer therapies by some means [3] . efforts have been devoted to determine how cellular and noncellular components of the tumor's surrounding environment may help it to acquire these characters. this environment and its cellular and noncellular components are called tumor microenvironment (tme) [4] [5] [6] [7] . the recognition that cancer cells need their microenvironment to efficiently display their phenotype has opened the door to hypothesize and implement new therapeutic strategies. today, the main tumor therapy strategies consist of surgery, radiological intervention, chemotherapy, and somatostatin analogs to control symptoms. however, it seems that tumor cells are particularly clever and elastic, and may adapt to treatments and environmental modifications quickly, i.e., once one component has been blocked, other mechanisms will rapidly follow. this may be one of the main factors that lead to poor cancer therapies [8] . it is why different obstructing mechanisms at the same time might lead to the best results of tumor development prevention [9] . the above facts illuminate the motivation for researches in the field of combination therapies. precision medicine refers to the tailoring of medical treatment to the individual characteristics of each patient. it often involves the application of system biology to analyze the cause of an individual patient's disease at the molecular level and then to utilize targeted treatments (often combinatorial) to address that individual patient's disease process. the branch of precision medicine that addresses cancer is referred to as "precision oncology" [3, [10] [11] [12] . tumors are encircled by extracellular matrix (ecm) and stromal cells, and the physiological state of the tme is closely connected to every step of tumorigenesis. evidence suggests that the vital components of the tme are (1) fibroblasts and myofibroblasts, (2) neuroendocrine cells, (3) adipose cells, (4) immune and inflammatory cells, (5) the blood and lymphatic vascular networks, and (6) extracellular matrix (ecm) [13] . the combinatorial complexity of possible combination therapies [14] and the expense and risks of trial and error experiments, as well as the lack of time for cancer patients, are the main reasons for combination therapies fail in clinical trials. in this circumstance, appropriate biologically realizable models and bioinformatics can be a solution. mathematical models and computer simulations can be good alternatives for preestimations and evaluations of effectiveness of drug therapies strategies. computational oncology [15] and in silico trials are good preclinical alternatives to predict the progress of the disease in individuals and suggest new diagnostic and therapeutic methods. patients with cancer are known to be at an increased risk for community-acquired respiratory viruses, such as sars cov-2. there is high proportion of patients who acquired the infection while already in the hospital for cancer treatment affairs. using bioinformatics, mathematical and computational models and in silico analysis are very safe and cost-effective tools for design and analyzing therapy strategies. in silico trials as precision medicine simulators can reduce patient commuters to hospitals and high-risk health centers [16] . cancerous system models can be categorized into three general groups: continuous, discrete, and hybrid, where each one may have deterministic or stochastic formalisms. continuous models describe the system by using ordinary differential equations (odes) or partial differential equations (pdes). several researchers have used odes to study the growth of tumors. to capture spatial structures of tumors, one should use pdes. pdes can better express the temporal and spatial properties of tumor growth at the same time [17] [18] [19] . in the family of agent-based models (abms), cells are considered as discrete elements, and the interaction between them is defined by biological-based rules [20] [21] [22] [23] [24] [25] [26] . abms can simulate emergent structures, i.e., structures that a number of not too complex components work together and form more complex behaviors as a group. it is noteworthy that in differential equation-(de-) based models, rules are applied to the whole system where in abms, the functional rules of each single agent can be specific and special to that agent. these make abms more appropriate for emergent behaviors like tumor growth and tme dynamics modeling. abm is also one of the most frequently used methods for modeling multiscale systems like cancers [27] . today, cancer therapy has dramatically changed, i.e., surgery and radiotherapy are not the only effective ways to fight tumor. novel methods and approaches are emerging, where the molecular and agent features of tumors seem to be the keystone of any therapy. new antibodies, small molecules, antiangiogenics, viral therapy, and precision medicine methods are typical examples. because of the abovementioned new therapies use microscopic or molecular level agents explicitly, so system biology-based cancer models can be the most satisfactory candidates for in silico experiments and studies. it is notable to remember that system biology is a term to describe the study of the interactions between the components of the biological systems and how these interactions give rise to the function and behavior of that system. however, although system biology-based mathematical models of cancer are very useful, but they have also limitations, because the recognition of the mechanisms governing cancerous systems has practical limitations as well [17, 18] . in this paper, we use our recently published agent-based stochastic tumor growth model (absm) as a cancer system. we design several combination therapies which can be hypothesized and regulated as precision medicine. we postulate five novel quantitative merits for comparing possible effectiveness of different combination therapies. we show how in silico experiments can help oncologists to conduct and design combination therapies, and test their ideas, biomed research international in this research, we have used our previously proposed absm model [28] as a cancer system, i.e., we may fit it to a given patient and use it for demonstrating our system biology-based approach for designing combination therapy in cancer precision medicine. agent-based modeling is a stochastic approach used to describe a population of interacting agents, where agents behave according to a set of rules that represent the dynamic features of system. in this way, our absm has been established on four bases: (1) biological assumptions, (2) physical structure, (3) agents and their states, and (4) states transition rules. the multilayer structure of the absm is shown in figure 1 . in absm, host tissue is assumed as a two-dimensional lattice composed of ncell × ncell squares as illustrated in figure 10 . here, each square of the lattice is called a cell. in the absm, two types of agents (immune (ia) and nonimmune (na) cells) are presumed, where three types of nas (normal cell or empty space (na_0), proliferating tumor cell (na_1), nonproliferating or quiescent cell (na_2), and necrotic cell (na_3)) are considered. in the absm, we assume two types of na_1 cells with different division probabilities: in the first type denoted by na_1_1, the cell division is not influenced by its neighbors, while in the second symbolized by na_1_2, the cell division probability (ρ pt ) is affected by its healthy neighbors (na_0s). in the absm, it is assumed that each na_1_1 may be divided into one na_1_1 and one na_1_2 daughter cells with the probability nmm or into two na_1_1 daughter cells with the probability (1 − nmm). it means the higher the value of nmm, the more susceptibility of cancerous cell division to its microenvironment. in table 1 , the basic elements of absm and their brief descriptions are summarized. as it was stated beforehand, the absm is a system biology-oriented model in the sense that it is constructed from several agents and interaction rules between them, and as is shown in [28] , these elements and interactions can give rise to the function and behavior of cancer growth system. with a conceptual and intuitive look at the elements of table 1 , some of them can be assumed individual-dependent, those are indicated by a star mark. you see all but one of them has probability dimensions. we will discuss more this matter in following sections. more details and descriptions about the absm can be found in appendix a and [28] as well. medicine. combination therapy, i.e., a treatment approach that combines two or more therapeutic agents, is a keystone of cancer therapy. the consolidation of anticancer drugs and therapies enhances efficiency compared to the monotherapy approach, because it targets key pathways in a characteristically synergistic or an additive manner. this approach potentially reduces drug resistance, while simultaneously providing therapeutic anticancer benefits, such as reducing tumor growth and metastatic potential, arresting mitotically active cells, reducing cancer stem cell populations, and inducing apoptosis [15, 29] . in this section, we perform in silico experiments as preclinical tests to design and suggest combination therapies with the use of absm. based on cancer drugs and new findings of cancer biology, we hypothesize relations between model parameters and cancer drugs, and therapies, i.e., we assume each drug in a target group can impact related parameter(s) and show by controlling combination of drugs (controlling parameters); we may simulate combination therapy strategies and control tumor size. having in mind system biology approaches, a survey of cancer therapy literature shows that we may classify five possible groups of target agents in cancer therapies as (a) new vessel formation agents, (b) progrowth signal amplification agents, (c) progrowth signal transmission agents, (d) dna replication-related agents, and (e) cell cycle activation agents. these groups and their relative levels are schematically illustrated in figure 2 . concerning the definitions and roles of the parameters of the absm, we have hypothesized the relations of four model parameters, p 01 , p 02 , age, and nmm, with above groups as is illustrated in table 2 . in absm, the parameters p 01 , p 02 are base probabilities of division of na_1_1 and na_1_2 cells, respectively, and n mm is the probability of production of a na_1_2 from the mitosis of a na_1_1. age is maximum allowable time duration to stay in na_1 mode without proliferation. we see p 01 , p 02 , and nmm are probability numbers; thus, their values can be on [0, 1] interval, where age is time or more precisely number of iterations [28] . progrowth signal transmission, as is depicted in figure 2 , sends division signals to the cell; after the first molecule in a pathway receives a signal, it activates another molecule; this process is repeated until the last molecule is activated and the cell function is carried out. therefore, the assumption that age may be considered as a control tool of the target group c is biologically plausible, i.e., "age" can control the delay of progrowth signal. as a drug in the group b, one may name "trastuzumab", an antibody drug conjugate (adc) consisting of the recom-binant antiepidermal growth factor receptor 2 (her2) monoclonal antibody trastuzumab conjugated to the maytansinoid dm1 via a nonreducible thioether linkage (mcc) with potential antineoplastic activity. the trastuzumab moiety of this adc binds to her2 on tumor cell surfaces; upon internalization, the dm1 moiety is released and binds to tubulin, thereby disrupting microtubule assembly/disassembly dynamics and inhibiting cell division and the proliferation of cancer cells that overexpress her2. all of these mean that it is reasonable to relate parameters such as p 01 p 01 = 0.81 ,p 02 that are related to cell division (proliferation) by any means to drugs like trastuzumab and treat them as tools for controlling the target groups (b, d, and e). in absm, the parameter nmm is related to proliferative cell type and quality; this is why we have attributed it to the target group d. on the one hand, all parameters in absm have exact mathematical definitions and biological interpretations, and on the other hand, all the abovementioned drugs and many others which are used in cancer therapy [35] are validated clinically [30] [31] [32] [33] [34] , so we can treat table 2 as a theoretical deduction which implicitly is supported clinically, i.e., the mapping from treatments to model parameters is implicitly validated. for simplicity and ease of graphical and qualitative analysis, in this research, we only consider combinations of two target groups for therapies, and because we have no delegated parameter in the group a, so the number of sets of two p 02 = 0.75 table 6 . biomed research international groups of four groups, b, c, d, and e, will be six as bc, bd, be, cd, ce, and de. reconsidering these six possible combinations and dismissing repeated parameter sets, i.e., combinations that have same players, we consider three distinct combinations of the groups, bc, bd, and cd, with the attributed players as are listed in table 3 . as we stated earlier, p 01 , p 02 , and nmm are probability numbers, so they range on the [0, 1] interval, and besides considering their definition in absm, they generally repre-sent the probability of (abnormal) proliferation of their respective agents (cells). therefore, a large value, i.e., near unit, means high probability of cell division where a small value (near zero) means low probability. generally speaking, anticancer drugs of the groups b, c, d, and e try to slow cell reproduction via their underlying mechanisms; therefore, it will be plausible if we assume a relation between drug dosage and its effect on cell division probability, i.e., the more the value of drug dosage, the less p 01 = 0.81 table 7 . 7 biomed research international the value of expected probability value will be. because maximum allowable dosage of a drug differs from case to case, we consider qualitative measures high (h), medium (m), and low (l) and assume that these values act against h, m, and l proliferation (division) probability, respectively. in this situation, we are so lucky because probability number interval is known to be on [0, 1] interval. in table 4 , some typical values are listed, although these values are chosen randomly for simulation investigations, but we may attribute them to individuals' variabilities in genes or biological characteristics and may also attribute them to high, medium, and low dosages by some means. this matter will be discussed more in discussion. in this strategy of combination therapies, we assume that therapy agents disturb (target) progrowth signal amplification and progrowth transmission at the same time. we assume that the tumor growth system is the same as the one given in figure 14 (in fact using medical records of a patient, a physician can use absm to simulate existing tumor growth in the patient, i.e., run a simulation like figure 14 and draw a table like table 15 , so she or he can have an estimate of parameters: p 01 , p 02 , nmm, and age). we see this (assumed) given patient has individual characteristics or personalized genetics as p 01 = 0:7, p 02 = 0:5, nmm = 0:2, and age = 1; we set up in silico combination therapy experiments and examine the tumor growth, with controlling the set (p 01 , age) via dosages. the results are depicted in figures 3(a)-3(i) . these figures show tumor structural details qualitatively in the day 12th. in all figures, the light grey represents normal tissue cells, the heavy grey represents outer region of tumor that is comprised of proliferating cells, the middle region of white color is nonproliferative (quiescent) cells, and the black region is necrotic cells. table 5 compares the tumor structures quantitatively; as you see, we have chosen necrotic fraction (nf), pure tumor growth fraction (pgf), and growth fraction (gf) [28] , as quantitative measures to judge the effectiveness of the therapies. as one sees, the size of tumor in this regime (pgf), on day 12 th , is a number between 38 and 40 percent of the considered tissue depending on the doses values, where the harmfulness of it (gf) varies between 40 and 55 percent; in this circumstance, the nf measure, that can be assumed as a therapy effectiveness feature after 12 days, is a number between 37 and 52 percent. as we will see later, the best result of therapy is seen in this set; it is highlighted in in figure 3 (d) and table 5 . another possible combination of players in the bc group is (p 02 , age). we repeat our in silico study as above; the qualitative and quantitative results are depicted in figures 4(a)-4(i) and table 6 , respectively. the size of tumor in this regime (pgf), on day 12th, is around 51 percent of the considered tissue, i.e., larger than the previous one. where the harmfulness of it (gf) varies between 40 and 54 percent, in this condition, nf, that can be assumed as a therapy effectiveness feature after 12 days, is a number between 38 and 51 percent. it seems that this set of players (p 02 , age) is weaker than its counterpart (p 01 , age) of the bc group in the fight against cancer. in this tactic of combination therapy, we assume that therapies' players disturb progrowth signal amplification and dna replication process at the same time. here again, it is assumed that the tumor growth system is the one that considered in figure 14 . tables 7-9 show the tumor structures quantitatively for each set, respectively. regarding table 7 , the size of tumor in this regime (pgf), on day 12 th , is around 60 to 61 percent of the considered tissue. the harmfulness measure of the tumor (gf) varies between 39 and 55 percent; in this condition, nf (the therapy effectiveness feature) is a number between 39 and 52 percent. this set of players has the worst results of fighting against cancer among the all considered sets, where it is highlighted in red color in figure 5 (a) and table 7 . figure 6 and table 8 illustrate results of therapies in the bd group, where (p 01 , nmm) are players. although these players are a bit better than previous players of this group, but they should be categorized as weak players still. another set of players in the bd group is (p 02 , nmm), where their play results against tumor growth are summarized in figure 7 and table 9 . results confirm that we may label these players as weak ones like the other players of the bd group. 3.3. group cd combined therapies. in this approach of combination therapies, we assume that therapy actors disturb progrowth transmission and dna replication process at the same time. for investigating the behavior and properties of this kind of combination therapies, we do the same as the two previous groups. this strategy has three sets of two players: (age, p 01 ), (age, p 02 ), and (age, nmm). it is noteworthy that the effects of players (age, p 01 ) and (age, p 02 ) have been investigated as players (p 01, age) and (p 02 , age) in the bc group beforehand; therefore, we only consider the player set (age, nmm) as the agents of the cd group. qualitative and quantitative measures of therapies, when (age, nmm) act against tumor growth, are summarized in figure 8 and table 10 , respectively. it is seen that although these agents are better in comparison with the bd group teams, but are not as well as bc teams. the tumor size of 56% can be reached after 12 days of therapy, where the score p 01 = 0.81 p 01 = 0.52 p 01 = 0.23 figure 6 : snapshots of tumor structure where therapy regime hits progrowth signal amplification and dna replication process. all tumor structures of cancer system on day 12, but at different dosages (a-i) of a combination therapy. 9 biomed research international of 40% of harmfulness will be realizable (the 52% of therapy effectiveness measure); nf can be accessible by this team. precision medicine and combination therapies can improve the life expectancy of most patients and diminish damages to the tissues surrounding the tumor [36] [37] [38] . in this way, mathematical models and in silico experiments are of great help. in this research, we used absm and hypothesized some relations between the model parameters and anticancer drug (agents) groups; besides, some parameters were attributed implicitly to individual variability. we performed in silico experiments and investigated the effects of some combinations of these parameters as therapy players. here, we analyze and discuss the postulated combination (and precision) therapy strategies and evaluate the results against recent findings in cancer biology and therapies. as stated beforehand, one of the intents of the researchers in the field of oncology is to take into account individual variability for each person (e.g., in genes, environment, and lifestyle) and understand and examine potential role(s) of any combinations of therapies on cancer cells' growth and spread [10, 12, 14, 15, 39, 40] ; in addition, with regard to cancer hallmarks named in introduction, we see that six out of ten hall-marks are related to cell division explicitly or implicitly (hallmark numbers: 1, 2, 4, 6, 7, 9) , and generally speaking (and setting aside environment and lifestyle), cancer is basically a genetic disease of cells; all of these mean that cell division probability (and cell cycle as well) is a genetic, i.e., individual representative. a dose refers to a specified amount of medication (therapy) taken at one time, and dosage is the prescribed administration of a specific amount, number, and frequency of doses over a specific period of time, i.e., a dosage guides a drug regimen. based on therapy type, doses are expressed in different metrics like mass units (e.g., milligrams), drops, and radiations. however, to avoid a special kind of metric or therapy, we use more general metrics (high, medium, and low doses) as it is common in medicine. in this situation, a physician can have his or her own interpretation from high, medium, and low metrics according to underlying cases. in this way, we may attribute [0, 1] interval as a mathematical representation (or normalized quantity) of [low, high] doses (and dosage as well). this issue is illustrated in figure 9 . note that figure 9 illustrates only the mapping between domains, and not the exact relations among variables of different domains; in fact, relationships may be complex and nonlinear. nevertheless, "high, medium, low" approach can be considered table 9 . biomed research international as an alternative for avoiding engagements in complex relations among variables of different domains. to evaluate the best combination therapy strategy, we not only used qualitative graphical structures of the tumors on day 12 th that are depicted in figures 3-8 but also used quantitative measures (pure tumor growth fraction (pgf), growth fraction (gf), and necrotic fraction (nf)) [28] . pgf, that is the fraction of the whole number of tumorous cells to the whole number of all cells in tissue, ncell × ncell, can be treated as a quantitative measure of the tumor size; a less pgf on day 12 th means a smaller tumor. gf, that is the fraction of the population value of tumor proliferative cells to the population value of all tumorous cells, can be treated as a measure of the aggression and invasion potential of tumor; therefore, if two tumors have the same size on day 12 th , we should look at their malignancy and prognosis for their future growth; in this case, gf can be used as a measure; the more the value of gf, the more harmfulness and aggression can be expected. nf, i.e., necrotic fraction, is the ratio of the number of necrotic cells to the whole number of tumorous cells; therefore, in spite of gf, nf can be treated as a measure of table 10 . 11 biomed research international manageability and controllability of the tumor; a larger value of nf on day 12 th means a less dangerous tumor. nf can be treated as a measure that shows how a therapy can prevent tumor cells to be proliferative. one of the main problems in the examining different combinations of drugs (agents) is the huge number of different alternatives [14] , e.g., when trying to identify the best combination of 10 drugs at 3 doses (e.g., high, medium, and low doses), one will have to test 3 10 combinations. in this research, by defining five new features, we introduce a general algorithm for prediction (or suggestion) of the number of possible more (less) effective combinations. the usefulness of this algorithm is that we can try the best expected combinations of therapies at first and consume the time and expenses in clinical trials. it is well established in oncology that different obstructing mechanisms at the same time (compound therapy) might lead to the best results of tumor development prevention [37] . in this circumstance, we expect therapies with more diverse players, i.e., the sets with different players from different groups and parameters that have different interpretations from cancer biology point of view will create more successful pairs of fighters against cancer. in this way, because different physical dimensions usually represent different action mechanisms, we give a positive score to therapy teams which have players with different physical dimensions; to deal with this favorite score, we have introduced a novel metric (dpd) in our research. to introduce our algorithm and attribute the above biological findings to combination sets, we reorganize table 3 as depicted in table 11 and add the following feature numbers to it: the number of different players of the considered target groups (dtg) in a set, the number of same players of the considered target groups (stg) in a set, the number of different mechanisms of parameter (dmp) actions, the number of same mechanism of the parameter (smp) actions, and finally, let us give an additional positive score to the teams with diversity number of the physical dimension (dpd) of the players. as an example for better description of table 11 , in the fourth row, the features of the combined group "cd" have been summarized, i.e., considered groups are c and d. reconsidering tables 2 and 3 , we see that parameters in each three sets, (age, p 01 ), (age, p 02 ), (age, nmm), are all from different target groups, so we attribute the number 2 to dtg feature of each set. we also see that in all three sets, (age, p 01 ), (age, p 02 ), (age, nmm), there is no parameter in common between target groups c and d; therefore, we assign the number 0 to stg feature of each set. regarding the definitions of each parameter in each three sets, (age, p 01 ), (age, p 02 ), (age, nmm), it is clear that they affect the tumor growth by different mechanisms; therefore, we assign the number 2 to dmp feature of each set. as reasoned above, we should give the number 0 to smp feature of each set. it is interesting to note that each parameter in each set of (age, p 01 ), (age, p 02 ), (age, nmm) has different physical dimensions (time or probability); so as you see in the last column of table 11 , we may assign an additional positive score to each team, i.e., the number 2 to dpd feature of each set for the cd group. considering definition of terms, dtg, stg, dmp, smp, and dpd, it is clear that in a considered therapy, the higher the value of a term that begins with letter d and the lower the value of a term that begins with letter s, the better feature that therapy has. inspecting tables 5-10, we can find the best and the worst therapies as follows: the best therapy result belongs to the combined group bc, where p 01 and age are treated as therapies agents; the tumor features of this therapy on day 12 th are highlighted in table 5 . the worst result can be assigned to the combined group bd, where p 01 and p 02 are therapies players; the tumor features of this therapy on day 12 th are highlighted in table 7 . the features of these two therapies are compared in table 11 . it is clear that p 01 and age are players from different target groups (dtg = 2, stg = 0) and affect tumor growth with different mechanisms (smp = 0) and having different physical dimensions (dpd = 2). it is where p 01 and p 02 are players that are common between target groups b and d (dtg = 0, stg = 2) and affect tumor growth with different mechanisms ( smp = 0) and having same physical dimensions (dpd = 0). in this research, the hypothesized algorithm has been discussed for the set of therapies with two players; nevertheless, it can be extended to three and more player sets in the future researches. in fact, the importance and meaning of presented quantitative merits (dtg, stg, dmp, smp, and dpd) are more realizable, when one faces the sets with three or more players. although the presented research can help one to broaden the conclusions drawn from existing medical data, suggest new experiments, test hypotheses, predict behavior in experimentally unobservable situations, but has some limitations that need further investigations. where the value of a parameter as a therapy player may be controlled by dose value, some of parameters may also be related to individuals' genetic contents. in this context, although table 5 represents the best therapy results, but it is seen that the best of the best results is where we set a high dose value to control p 01 and an intermediate value for age; in table 7 , it is seen that the worst results are for high doses for controlling p 01 and p 02 when we deal with a given patient with personal characteristics like table 15 . the implementation of the roles of dose values in our presented algorithm should be more investigated. biological systems are complex; they are composed of several agents and sophisticated interactions (which look to be stochastic) among them. this is where stochastic system biology-based models can help us. in fact, in this circumstance, models such as our absm seems to be more compatible with the biology of cancer than ode or pde ones; in addition, although personalization and relationship between dosages and parameter values in absm look like to be difficult and a challenging issue, but proposed "high, medium, low" approach which is pharmacologically (biologically and therapeutically) plausible seems to be satisfactory; however, this matter should be more investigated in the future researches. one of the current limitations of our model is that we have not considered angiogenesis in it, and therefore, it has no delegated parameters in the target group a. we are hoping to add angiogenesis to absm in our future work. in this research, because the number of model parameters that are attributed to players of different target groups is limited, therefore, we could not investigate all possible combination sets of group players, e.g., be, ce, and de. we think about making a training and teaching software tool, using our proposed model and method. the results of this work should be more explored from clinical applications' point of view, and our research colleagues in cancer institute of iran are looking for more biological validations of our findings by implementing in vitro experiments. an interesting direction for future research can be the investigation of side effects of each combined groups. a side effect is an important issue in cancer drug therapy [38] . it can decrease the life quality of a patient. it seems reasonable that combination therapies that can employ lower doses of each therapy element have the potential to milder side effects. it means that for more precious deductions from tables 5-10, we may introduce a new merit that involves dosedependent side effects of each combined groups. in addition, because side effect, i.e., damage to na_0 agents due to drugs, is not involved in absm, we hope to implement the modified version of the model in the future. our colleagues in the cancer research institute of iran [41] are working on the setting up of statistical studies to test the proposed combination precision cancer therapy. however, although the quality of a scientific field depends on how well the mathematical descriptions developed on the theoretical side agree with results of experiments, possible lack of agreement between theoretical mathematical explanations and experimental measurements often leads to important advances as better theories are developed. it should be noted that absm like other agent-based models may suffer from computational bottleneck for large numbers of cells [27] . this matter should be investigated, and possible use of "hybrid" approaches can be considered in the modified version of absm. treatment schedule and therapies strategies can improve the life expectancy of cancer patients. although precision medicine along with consolidation of cancer therapies, combination therapy enhances efficiency compared to the monotherapy approach, but which combination for which person is the most effective one is an open question. in this study, we presented a method and novel quantitative merits-dtg, stg, dmp, smp, dpd-that an oncologist can use to estimate and predict effectiveness of therapies for each individual and make quantitative rankings among therapies. in silico experiments showed that combined therapies are more effective when players affect tumor via different mechanisms and have different physical dimensions. our absm is founded on four bases: (1) biological assumptions, (2) physical structure, (3) agents and their states, and (4) states transition rules. in absm, host tissue is assumed as a two-dimensional lattice composed of ncell × ncell squares as illustrated in figure 10 . here, each square of the lattice is called a cell. as is shown in figure 10 , we assume that a tumor consists of three layers: (1) the outer layer of proliferative tumor cells (dotted region), (2) the middle layer of nonproliferating or quiescent tumor cells [34] (dashed grey region), and (3) the inner layer of necrotic cells (dark grey core). in the absm, with the physical neighborhood, we mean moore neighborhood as is depicted in figure 10 (b). in the absm, two types of agents (immune and nonimmune cells) are presumed. definitions of agents and their states are summarized in table 12 . each agent represents a biological cell placed at (i, j) coordinate, where 0 < i, j < n − cell. the ia is a moving agent and may accidentally collide with nas. in this paper with the total number of tumor cells, we mean the sum of na_1, na_2, and na_3. in the absm, it is supposed that each ia has the ability to call other immune cells. in absm, na_0 cells play the role of empty places for na_1 or ia cells. for example, the ia cells that are looking for cancer cells or recalled to a specific location may substitute the na_0 cells, i.e., possess na_0 sites. to our knowledge from cancer biology, if na_1 cells are surrounded by more healthy cells, they will access more nutrients and oxygen; it means that the higher is the number of healthy cells, na_0 (empty space) around a proliferative tumor cell (na_1), the greater is the likelihood of division of the na_1 cell. two types of pt cells with different division probabilities are considered. in the first type denoted by na_ 1_1, the cell division is assumed independent from the environmental conditions, i.e., the division probability (ρ pt is constant), while in the second type is symbolized by na_1_ 2, i.e., the cell division probability (ρ pt is a function of its healthy neighbors (na_0s)). in the absm, it is assumed that each na_1_1 may be divided into one na_1_1 and one na_1_2 daughter cells with the probability nmm and into two na_1_1 daughter cells with the probability (1 − nmm). however, it is assumed that each na_1_2 cell can only be divided into two na_1_2 daughter cells. at every simulation time step, the chance of each na_1 (either na_1_1 or na_1_2) for proliferation (division) is checked, i.e., the existence of at least one empty place in the neighborhood of that parent cell is checked. if there is at least one empty space (na_0) in its neighborhood, the na_1 cell will divide with the probability ρ pt into two na_1 daughter cells, so that one of the daughter cells will stay in the position of her parent and the other daughter will place in that empty neighbor. if there is more than one empty place in the neighborhood, the second na_1 daughter cell will pick one of the empty spaces randomly (with the same probability). if the na_1 cell cannot find any empty place in its neighborhood or if it cannot proliferate (with the probability 1 − ρ pt ), it would stay at na_1 state. the maximum of time length at which na_1 can stay without any division is limited to age. if na_1 has no chance for proliferation for a time length (age), then its state will be changed to nonproliferating (quiescence) tumor (na_2). researches indicate that there are many levels of quiescence. various signals such as serum starvation or loss of nutrients, loss of adhesion, and cell contact inhibition at high cell density can induce quiescence to a tumor cell [42] [43] [44] . in the absm, the above fact has been considered by introducing the lifetime parameter age. if a na_2 cell is placed at a radial distance less than the radius of the necrotic core (r n in figure 10 ), then it will turn to na_3 (necrotic cell) at the next time step. if a na_2 cell is at a radial distance less than w p from the external edge of the tumor (see figure 10 ), it may turn to a na_1 cell due to accessing nutrient. the na_3 cells accumulate in the inner zone of the tumor and form a necrotic layer. their state will not ever change to any other states since they are already dead. researchers have found that tumor cells release signals [43] in the host tissue which can be detected by immune cells via specific receptors. the immune cells will receive these signals and search for the tumor cells. for more compatibility of the absm with cancer biology, we introduced pure tumor growth fraction ðpgfðmþþ and growth fraction ðgfðmþþ concepts. pgfðmþ refers to the ratio of the total number of tumor cells at time m to the total number of cells in the studied tissue (equation (a.1) ). gfðmþ denotes to the fraction of at the start of the tumor growth in a tissue, when pgfðmþ is negligible, ias enter the tissue from one corner of the lattice figure 10 , where they have long distance from the center of the tumor. in this circumstance, each ia is assumed to move directly towards the center of the tumor and will take the place of an empty space in its moore neighborhood. this empty space neighbor is one that has the shortest euclidean distance from the center of the tumor. figure 11 shows a typical simulation result of the absm. it can be seen that at time m ≥ 60, the growth fraction factor (gfðmþ) remains almost constant, which means that the necrotic layer is formed and is noticeable. after this time, the ias can walk randomly in any direction to meet tumor cells. in the absm, we implement short-range or contactdependent biological communication [44] . it means that any interaction between ia and na agents takes place only when they are in physical contact, i.e., when they are neighbors. when an ia encounters an na, one of the following events may take place: (1) antitumor action. in this case, the na will fail. if ia_ 1 encounters the na_1, the na_1 cell may die with the probability ρ i (equation (a.3)) and will change to na_0 state at the next iteration in equation (a.3), k dt is tumor death constant;ni i,j and npt i,j are the numbers of ia_1 cells and na_1 cells, respectively, in the neighborhood of the studied cell (cell located at position (i, j)). in other words, the ia_1 will substitute the na_1 which is located in its neighborhood with the probability k dt , and the na_1 will be dismissed from the microenvironment. if there are more than one na_1 in the neighborhood of the ia_1, the ia_1 will kill all na_1 cells and randomly replace one of them. the other na_1 cells will change into na_0. if the collision agent is ia_0, the appropriate rules will apply only to one of the randomly chosen (with a uniform density function) na_1 cells in its neighborhood. (2) null action. in this case, nothing happens, i.e., both agents; na_1 and ia will survive and remain in their states. meanwhile, the ia will continue its search to find another na_1 (3) protumor action. in this case, the ia will fail in killing na_1 and die with probability ρ t (equation (a.4) ). the ia will become an empty space, i.e., the ia will be removed from the tissue and will be replaced by a na_0 where ntðmþ and nptðmþ are the sum of the number of tumor cells and the total number of na_1 cells, respectively, in the studied tissue at time m. in fact, the more immune cells are successful to fight against tumor cells (v − f > 0), the more will be recalled. however, if v − f < 0, there will be no newborn immune cell in the studied tissue. the newborn immune cells will enter from the four corners of the lattice into the tissue randomly. the transition rules of ia will be applied to each of the newborn ias. the basic absm elements and their brief descriptions are summarized in table 1 . as it is deduced from table 1 and above discussions, the absm is a system biology oriented one, i.e., it is constructed from several agents and interactions rules between them and as we will see these elements and interactions can give rise to the function and behavior of cancer growth system. with a conceptual and intuitive look at the elements of table 1 , some of them can be assumed individual-dependent; those are indicated by a star mark. for better realization of our model, we hypothesize possible connections of each parameter to cancer hallmarks and tme follows. the hypothesized relations that are based on biological findings and arguments are listed in table 13 . in this table, the number of hallmarks or tmes is same as the number that was given to them in introduction. for example, in the first and second columns of the first row of table 13 , we have listed the possible effect of the valve of parameter p 01 in cancer hallmarks and possible tme players which are delegated by p 01 ; here, the number 1 means that we hypothesize that parameter p 01 will influence the hallmark number 1 (unlimited multiplication) and tme player number 1 (fibroblasts and myofibroblasts) as well. here, our approach is based on our interpretations from oncology literature, following discussions, and in silico experiments. as we mentioned earlier, fibroblasts and myofibroblasts are the number one component of tme. researches have confirmed that only the activated fibroblasts are required to initiate and endorse tumor growth [44] [45] [46] [47] . when the fibroblasts remain activated after the initial insult has regressed, these activated fibroblasts may work with other molecular pathways to boost neoplasm initiation, where it has a significant impact on cancer progression through remodeling ecm, inducing angiogenesis, employing inflammatory cells, and directly stimulating cancer cell proliferation via the secretion of growth factors, immune suppressive cytokines, and mesenchymal-epithelial cell interactions [48, 49] . experimental evidences prove that neuroendocrine cells, the number two component of tme, exhibit a combination of neuronal and endocrine features [50] . neuroendocrine cells are the accomplices of tumor formation [51] . it is proven that the neuroendocrine system strongly influences the function of the immune system. neuroendocrine cells could stimulate the proliferation of prostate carcinoma cells and increase their aggressiveness [52] , while in the development of neuroendocrine cell tumors, they may play a principal role [53] . some features of adipose tissue, the number three component of tme, are associated with cancer. adipose cells secrete more than 50 different cytokines, chemokines, and hormone-like factors [54, 55] . these factors, whose production may upregulate in obesity, may be assistants in tumor initiation. obese adipose tissue hypoxia establishes a highly proinflammatory microenvironment, which is more likely to breed tumors [56] [57] [58] . adipose tissue reprogramming and the associated systemic secretion may have an effect on cancer growth and progression [59] . excess adiposity leads to high circulating blood estrogen [60] and chronic, lowgrade inflammation, which is involved in cancer development, a major energy source, relating with inflammation, recruiting immune cells, and supporting vasculogenesis [54, 61, 64] . the mammalian immune system monitors tissue homeostasis to protect against invading infectious pathogens and to eliminate damaged cells [62] . findings recommend that immune surveillance has significant roles in recognizing and eradicating a large part of emerging tumor cells [2] . however, unlike normal functions, immune inflammatory cells, the number four component of tme, would persist in sites of chronic inflammation, linked to diverse tissue pathologies, including fibrosis, aberrant angiogenesis, and neoplasia [63] . recent discoveries in immune system research illuminate that it is difficult to ignore the crucial issue that immune inflammatory cells may be the early support structure of cancer, having a double effect on tumor formation [64] [65] [66] [67] [68] [69] [70] . blood and lymphatic vascular networks, the number five component of tme, help tumor cells escape immune surveillance in the process of tumor progression. escape measures are mainly divided into two categories. directly, the lymphatic microenvironment will weaken or eliminate the normal function of immune cells. for instance, the myeloidderived suppressor cells could restrict the normal operation of t cells [71] [72] [73] . when the metastatic tumor enters a new environment, cd4 + and cd8 + t cells may help them to evade the host immune system [74, 75] . indirectly, the remodeling of unusual endothelial venules can influence immune cells to move into the lymph nodes [76] . ecm, the number six component of tme, is a dynamic and complicated environment. evidences show that ecm can help neoplasms to get away from immune surveillance [77] . ecm contains all the cytokines, growth factors, and hormones secreted by stromal and tumor cells. a model indicated that ecm heterogeneity is crucial for controlling collective cell-invasive behaviors and determining metastasis efficiency [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] . ecm may affect tumors through extracellular secretion. in addition, ecm may alter the phenotype type of stromal cells or tumor cells [88] . the ecm of tumor will provide a hypoxic or acidic environment in which the tumor cells have greater survival advantages than normal cells. ecm will select survival cells to aid in tumor growth and invasion at the fastest rate. using matlab codes, the absm was implemented. in following simulations, at first, it is supposed that the entire tissue (lattice in figure 10 ) is covered with na_0s. the state of one cell in the center of the tissue is changed into na_1 at time m = 0. the 0.1% of total number of cells (i.e., 0:001 × ncell × ncell), where ncell = 100, in the studied tissue, are entered as ias from a corner of the lattice randomly. figure 12 shows the initial condition for a typical simulation, where table 14 depicts typical value of parameters. the absm was evaluated qualitatively and quantitatively considering the following criteria: the average radius of the external edge of tumor at iteration mðr t ðmþþ, the average radius of the necrotic zone at iteration mðr n ðmþþ, growth fraction at iteration m ðgfðmþþ, necrotic fraction at iteration m ðnfðmþþ, and the number of na_1, na_2, na_3, and ia cells at iteration m. besides, the graphical illustration of the growth is used as qualitative evaluation in some cases. figure 13 shows the number of na_1, na_2, na_3, and ia vs. iterations. it is seen that the number of ia enhances due to recruitment which is qualitatively consistent with cancer biology. in this simulation, although ia cells enter the tissue to fight against tumor cells, but the permanent presence of 400 cells of na_1 in tissue is observed. figure 14 compares the size of the simulated tumor growth using the absm to an in vivo experimental result [90] , where the model parameters are as table 15 . the reported in vivo data is for balb/c mice that have been involved with ct26-tk cells. the sizes were normalized in order to check the results more accurately. the simulation starts at time m = 6 and iteration time step is 2-hours. it is seen that the simulation results using the absm are consistent with the reported in vivo result, that is, the absm can quantitatively simulate a real tumor growth. quantitative measures are listed in table 5 . quantitative measures are listed in table 8 . no data were used to support this study. bioengineering models for breast cancer research hallmarks of cancer: the next generation use of big data in drug development for precision medicine: an update targeting the epithelial to mesenchymal transition in glioblastoma: the 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carcinoma epstein-barr virus-encoded mir-bart6-3p inhibits cancer cell metastasis and invasion by targeting long non-coding rna _loc553103 epstein-barr virus encoded mir-bart11 promotes inflammation-induced carcinogenesis by targeting foxp1 genome-wide analysis of epstein-barr virus (ebv) integration and strain in c666-1 and raji cells ebv-mir-bart10-3p facilitates epithelial-mesenchymal transition and promotes metastasis of nasopharyngeal carcinoma by targeting btrc upregulated long non-coding rna linc00152 expression is associated with progression and poor prognosis of tongue squamous cell carcinoma afap1-as1, a long noncoding rna upregulated in lung cancer and promotes invasion and metastasis hyou1, regulated by lplunc1, is up-regulated in nasopharyngeal carcinoma and associated with poor prognosis enhanced invasion of metastatic cancer cells via extracellular matrix interface long non-coding rnas function as competing endogenous rnas to regulate cancer progression sulfated glycosphingolipid as mediator of phagocytosis: sm4s enhances apoptotic cell clearance and modulates macrophage activity toward an ising model of cancer and beyond necrotic tumor cell death in vivo impairs tumor-specific immune responses special thanks are due to dr. alizadeh (cancer biology research center, cancer institute of iran, tehran university of medical sciences, tehran, iran) and dr. esmati (department of radiotherapy, imam khomeini hospital, tehran university of medical sciences) for their valuable guidance and advice. the author declares that there is no conflict of interest regarding the publication of this paper. key: cord-265260-n6wm54wz authors: cuong, hoang quoc; hai, nguyen duc; linh, hoang thuy; anh, nguyen hoang; hieu, nguyen trung; thang, cao minh; thao, nguyen thi thanh; lan, phan trong title: comparison of primer-probe sets among different master mixes for laboratory screening of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) date: 2020-09-25 journal: biomed res int doi: 10.1155/2020/7610678 sha: doc_id: 265260 cord_uid: n6wm54wz background: there is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) diagnosis and a surge of sars-cov-2 cases, especially in limited-resource settings. therefore, the combination of an optimal assay kit is necessary. methods: we compared the ability to screen sars-cov-2 among three primer-probe sets in two different master mixes, invitrogen™ superscript™ iii one-step rt-pcr and lightcycler multiplex rna virus master. results: the assay with tib-molbiol, idt, and phu sa sets for lightcycler multiplex rna virus master or invitrogen™ superscript™ iii one-step rt-pcr showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. r squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. conclusions: our findings indicated that tib-molbiol, idt, and phu sa primer-probe sets could be beneficial for the laboratory screening of sars-cov-2 by rt-qpcr assay of e gene. there is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for covid-19. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has a threat to human health which involves over 7,273,958 confirmed cases and 413,372 deaths [1] . while waiting for the coronavirus vaccine approval, molecular testing for sars-cov-2 is one of the important strategies to prevent and reduce the rate of infection by case identification, isolation, social distancing, and proper treatment [2, 3] . many factors are leading to the low sensitivity of sars-cov-2 such as (a) the detection that depends on the location of clinical specimens, (b) low patient viral load, (c) sporadic shedding, and (d) discrepancy in detection kits from various producers [4, 5] . however, the molecular diagnosis of sars-cov-2 using rt-qpcr assay is a gold standard method [6] [7] [8] . consequently, the combination of an optimal assay kit is necessary because of the shortage of chemical reagents for sars-cov-2 diagnosis and the surge of sars-cov-2 cases, especially in limited-resource settings. in the present study, we aim to analyze the commonly used primer-probe sets, targeting e gene of sars-cov-2 by the rt-qpcr assay for laboratory screening to increase testing capacity in the context of thousands of overseas travelers returning to their countries. 2.1. primer-probe information. in this study, these three primer-probe sets based on the sequence information received from three different companies, tib-molbiol (berlin, germany), idt (integrated dna technologies, skokie, illinois, usa), and phu sa (phu sa biochem, vietnam), were used for comparative analysis [8] (supplementary table 1 ). the infection assays were performed in a biosafety level 3 laboratory. vero e6 cell lines were infected with a clinical isolate sars-cov-2 [9] . after 72 hours, the virus medium was inactivated at 65°c for 1 hour. viral rna was then isolated from the culture medium using the qiaamp viral rna extraction kit (qiagen, hilden, german) following the manufacturer's instructions. the copy number of rna extracted from sars-cov-2 strain was estimated through a standard curve, which was published in a previous study [10] . sars-cov-2 strain isolated in this study is mt192773. the length of the genome sequence was 29,890 bp without gaps and high coverage 1,897×. this strain belonged to betacoronavirus b type and of 99.98% sequence similarity at the nucleotide level which was isolated in wuhan (mt019529) and >90.56% similarity with sars-cov isolated from pangolin (epiisl410721). there have been four mutations classified as nonsynonymous mutations such as g8388a (serine to asparagine), a8987t (isoleucine to phenylalanine), and c10232t (arginine to cysteine), and a synonymous mutation is g3778a, which was published in a previous study [9] . rna-extracted specimens from the inactivated virus were tested for comparative assay of sars-cov-2 by rt-qpcr on a lightcycler 480 or abi 7500 system following the manufacturer's protocol (invitrogen™ superscript™ iii one-step rt-pcr system or lightcycler multiplex rna virus master). in this study, the reaction combination was prepared by multiplying the volumes of each reagent in table 1 . rt-qpcr conditions were applied in the present study with details described in table 2 . a cycle threshold value (ct value) of ≥40 was defined as a negative test [8] . 2.4. analysis. in this study, data were entered using epi-data version 3.1 (epidata association, odense, denmark, 2005), and all statistical analyses were performed using stata version 13.0 (statacorp, tx, 2013). the results were summarized using means and standard deviation (sd) for continuous variables. linear regression analysis was performed to estimate the r square. amplification efficiency (ae) was calculated using the equation ae = −1 + 10 ð−1/slopeþ [11] . in this study, the assay with tib-molbiol, idt, and phu sa sets for lightcycler multiplex rna virus master showed positive results from a single reaction of triplicate in the three days of 4.8 copies/reaction ( table 3) . the ct values (mean ± sd) of e gene (tib-mobiol), idt, and phu sa at 1 : 10 8 ½ were 39:02 ± 0:34, 39:17 ± 0:34, and 39:23 ± 0:10, respectively. r 2 value from tib-mobiol, idt, and phu sa showed equal values of 0.97. similarly, the ae of each set was also showing the same value, for the figures of tib-mobiol and idt were 107, and phu sa was 108 ( table 4) . the assay with tib-molbiol, idt, and phu sa primerprobe sets for the invitrogen™ superscript™ iii one-step rt-pcr system exhibited positive results from a single reaction of triplicate in the three days of 4.8 copies/reaction (table 5 ). furthermore, these three primer-probe sets showed the equivalent sensitivity in low concentrations for lightcycler multiplex rna virus master. in this study, we found that the r-square values from tib-molbiol, idt, and phu sa were the same (0.97). similar to the results of lightcycler multiplex rna virus master, the ae of idt and phu sa were also the same (108) and 107 for tib-molbiol (table 6 ). in this study, we reported the comparative analysis of laboratory screening for sars-cov-2 among three primer-probe sets in two different master mixes (invitrogen™ superscript™ iii one-step rt-pcr and lightcycler multiplex rna virus master). the initial analysis showed that the combination of tib-molbiol, idt, and phu sa primer-probe sets was quite sensitive to positive results (4.8 copies/reaction) among invi-trogen™ superscript™ iii one-step rt-pcr system (table 7 ). in terms of lightcycler multiplex rna virus master, tib-molbiol, idt, and phu sa primer-probe sets also showed the same sensitivity (4.8 copies/reaction). the r square of each primer-probe set among the different master mixes was around 0.97, and the findings were compatible with a previous study [12] . also, the values of ae in each primer-probe set among different master mixes reach the accepted criteria of ae ranged from 90 to 110% [11] . these findings could also contribute to gain better understandings of the combination of the best reagents for sars-cov-2 screening to select the most optimum reagents to effectively halt covid-19. the need for the optimum strategies which is aimed at enhancing the testing capacity is a prerequisite because there has been an increasing figure of false-positive results of rt-qpcr that were reported in the performances of sars-cov-2 diagnosis for recovering patients and asymptomatic infected patients [3] . several recent studies reported the analytical sensitivity and efficiency comparisons of sars-cov-2 detection by several molecular assays with multiple primerprobe sets [3, [12] [13] [14] . although rt-qpcr is appropriate for the large-scale diagnosis of viral infection in normal viral load specimens, the rt-qpcr performance of each primerprobe set is different from others, and various primer-probe sets have contextual amplification with sars-cov-2 negative nasopharyngeal swabs [4] , resulting in the inconclusive results. because of the different performances of each primer-probe set in rt-qpcr, the optimization for the design or selection of the proper primer-probe set, the 2 biomed research international [3] . on the other hand, rt-qpcr assays targeting sars-cov-2 depended on the high similarity of sars-cov-2 to sars-cov as cross-react occurred. furthermore, there is less sensitivity of the assay when collecting specimens in the early time points after admission [12] . consequently, almost all laboratories should locally validate diagnostic sensitivities and limit of detection values when beginning these assays [13] . a recent study showed that the droplet digital pcr method could diminish the inaccurate results in the low viral load specimens compared to rt-qpcr [15] . however, the possible false-positive results or false-negative results are arising from the current standard rt-qpcr detection of sars-cov-2; therefore, the improved choice of clinical practice could be a comprehensive approach about molecular diagnosis, x-ray or computed tomography scan, and serology test, along with the decision by experienced clinicians in place of exclusively depending on rt-qpcr [15] . several previous studies have also shown that the selection of the best primer-probe sets and equipment for sars-cov-2 screening and diagnosis was an urgent and important solution for prevention and control covid-19 [12, 14] . our findings found that these primer-probe sets in two different master mixes were sensitive and reliable for laboratory screening of sars-cov-2. hence, these primer-probe sets could be beneficial for the laboratory screening of sars-cov-2 by rt-qpcr assay of e gene. it is crucial to improve the capacity of suspected case screenings and to reduce the affected performance of the testing that yield false-negative results [16] . the results of this study are a prelude for other studies to improve the testing capacity of screening suspected cases. our findings indicate that tib-molbiol, idt, and phu sa primer-probe sets could be beneficial for the laboratory screening of sars-cov-2 by rt-qpcr assay of e gene. there is a need for considering the combination of these reagent sets as a new strategy to increase the testing capacity for screening programs for covid-19. quantitative reverse transcription pcr sars-cov-2: severe acute respiratory syndrome coronavirus 2 covid-19: corona virus-infected diseases 19. the data used to support the findings of this study are available from the corresponding author upon request. the authors have no conflicts of interest. world health organization feasibility of controlling covid-19 outbreaks by isolation of cases and contacts risk-adapted treatment strategy for covid-19 patients evaluation of who listed covid-19 qpcr primers and probe in silico with 375 sers-cov-2 full genome sequences, medrxiv comparative performance of sars-cov-2 detection assays using seven different primer/probe sets and one assay kit identification of a novel coronavirus in patients with severe acute respiratory syndrome a one step quantitative rt-pcr for detection of sars coronavirus with an internal control for pcr inhibitors detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr clinical features, isolation, and complete genome sequence of severe acute respiratory syndrome coronavirus 2 from the first two patients in vietnam development of standardized specimens with known concentrations for severe acute respiratory syndrome coronavirus 2 realtime-rt-pcr testing validation amplification efficiency: linking baseline and bias in the analysis of quantitative pcr data comparative analysis of primer-probe sets for the laboratory confirmation of sars-cov-2 analytical sensitivity and efficiency comparisons of sars-cov-2 rt-qpcr primer-probe sets comparative performance of sars-cov-2 detection assays using seven different primer/probe sets and one assay kit, medrxiv analytical comparisons of sars-cov-2 detection by qrt-pcr and ddpcr with multiple primer/probe sets the effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5′ nuclease assay we would like to thank colleagues at pasteur institute, ho chi minh city for supporting the study. thanks are due to mr. alan tan, sydney, australia, for editing and proofreading the manuscript. supplementary table: information of primers and probes were used in this study. (supplementary materials) key: cord-330276-qvmhuid3 authors: giorgi, gabriele; leon-perez, jose m.; pignata, silvia; topa, gabriela; mucci, nicola title: addressing risks: mental health, work-related stress, and occupational disease management to enhance well-being 2019 date: 2020-06-19 journal: biomed res int doi: 10.1155/2020/1863153 sha: doc_id: 330276 cord_uid: qvmhuid3 nan the importance of a contextualized health approach with a focus on organizational environments is becoming more strategic than ever due to covid-19 and the ensuing difficult situation that employees are experiencing worldwide. the prevention of workers' mental health problems is complex and multidimensional, and it is not always possible to protect the person by analyzing personality, psychopathology, and psychiatric syndromes. accordingly, a peer review process involving international experts, with 21 papers accepted in this special issue, considered the important concept of work contextualized health. from this perspective, this special issue had the power to establish a dialogue between the multiple disciplines completing the majority of research on mental health constructs within clinical, neuroscientific, and psychiatric contexts, which usually led to a person-centered analysis or research conducted in artificial laboratory settings. as stated by giorgi et al. [1] , trauma and diseases related to stress and mental health that originate in the workplace may have a different pattern of development or require an organization-centered treatment approach, including field and intervention studies. in addition, this special issue followed the united nations agenda for developing 17 sustainable goals by 2030 [2] and tried to contribute to two of these goals: (1) promoting well-being at all ages, including mental health, and (2) promoting safe and secure working environments to create a decent work for all. in doing so, this special issue assumed that addressing such goals required an interdisciplinary approach involving scientific fields ranging from occupational medicine to organizational psychology. regarding the promotion of well-being at all ages, g. giorgi et al. concluded in their narrative review that stress management strategies at work need to include "aging" as a crucial variable to tailor interventions and prevent workers' cognitive impairment processes. also, m. ziarko et al. pointed out the necessity to consider the health and wellbeing of workers with chronic disease. in particular, their paper analyzed the mental health consequences of the type of treatment received by 85 participants affected by rheumatoid arthritis and confirmed the assumption that pain intensity, coping strategies, and ego resiliency depend on the severity of their levels of anxiety and depression. similarly, two papers emphasized the need to consider all agents involved in an organization, including students, as targets of safety and well-being measures. k. gerreth et al. analyzed the anxiety of dental students during their first clinical class involving performing a prophylactic procedure in a pediatric patient. their results indicated that more than 51% of students reported high levels of anxiety. these findings emphasize the need for students to be trained to deal with stress as a part of their academic curriculum. in addition, k. frömel et al. explored whether students reporting academic stressors differ in physical activity after school compared to those students that did not report being exposed to academic stressors. although their hypotheses were not supported, it seems that gender should be taken into account when promoting physical activity to reduce stress: girls in the academic stressor group walked more (steps/hour measured with accelerometers) than girls in the nonacademic stressor group. also, their study is a good example on how new devices such as accelerometers can be used to collect information in occupational health and safety research. with regard to promoting safe and secure working environments to create a decent work for all, some papers published in this special issue introduce advances in measuring psychosocial risk factors, mental health, and work-related issues. for example, n. tao et al. conducted a study in which they analyzed the relationship between occupational stress and secretory immunoglobulin a (siga) in a sample of 625 military recruits during their basic military training period. as expected, siga measured in saliva and quantified by enzyme-linked immunosorbent assay presented higher levels in the high occupational stress group than that in the low stress group. furthermore, the salivary siga level was also associated with perceived personal strain. another work using innovative approaches to assess psychosocial risks and their consequences is the paper by j. r. lópez-garcía et al. they proposed using bayesian networks to determine the probability of an occupational accident in a certain productive sector depending on the relationship between ergonomic and psychosocial factors. they used data from a national survey of working conditions in spain (n = 8,892) to illustrate their approach. their results suggest that ergonomic risks associated with physical strains and a lack of job satisfaction are associated with a higher probability of being involved in an occupational accident. in contrast to these new approaches, traditional approaches to conduct psychosocial risk assessments are based on self-rated scales. in that sense, it is important to validate well-known scales to facilitate cross-cultural comparisons. this is the case of the study conducted by a. s. n. isha et al. who validated the copenhagen psychosocial work environment questionnaire (copsoq) in malaysia. they also proposed the inclusion of physiological measures (blood pressure and body mass index) to monitor workers' health. similarly, v. katsari et al. validated the jefferson scale of patient perception of physician empathy in greece, which may be useful for monitoring both physicians' health and the quality of service that they provide. a noteworthy aspect of this study was the comparison between self-rated empathy and their patients' ratings. a similar approach was followed by i. schneider et al. in their research on the degree of agreement between self-rated and observer-rated occupational psychosocial risks. they compared the ratings of workers and occupational safety and health committees to occupa-tional psychosocial risks measured with the same instrument (n = 669). their findings showed that observer ratings and self-ratings provided comparable results. therefore, they concluded that (a) the observer rating approach is especially suitable for small-to-medium enterprises that do not have access to a large anonymous survey assessment and (b) aggregation of item means at the group level is justified because their results showed a reasonable agreement and excellent reliability in workers' self-ratings, and therefore, the self-rating approach can be very useful for large enterprises. another way to improve existing scales to measure psychosocial risks at work is to add relevant dimensions that are associated with employees' health and well-being. in that sense, the work by k. kowalczuk et al. attempted to identify the most arduous and frequently occurring burdens in nursing workplaces. they found that ward type predicted the level of work arduousness beyond other factors such as age or gender, suggesting that trauma and diseases related to stress and mental health that originate in the workplace may have a different pattern of development or require an organizationcentered treatment approach that complements the personcentered approach derived from research conducted in clinical and psychiatric contexts. in a similar vein, m. martini et al. highlighted the importance of including both demands and support derived from interactions with students when conducting psychosocial risk assessments in higher education. with a sample of 550 professors from a large public university, their results revealed that relationships with students can play a crucial role in how academics experience emotional exhaustion and engagement at work. also, findings from the study conducted by m. del mar molero jurado et al. in the education sector reaffirm that burnout is a pivotal psychosocial risk that requires prevention within the sector. they proposed that measures to prevent burnout need to consider the educational context when implementing preventative actions both at the individual (i.e., increasing self-efficacy) and organizational level (i.e., improving the education system). moreover, organizational level measures should include the promotion of healthy behaviors as emphasized by research on public health initiatives to prevent noncommunicable diseases [3] . this is clearly exemplified in the study by a. habib et al. who analyzed the risk factors of noncommunicable diseases in a sample from saudi arabia (n = 1,070) . their findings revealed the need to promote healthy behaviors as a suitable public health strategy to reduce noncommunicable diseases such as cardiovascular disease or diabetes. in addition to these potential measures to promote health and well-being, the literature has indicated that active coping and recovery from work are crucial to avoid stress-related problems [4, 5] . in that sense, the paper by y. hsu et al. reported that working more hours was associated with higher levels of occupational stress, which was related to lower levels of work-family balance and job satisfaction. they found that perceived control over time plays a protective role because it was associated with increased recovery-related self-efficacy. in addition, a focus on coping strategies by x. wang et al. revealed that 2 biomed research international depressive symptoms in military institutions is a matter that needs to be considered, as they found that the relationship between coping (i.e., hardiness) and depressive symptoms is mediated by motivational dispositions. addressing psychosocial risks and introducing preventive measures at work are equally important as identifying who is exposed to the risks and what are the potential negative consequences on employees' health and well-being. first, a. przystanska et al. explored the psychosocial predictors of bruxism. they concluded that perceived stress is a crucial somatic factor in the occurrence and maintenance of awake bruxism. second, k. golonka et al. went beyond the usual negative effects of burnout and explored potential brain activity differences between burned-out and nonburned workers (control group). their results suggest that participants in the burnout group showed cortical hyperactivity, which results in reduced alpha power compared to participants in the control group. finally, t. mitake et al. analyzed the stigma related to mental illness in the workplace, such as the psychological consequences derived from burnout. this relationship is important to examine because being stigmatized at work due to mental illness can result in experiencing discriminative behaviors. following the abovementioned findings, another factor that deserves special attention to create a decent work for all is the promotion of working environments free from discrimination and violence, including sexual and psychological violence (i.e., sexual harassment or workplace bullying). the paper by s. a. jahnke et al. addressed the prevalence of chronic work discrimination and the harassment of women firefighters (n = 1,773) and its psychosocial consequences. their results revealed that a considerable percentage of women firefighters reported that they had experienced verbal harassment (37.5%) and unwanted sexual advances (37.4%) in their fire service work. furthermore, this discrimination and harassment at work were related to increased alcohol consumption and mental health problems, including depressive symptoms, anxiety, and posttraumatic stress symptoms. similarly, s. berlanda et al. analyzed the experiences of violence (emotional, physical, and sexual) perpetrated by patients and visitors against healthcare professionals working in emergency units. they found that greater age and higher scores in secure attachment are associated with reduced experience of emotional violence from patients and visitors, and the relationship between secure attachment and the amount of patient-and-visitor-perpetrated emotional violence experienced is mediated by levels of job satisfaction. finally, with regard to the ongoing situation and the economic crisis caused by the covid-19 pandemic, employee welfare and social support may not be the current priorities for companies as they attempt to maintain their survival by staff layoffs and budget reductions [6, 7] . moreover, studies have shown that turbulent economic periods, in which job uncertainty is the norm, create a fertile soil for the increase of violence at work and stress-related mental health problems [6, 8] . in this regard, s. de sio et al. studied the role of job insecurity in the perception of psychosocial risks at work in a sample of 338 administrative technical workers and found that workers with temporary contracts perceived higher exposure to psychosocial risks at work than their colleagues with permanent contracts. the contributions to this special issue highlight the essential need to consider organizational practices and culture in the management of mental health problems linked to the workplace as organizational causes are often more harmful than individual antecedents. raising awareness of the organization's intervention politics, of organizationworker health relationships at work, and of an organizational science of mental health appears necessary. overall, the manuscripts included in this special issue reported the perspectives of 123 authors, reflecting a valuable cross-cultural point of view on health prevention and promotion. in conclusion, we would like to share a reflection on what we have seen during the covid-19 pandemic as workers' mental health still represents a point of fragility of the systems-countries, where an overly medicalized and pathologizing model of mental health risks hiding not only organizational causes and responsibilities creating an image of stigmatized workers but also hiding potential and successful organizational interventions in prevention, safety, and health areas. there is therefore a disharmonious relationship between business and health while, as strongly supported by the business@health laboratory of the european university of roma, there is no business without employee health, and in the same way, employee health becomes business. addressing risks: mental health, work-related stress, and occupational disease management to enhance wellbeing sustainable development goals knowledge platform social determinants and noncommunicable diseases: time for integrated action recovery from job stress: the stressor-detachment model as an integrative framework after work is done: psychological perspectives on recovery from work fear of nonemployability and of economic crisis increase workplace harassment through lower organizational welfare orientation psychosocial factors and economic recession: the stormont study going beyond workplace stressors: economic crisis and perceived employability in relation to psychological distress and job dissatisfaction the editors declare that they have no conflicts of interest regarding the publication of this special issue. silvia pignata gabriela topa nicola mucci key: cord-298185-w37nvorf authors: cao, kai; kline, brad; han, ying; ying, gui-shuang; wang, ning li title: current evidence of 2019 novel coronavirus disease (covid-19) ocular transmission: a systematic review and meta-analysis date: 2020-10-24 journal: biomed res int doi: 10.1155/2020/7605453 sha: doc_id: 298185 cord_uid: w37nvorf objective: to estimate the prevalence rate of ocular symptoms and the positive rate of conjunctival swab samples of patients diagnosed with 2019 novel coronavirus disease (covid-19). methods: we performed a systematic review and meta-analysis. a comprehensive literature search was done based on pubmed, embase, medrxiv, and the cochrane library. the primary outcomes are the prevalence rate of conjunctivitis/conjunctival congestion and the positive rate of conjunctival swab samples. rates were expressed as proportions with 95% confidence intervals (cis). results: a total of 12 studies with 1930 participants were included for meta-analysis. the pooled prevalence rate of conjunctivitis/conjunctival congestion was 8% (95% ci: 5%-12%). 1% (95% ci: 1%-4%) of covid-19 patients were diagnosed with conjunctivitis/conjunctival congestion as the initial symptom. the pooled positive rate of conjunctival swab samples was 3% (95% ci: 2%-5%). we also assessed other ocular symptoms reported in the 12 studies, including foreign body sensation, increased secretion, and eye itching. the pooled prevalence rates were 6% (95% ci: 3%-10%), 10% (95% ci: 8%-12%), and 9% (95% ci: 7%-10%), respectively. conclusions: the evidence on the positive rate of conjunctival swab samples and the prevalence rates of ocular symptoms indicated that covid-19 ocular transmission was possible but less likely. humans have battled viral infections throughout history. from measles and smallpox outbreaks to the 1918 influenza pandemic, countless lives have been lost. in december 2019, a viral pneumonia of unknown etiology quickly spread worldwide. the world health organization declared the disease 2019 novel coronavirus disease a global pandemic that is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov2) [1] . through 8 sep 2020, the cumulative number of infected people stood at more than 27.25 million globally with over 891,285 deaths. during the covid-19 pandemic, two cases drew the attention of ophthalmologists. in one, a chinese ophthalmologist, dr. wenliang li, who raised the earliest alarm about covid-19 in china, unfortunately died from this disease. it is not known whether contacting the eyes of patients who had contracted covid-19 played a role in his own infection [2] . in another case, dr. guangfa wang, a chinese pneumonologist, was infected with covid-19 in wuhan while wearing full personal protection equipment (ppe) at all times except goggles. he complained of redness of the eyes and speculated that the virus might have attacked him via the ocular surface [3] . through today, whether covid-19 can transmit through the ocular surface is still controversial. herein, we performed a comprehensive systematic review and meta-analysis to quantitatively analyze current evidence on covid-19 ocular transmission. criteria. four english electronic data sources (pubmed, embase, medrxiv, and the cochrane library) were comprehensively searched following prisma guidelines (from 1 december 2019 to 7 sep 2020), and only studies in english would be screened. the following search terms and their combinations were used: "covid-19," "2019-ncov-2," "coronavirus," "sars-cov-2," "novel coronavirus," "ocular manifestation," "ocular symptom," "conjunctival swab," "conjunctivitis," "conjunctival congestion." the eligibility of studies was independently determined by two authors, and discrepancies in study selection were resolved by team discussion. all studies included in the metaanalysis met the following criteria: (1) the study population consisted of covid-19 patients; (2) covid-19 patients were diagnosed using a laboratory method; and (3) at least one of the two primary outcomes (conjunctivitis/conjunctival congestion, positive conjunctival swab samples) was assessed, and the number of events was reported. studies were excluded if (1) they included no data on ocular manifestations and no detection of viral rna in conjunctival swab samples; (2) they involved animal or in vitro research; or (3) the study design was a small case report, letter, editorial, or review. a predefined excel form was utilized for data collection. the following information was extracted: the first author's name, publication year, country or area where the study was conducted, sample size, mean age of subjects (in some studies, only median age or range of age was reported), and the method used to diagnose covid-19. most importantly, we extracted the number of events of ocular symptoms (conjunctivitis/conjunctival congestion, foreign body sensation, increased secretion, and eye itching) and the number of positive viral rna detections in conjunctival swab samples. any disagreement was resolved by consensus. analysis. the pooled prevalence rates of ocular symptoms, such as conjunctivitis/conjunctival congestion, were expressed using proportions with 95% confidence intervals (cis) estimated from either a fixed-effect model or a random-effect model. model selection was decided by the heterogeneity across included studies. the i 2 statistic was used to measure the heterogeneity quantitatively. i 2 describes the percentage of variability that was caused by heterogeneity rather than chance. if the i 2 was below 50%, a fixed-effect model was applied, otherwise a random-effect model was used [4] . the egger's test was used to check publication bias [5] . sensitivity analysis was performed to explore the potential source of heterogeneity, and pooled estimations by sensitivity analysis were reported as the final results. a two-tailed p < 0:05 was considered statistically significant. all statistical analyses were conducted with the open source r software, version 4.0.0. a total of 734 articles were identified after an initial database search. of these, 258 duplications were removed and another 441 publications were further excluded by screening the title and abstract. subsequently, 35 full-text records were evaluated for eligibility. after screening the full text, 23 articles were excluded ( figure 1 ). finally, 12 studies [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] involving 1930 participants were included for meta-analysis. table 1 describes the detailed characteristics of the included studies. eight studies were carried out in china and one in iran. the sample size varied from 30 to 534. most studies enrolled patients with a mean age of around 50 years old. the assessed ocular symptoms included conjunctivitis/conjunctival congestion (11 studies), foreign body sensation (six studies), increased secretion (three studies), and eye itching (five studies). all included studies used reverse transcription-polymerase chain reaction (rt-pcr) to make a diagnosis of covid-19. six studies reported the number of positive conjunctival swab samples. symptoms. the pooled prevalence rate of conjunctivitis/conjunctival congestion estimated by the random-effect model was 8% (95% ci: 5%-12%, figure 2 ) because the heterogeneity was relatively large (i 2 = 84%, p < 0:01). we further performed a sensitivity analysis. after the study of wu et al. in 2020 [15] was omitted, the i 2 dropped sharply, indicating that the study of wu et al. in 2020 was the source of heterogeneity. the reason was that wu et al. took all the following symptoms into account when defining conjunctivitis: conjunctival hyperemia, chemosis, epiphora, and increased secretion. this resulted in a much higher prevalence rate of conjunctivitis/conjunctival congestion than those of the other studies. the pooled prevalence rate of conjunctivitis/conjunctival congestion was 8.3% (95% ci: 7.1%-9.7%) by sensitivity analysis. notably, four studies [6, [10] [11] [12] 14] reported 17 covid-19 patients whose initial symptom was conjunctivitis/conjunctival congestion ( figure 3 ). the pooled prevalence rate was 1% (95% ci: 1%-4%). the corresponding i 2 was 63% across the four studies. we also assessed other ocular symptoms reported in the 12 studies, including foreign body sensation (see figure 4 ), increased secretion (see figure 5 ), and eye itching (see figure 6 ). the pooled prevalence rates were 6% (95% ci: 3%-10%), 10% (95% ci: 8%-12%), and 9% (95% ci: 7%-10%), respectively. a heterogeneity test of the pooled prevalence rate of foreign body sensation showed a relatively large heterogeneity (i 2 = 81%, p < 0:01), thus a sensitivity analysis was applied. the pooled prevalence rate of foreign body sensation was 5.5% (95% ci: 4.1%-7.4%). six studies reported positive conjunctival swab samples. the pooled positive rate of conjunctival swab samples was estimated using a fixed-effect model (figure 7) since there was no heterogeneity across the six studies (i 2 = 0%, p = 0:62). the pooled positive rate of conjunctival swab samples was 3% (95% ci: 2%-5%). results of egger's test (see table 2 ) showed no publication bias for the following models: the pooled prevalence rate of conjunctivitis/conjunctival congestion (t = −0:975, p = 0:355), the pooled prevalence rate of conjunctivitis/conjunctival congestion as initial symptom (t = this systematic review and meta-analysis is aimed at quantitatively evaluating the current evidence for the possibility of covid-19 ocular transmission. we identified and analyzed 12 studies with 1930 covid-19 participants. the common ocular symptoms involved conjunctivitis/conjunctival congestion, increased secretions, eye itching, and foreign body sensation. interestingly, 1% of covid-19 patients were diagnosed with conjunctivitis/conjunctival congestion as the ini-tial symptom. in addition, our meta-analysis revealed that the pooled positive rate of conjunctival swab samples was 3%. these results suggested that ocular transmission of the covid-19 virus was possible but unlikely. laboratory studies had been conducted to explore the mechanisms of covid-19 ocular transmission. the sars-cov-2 infection has a high degree of similarity to sars-cov, which infects the host receptor of angiotensinconverting enzyme 2 (ace2), resulting in cross-species and human-to-human transmissions [20] . during the sars pandemic, a study confirmed that ace2 was a functional receptor for sars-cov [21] . a recent study revealed that ace2 is expressed in human cornea tissues and that a high and consistent expression of ace2 in the cornea poses a high potential for infection by sars-cov-2 [10, 11] . ace2 expression has also been reported in human aqueous humor [22] [23] [24] . a recent genomics study demonstrated that the ace2 gene was expressed in corneal epithelial cells, which suggests that the eyes could be vulnerable to covid-19 infection [25] . in addition, from the perspective of human anatomical features, the nasolacrimal system forms a bridge between the ocular surface and the respiratory tract. therefore, conjunctival secretions containing the virus can drain into the nasopharyngeal space and infect the human body. consistent with this perspective, a recent study revealed that rhesus macaques can be infected with covid-19 through ocular conjunctival inoculation [26] . in support of the laboratory data, much clinical evidence indicates the possibility of ocular surface transmission. studies have reported ocular manifestation as the first sign of covid-19 in patients [27] [28] [29] . in one covid-19 case [29] , conjunctivitis was even reported to be the only presenting sign and symptom of covid-19. recently, xia et al. isolated sars-cov-2 in tears [16] . similarly, our meta-analysis found that up to 10% of covid-19 patients had ocular symptoms and 3% of conjunctival swab samples were positive in covid-19 patients, supporting the possibility of transmission through the ocular surface. however, the prevalence rates of ocular symptoms and the positive rate of conjunctival swab samples were relatively low based on our meta-analysis. meanwhile, studies reported that 94% to 98% of covid-19 patients showed fever [18, 19] , 79% showed cough [18, 19] , and 44% showed myalgia or fatigue [30] ; these manifestations of covid-19 were more common than ocular manifestations. the current evidence indicates that the risk of contracting covid-19 via ocular tissue is relatively low, although this could be due to limitations of current detection methods. the strength of our study is that it is the first meta-analysis to summarize the rapidly emerging yet controversial publications reporting the prevalence rates of ocular symptoms and the positive rate of conjunctival swab samples in covid-19 patients. however, there are several limitations of our metaanalysis. firstly, in many studies, it was difficult to determine whether patients' ocular symptoms showed up before or after they were diagnosed with covid-19, which might cause bias in the estimation of the prevalence rates. secondly, ocular symptoms were reported in some but not all studies, which could lead to relatively low pooled prevalence rates. in summary, this meta-analysis showed that the pooled prevalence rate of conjunctivitis/conjunctival congestion was 8% in patients with covid-19. about one percent of covid-19 patients were diagnosed with conjunctivitis/conjunctival congestion as the initial symptom. the pooled positive rate of conjunctival swab samples was 3%. ocular transmission of covid-19 may be possible but seems 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decade-long structural studies of sars coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus antiglaucomatous effects of the activation of intrinsic angiotensinconverting enzyme 2 angiotensin (1-7) and ace2, "the hot spots" of renin-angiotensin system, detected in the human aqueous humor many faces of renin-angiotensin system-focus on eye atlas of ace2 gene expression in mammals reveals novel insights in transmission of sars-cov ocular conjunctival inoculation of sars-cov-2 can cause mild covid-19 in rhesus macaques ocular manifestation as first sign of coronavirus disease 2019 (covid-19): interest of telemedicine during the pandemic context unilateral conjunctivitis as first presentation of coronavirus disease 2019 (covid-19): a telemedicine diagnosis conjunctivitis can be the only presenting sign and symptom of covid-19 clinical features of patients infected with 2019 novel coronavirus in wuhan, china the authors declare no conflict of interest. ning li wang and kai cao contributed in designing and conceptualizing the study. kai cao and ning li wang contributed in database search and data extraction. kai cao contributed in data analysis. kai cao and gui-shuang ying contributed in manuscript writing. brad kline, gui-shuang ying, and ying han contributed in polishing the english language of the manuscript. brad kline, gui-shuang ying, ying han, and ning li wang contributed in revising the manuscript. key: cord-330602-g0xaonxv authors: sugiura, hiroaki; fujimoto, tsuguto; sugawara, tamie; hanaoka, nozomu; konagaya, masami; kikuchi, kiyoshi; hanada, eisuke; okabe, nobuhiko; ohkusa, yasushi title: prescription surveillance and polymerase chain reaction testing to identify pathogens during outbreaks of infection date: 2013-02-07 journal: biomed res int doi: 10.1155/2013/746053 sha: doc_id: 330602 cord_uid: g0xaonxv syndromic surveillance, including prescription surveillance, offers a rapid method for the early detection of agents of bioterrorism and emerging infectious diseases. however, it has the disadvantage of not considering definitive diagnoses. here, we attempted to definitively diagnose pathogens using polymerase chain reaction (pcr) immediately after the prescription surveillance system detected an outbreak. specimens were collected from 50 patients with respiratory infections. pcr was used to identify the pathogens, which included 14 types of common respiratory viruses and mycoplasma pneumoniae. infectious agents including m. pneumoniae, respiratory syncytial virus (rsv), rhinovirus, enterovirus, and parainfluenza virus were detected in 54% of patients. for the rapid rsv diagnosis kit, sensitivity was 80% and specificity was 85%. for the rapid adenovirus diagnosis kit, no positive results were obtained; therefore, sensitivity could not be calculated and specificity was 100%. many patients were found to be treated for upper respiratory tract infections without the diagnosis of a specific pathogen. in japan, an outbreak of m. pneumoniae infection began in 2011, and our results suggested that this outbreak may have included false-positive cases. by combining syndromic surveillance and pcr, we were able to rapidly and accurately identify causative pathogens during a recent respiratory infection outbreak. japanese traditional surveillance is based on definitive diagnosis and is enforced by the infection control laws in japan for the early detection of agents of bioterrorism and outbreaks of emerging infectious diseases. after the infectious disease is diagnosed at sentinel medical institutions, at least 10 days are required until it is announced nationwide. therefore, a major fault of this surveillance system is the delay in disseminating information. a surveillance system that can identify the early stages of an outbreak of infectious disease is necessary. therefore, syndromic surveillance systems have been implemented in many countries since 1995 [1] . syndromic surveillance monitors changes in the number of patients according to symptoms such as fever, vomiting, diarrhea, and rash for further investigations. information regarding the identification of local infectious disease outbreaks, such as school absenteeism, emergency room visits, and prescriptions of therapeutic drug against infectious diseases, are also subjects of the survey [2] . syndromic surveillance can offer a rapid method to detect an outbreak of infection compared with traditional surveillance; such surveillance systems are currently used worldwide [3, 4] . in some cases, an infectious outbreak can be detected on the same or the following day. although syndromic surveillance provides rapid results, it has the disadvantage that there are approximately 500 sentinel medical institutions in japan, which are selected from those equipped with departments of pediatrics and internal medicine and with more than 300 beds. definitive diagnoses are not considered. in other words, in general, its specificity may be lower than that of traditional surveillance systems. laboratory testing performed on all symptomatic patients can yield a very high specificity, but is cost prohibitive, whereas laboratory testing on selected patients for syndromic surveillance can detect some specific aberrations at a lower cost, thereby overcoming the shortcomings of both systems. the current study highlights an example to further analyze this possibility. in the fall of 2011, the number of patients with symptoms of upper respiratory tract infections markedly increased in japan. infectious disease weekly reports (idwrs) (http:// www.nih.go.jp/niid/ja/idwr.html in japanese), which constitute the traditional and official japanese sentinel surveillance system, reported a higher incidence of respiratory syncytial virus (rsv) ( figure 1 ) and m. pneumoniae infections ( figure 2) . a primary feature of m. pneumoniae respiratory infections is the degree of the symptom worsening from mild upper respiratory tract inflammation to pneumonia. m. pneumoniae infection is associated with exanthem, hemolytic anemia, gastrointestinal damage, arthritis, and various neurological symptoms [5] . outbreaks of m. pneumoniae persisted throughout june 2012, although it is unclear why this organism has continued to be responsible for such a widespread national outbreak in japan since the fall of 2011 [6] . koike used, but during screening, a positive result in the test does not always indicate acute infection by this organism. we suspect that the outbreak of m. pneumoniae infection included false-positive cases [7, 8] . idwrs are very important for clinicians, enabling them to identify the seasonal prevalence of known diseases. however, these reports become available after a minimum of 10 days following patient examinations. therefore, traditional and official surveillance systems have the distinct disadvantage of being slow and are limited to reporting pathogens chosen in advance. in the fall of 2011, by monitoring the increase in the number of combination cold medications (active ingredients: salicylamide, acetaminophen, anhydrous caffeine, and promethazine methylene disalicylate) prescribed since 2009, the prescription surveillance system detected an increase in the number of patients with symptoms of upper respiratory tract infections. on september 26, 2011, we noticed the first unusual peak and began to carefully monitor the realtime prescription surveillance system and observed a second peak on october 3, 2011 ( figure 3 ). however, monitoring prescriptions for combination cold medications does not lead to the identification of the pathogens responsible for the illnesses being treated. thus, we conducted pathogen identification using the pcr method after being alerted by the prescription surveillance system on october 4, 2011 (the following day). the purpose of the present study was to evaluate whether the pcr method triggered by the results of the prescription surveillance system can rapidly and accurately identify causative pathogens of local outbreaks of infection. our results allowed for earlier diagnoses at medical facilities and the dissemination of this information among other institutions to avoid inappropriate use of antibiotics and instigate measures against the spread of infectious diseases. we confirmed this abnormality and began this study on the following day (october 4, 2011). although very common in the us and other countries, there is no nationwide syndromic surveillance system to electronically monitor medical records in japan. because of the low prevalence of electronic medical records and a restrictive privacy policy, we perform prescription surveillance nationwide for syndromic surveillance by monitoring the number of prescriptions for certain types of drugs such as anti-influenza medications. there are approximately 45,000 pharmacies that deliver almost half of the prescribed drugs nationwide and almost all record prescriptions electronically. the prescription surveillance system was developed by the infectious disease surveillance center of the national institute of infectious diseases in collaboration with em systems co. ltd. (osaka, japan), a leading provider of prescription surveillance used by pharmacies through the application server provider (asp) system. the asp system is very useful for syndromic surveillance because data transfer is unnecessary. thus, it can dramatically decrease costs and maintain a high level of confidentially. its widespread use started in april 2009, and approximately 6,300 (13%) japanese pharmacies actively participated in the program as of october 2011. the asp system tracks prescription information, but patient symptoms and diagnoses are not recorded. categories of syndromic surveillance include the type of prescribed drugs. currently, the syndromic surveillance system monitors several types of drugs, including those for relief of fever and pain due to common colds, as well as antiviral agents, anti-influenza medications (except amantadine), and antivaricella zoster virus (vzv) drugs. the surveillance of the last two is also classified by age: <15, 16-64, and >65 years. data collection and analysis are automatically performed every night, and the results are available on the home page of a secure internet site early the next morning. monitoring the usage of anti-influenza and anti-vzv drugs is particularly useful for early detection of outbreaks of infection because these drugs are used only to treat specific viral infections. between october 4 and 28, 2011, 50 patients were included in the present study who either presented at a single clinic with a chief complaint of respiratory symptoms or fever or were suspected of having respiratory tract infections after being identified through the syndromic prescription surveillance system. in japan, a rapid diagnosis kit suitable for use at outpatient clinics is currently available, and the costs are covered by the national health insurance program. the tests allow for rapid detection of infections caused by the influenza virus, rsv, and adenovirus. a total of 18 pharyngeal swabs to screen for adenovirus infections and 32 nasal swabs to screen for rsv and influenza viral infections (rapid rsv) were collected [9] . viruses were extracted from the swabs using immunochromatography (ic) kits with approximately 500 l of a mucolytic agent provided by the manufacturer. after the assay, approximately 200 l of the agent remained in the ic-kit tubes. this medical waste was transferred to universal transport medium (359c; copan italia s.p.a, brescia, italy) and analyzed using [12] . in addition, the presence of coronavirus infection was tested in patients from whom no infectious agents were detected [13] . this study only collected anonymous information that cannot be associated with individual patients. patient samples were collected during the course of medical care provided at the participating facilities, and all examinations and testing for pathogens occurred at the request of the medical facilities for the purposes of diagnosis and treatment. this study used only existing medical records and documents, and oral informed consent was obtained from all patients. after testing the specimens, we provided the results to a medical institution within 4 days including the conveyance period. the 50 patients tested in this study included 2 infants (1 male and 1 female, aged <1 year), 25 children (12 males and 13 females, aged 1-6 years), 10 elementary school pupils (6 males and 4 females, aged 7-12 years), 4 minors (2 males and 2 females, aged 13-18 years), 8 adults (3 males and 5 females, aged >18 years), and 1 patient (age unavailable). table 2 lists the pathogens detected by the pcr analysis stratified by age in the 27 patients. in children, enterovirus, rhinoviruses, rsv, and parainfluenza viruses were detected, whereas m. pneumoniae was detected only in elementary school pupils and minors. in the remaining 23 patients, no pathogens were detected. these 23 patients were also found to be negative for coronavirus. pcr was used to obtain definitive viral diagnoses via rapid rsv and adenovirus diagnosis kits, and the sensitivity and specificity were calculated for these test kits. for the rapid rsv diagnosis kit, sensitivity was 80% and specificity was 85%. for the rapid adenovirus diagnosis kit, no positive results were obtained; therefore, sensitivity could not be calculated and specificity was 100%. rsv infections were detected using the rapid diagnosis kit, but rhinovirus, enterovirus, and parainfluenza virus infections were not. the causative pathogens were unknown in many patients, although they were nevertheless treated for upper respiratory tract infections. evaluation of the incidence of various symptoms in patients infected with different pathogens showed that rhinoviruses were detected in nasal swab specimens more often than other viruses and patients with rhinovirus infections were less likely to present with fever ( table 3) . all rsv-positive patients were children, 80% of whom presented with coughing. all patients who were tested using the rapid adenovirus detection kit showed negative results. however, all these patients also tested negative for adenovirus using sensitive pcr tests. thus, adenovirus was not considered to be the causative organism of this suspected outbreak. here, we examined a combination of syndromic surveillance and pcr testing and showed the potential to identify pathogens during the early stage of an outbreak of respiratory infections. in the future, it would be desirable to develop an m. pneumoniae diagnosis kit that can diagnose pathogens from nasal or pharyngeal swabs at outpatient clinics or the bedside of patients. in japan, two official pathogen surveillance methods have been conducted under the infection control laws: sentinel pathogen surveillance and active surveillance. the official pathogenic surveillance has been conducted at sentinel medical institutions regardless of outbreaks. on the other hand, in patients with serious diseases, active pathogenic surveillance has sometimes been conducted on the basis of notifications by medical institutions. however, active surveillance is conducted only when an infection spreads widely enough to cause serious problems in a particular region and the surveillance of pathogens may not be timely enough to mount a response to control outbreaks. pathogenic surveillance for all patients with signs of an infection would detect agents of bioterrorism and emerging infectious diseases; however, the cost would be prohibitive. therefore, system coordination to perform pathogen surveillance based on early detection of outbreaks is necessary. the scheme proposed by the present study uses pcr testing triggered by detection alerts from syndromic surveillance systems. in general, syndromic surveillance offers earlier detection of infectious diseases than traditional surveillance. moreover, if the pathogen remains unknown following bedside testing using several rapid tests or other typical examinations, the proposed scheme requires the collection of specimens as soon as possible and sending them to a laboratory for definitive diagnoses. however, it takes a few days to transfer the specimens and a few extra days for the information of the identified pathogen to be shared among medical facilities, public health centers, and local governments in the involved areas. in the proposed scheme, we can use pathogenic information to control ongoing outbreaks and, hopefully, decrease the number of potential infections. thus far, syndromic surveillance with pathogenic testing has been conducted by collecting samples from patients receiving telephone consultations [18] and those receiving emergency department consultations [19] . syndromic surveillance using electronic medical records has been combined with testing for the influenza virus [20] . however, these systems have focused only on rapid testing and are mainly used for influenza monitoring [20, 21] . therefore, syndromic surveillance trials for nonspecific pathogens using pcr for undiagnosed infectious diseases, similar to the present study, have not been performed before. in the present study, an outbreak was detected by routine syndromic surveillance, in which samples were regionally collected for pcr analysis. these tests for viral infections allowed for differentiation between bacterial and viral infections, thus facilitating treatment without the unnecessary use of antibiotics. although the present laboratory tests cannot be performed for all individual clinical diagnoses, the results were immediately made available to clinicians for the treatment of other patients with similar symptoms. the symptoms reported in the present study were rather mild; therefore, no patient required hospitalization, and no further testing was performed in undiagnosed patients. however, if severe cases were to occur, careful identification of pathogens would be desirable. rhinoviruses were detected in nasal swab specimens more frequently than other viruses. therefore, it is likely that children who present with nasal discharge and mild fever may be reservoirs for rhinoviruses [22] . testing for respiratory viral infections in emergency room outpatients by pcr analysis showed that the most frequently detected viruses were picornaviruses, including rhinoviruses [23] . when children present with coughing as the main symptom, rsv should be considered as the most likely pathogen. the finding that m. pneumoniae infection was not detected in infants and children, but rather in elementary school pupils and minors, was consistent with reports that m. pneumoniae may often cause asymptomatic infections before the age of 5 years, after which immunity decreases as children become susceptible to symptomatic m. pneumoniae infections [5] . in japan, nationwide outbreaks of m. pneumoniae began in 2011 and continued as of january 2012, during which time m. pneumonia, rhinovirus, enterovirus, parainfluenza virus, and rsv have been identified. our results suggested that this outbreak may include false-positive cases and subsequent inappropriate prescriptions of antibiotics. an increased frequency of macrolide-resistant m. pneumoniae became widely reported in the japanese media in the fall of 2011 [24] . therefore, this news may have induced an abnormal increase in the number of patients ( figure 2 ). the rapid test available in japan for m. pneumoniae uses sera samples [25] . although general clinics may outsource m. pneumoniae antibody testing and cold hemagglutinin testing, blood testing is usually not performed in cases of mild pediatric illnesses. in the future, it would be desirable to develop m. pneumoniae diagnostic kits using nasal or pharyngeal swabs at outpatient clinics or bedside. until such kits for the diagnoses of m. pneumoniae and other infectious diseases are developed, syndromic surveillance with pcr testing offers a useful countermeasure against infectious outbreaks. in this study, we could not detect single infectious agents that explained the outbreaks; however, our results excluded m. pneumoniae. the present study was limited to a single clinic. therefore, further studies involving more facilities should be undertaken. it is also necessary to develop a network and sample transportation system among the facilities partaking in the syndromic surveillance system and to adequately staff laboratories with experienced technicians. syndromic surveillance data has been mathematically or statistically analyzed in many studies. however, when an abnormal value is reported by syndromic surveillance, there are many cases in which the pathogens cannot be identified by the calculations introduced in these articles. when m. pneumoniae and rsv infections were prevalent nationwide during the fall of 2011, we observed an abnormal increase in common cold prescriptions through the japanese surveillance system and were able to evaluate the incidence of various pathogens via pcr testing. what is syndromic surveillance? real-time prescription surveillance and its application to monitoring seasonal influenza activity in japan syndromic surveillance and bioterrorism-related epidemics syndromic surveillance practice in the united states: findings from a survey of state, territorial, and selected local health departments textbook of pediatric infectious diseases mycoplasmal pneumonia as of macrolide resistance and detection in mycoplasma pneumoniae at kansai medical university hirakata hospital utility and limitation of the rapid igm antibody detection test for the diagnosis of mycoplasma pneumoniae infection evaluation of a bedside immunochromatographic test for detection of adenovirus in respiratory samples, by comparison to virus isolation, pcr, and real-time pcr comprehensive detection of causative pathogens using realtime pcr to diagnose pediatric community-acquired pneumonia novel highspeed real-time pcr method (hyper-pcr): results from its application to adenovirus diagnosis detection of mycoplasma pneumoniae by two polymerase chain reactions and role of m. pneumoniae in acute respiratory tract infections in pediatric patients a pancoronavirus rt-pcr assay for detection of all known coronaviruses a novel real-time pcr system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus a and b by real-time pcr development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses usefulness of realtime reverse transcription-polymerase chain reaction for the diagnosis of echovirus aseptic meningitis using cerebrospinal fluid linking syndromic surveillance with virological self-sampling evaluation of alternative respiratory syndromes for specific syndromic surveillance of influenza and respiratory syncytial virus: a time series analysis next generation syndromic surveillance: molecular epidemiology, electronic health records and the pandemic influenza a, (h1n1) virus impact of influenza during the post-pandemic season: epidemiological picture from syndromic and virological surveillance rhinovirus infections in the upper airway etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in beijing macrolideresistant mycoplasma pneumoniae: characteristics of isolates and clinical aspects of community-acquired pneumonia utility of a rapid diagnosis kit for mycoplasma pneumoniae pneumonia in children, and the antimicrobial susceptibility of the isolates the present study was funded by a research grant from the ministry of health, labour and welfare of japan. the authors contributed equally to this paper. key: cord-351685-n70tkf38 authors: altamimi, asmaa; abu-saris, raghib; el-metwally, ashraf; alaifan, taghreed; alamri, aref title: demographic variations of mers-cov infection among suspected and confirmed cases: an epidemiological analysis of laboratory-based data from riyadh regional laboratory date: 2020-02-19 journal: biomed res int doi: 10.1155/2020/9629747 sha: doc_id: 351685 cord_uid: n70tkf38 introduction. middle east respiratory syndrome coronavirus was first recognized in september 2012 in saudi arabia. the clinical presentations of mers and non-mers sari are often similar. therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of mers-cov is essential. however, the real challenge is to flag these patients through some demographic markers. the nature of these markers has not previously been investigated in saudi arabia, and hence, this study aims to identify them. methods: it was a surveillance system-based study, for which data from a total of 23,646 suspected patients in riyadh and al qassim regions were analyzed from january 2017 until december 2017 to estimate the prevalence of mers-cov among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. results: of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. these confirmed cases (67.2% of which were males) had a mean age of 43.23 years (sd ± 22.8). around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. the study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. conclusion: the study provides evidence that the mers-cov epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. future studies should aim to confirm such findings in other regions of saudi arabia as well and explore potential preventable risk factors. a respiratory viral disease caused by the middle east respiratory syndrome coronavirus (mers-cov) was first isolated in 2012, in a 60-year-old man who died in jeddah, ksa due to severe acute pneumonia and multiple organ failure [1] . since then, 27 countries have reported the presence of this virus, including the 12 countries of the eastern mediterranean region. several outbreaks have occurred in multiple countries including saudi arabia, the united arab emirates and the republic of korea [2] . recent fatality rate (cfr) of 21% [5, 6] . very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that mers-cov infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . males with a history of serious medical conditions are highly susceptible to this infection. moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. dromedary camels are the major animal source of mers-cov transmission to humans. interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in icus across different healthcare settings [4] . studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rrt-pcr) assays [14] . ere is no specific treatment for mers-cov. like most viral infections, the treatment options are supportive and symptomatic [2] . at present, no vaccine exists for preventing the infections of mers-cov. e cdc indicated that preventative actions should be taken for any type of respiratory illness [4] . such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. one must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . many studies have been conducted in recent years in saudi arabia to combat this deadly disease. a large multicentre study showed that it is nearly impossible to differentiate between patients of mers-cov and non-mers-cov just on the basis of clinical presentation [16] . another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . similarly, two more single-centre case control studies reported that the presenting symptoms of mers-cov infection were not specific [18, 19] . physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . mild symptomatic cases, which result in a positive pcr, may be isolated at home. severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of mers-cov infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of mers-cov. e nature of these markers has not been investigated in saudi arabia, and hence this study aims to identify them. a cross-sectional study was conducted at the regional laboratory and blood bank, located at shumaisi hospital in riyadh, ksa. e laboratory has received the central blood banks and reference laboratories accreditation program saudi central board for accreditation of healthcare institution (cbahi) 2018 [21] . technique. data were collected during the period of january 2017 to december 2017. all patients in riyadh and al-qassim regions who had their samples tested at riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. for the descriptive aim, we estimated the prevalence of mers-cov. for the analytical aim, a binary logistic regression model was developed. in this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. data were cross-checked with a labcomputerized database. further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. we collected data from 25,400 cases, of which 23,646 suspected cases of mers-cov were included in the final analysis. data were cleaned, entered, stored, and managed with an excel database and ibm spss version 25. e statistical analyses consisted of descriptive counts and percentages. for those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. a logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. at first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. to determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. a confirmed case is defined as a suspected case with laboratory confirmation of mers-cov infection [20] . a total of 23,646 of mers-cov suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of mers-cov remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (a.or: 3.11, 95% ci: 1.104-8.76, p value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of mers-cov infection laboratory-based data was conducted in riyadh over a one-year period (2017). a total of 23,646 suspected cases were included in the results. of the total suspected cases, 119 cases had been confirmed via laboratory results. all the confirmed cases are reported to moh through hesn (health electronic surveillance networks) and to the world health organization (who) through the international health regulations (ihr), national focal point of saudi arabia. we found that mers-cov infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. during the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of mers-cov infection cases has decreased in riyadh and al-qassim regions in comparison to that of the last three years. from 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of da'ar and ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in ksa (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . it is worth mentioning that saudi arabia defines age categories differently from the who (children: 0-14, adult: otherwise) [20] . however, unlike the classification used in saudi arabia, we have followed the who categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in who reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by aly and his collaborators in their recent paper published in 2017 [14] . adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of mers-cov were reported among people aged 40 and above [24] . in 2016, only 9 of 552 cases (1.6%) of mers-cov infection were found among pediatric patients. moreover, the study which was conducted in king fahad medical city in riyadh (kfmc) between january 2012 and december 2013 did not report any mers-cov cases among children [26] . e study which was conducted across the gulf countries for four years by mahmoud aly et al. between 2012 and 2016 suggests that the prevalence and distribution of mers-cov were the highest-risk in elderly aged 60 years or above [14] . similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by ahmad to estimate the survival rate in mers-cov globally prior to 26 january 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . our data reported a higher prevalence of infection among saudi nationals as compared with non-saudi. another study also showed similar results but with a much higher percentage among saudis, which may be due to the fact that it included saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the riyadh and al qassim regions only. in our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the real star kit from altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. in fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. to the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. however, it is worth mentioning that ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (ast) [21] . however, further investigations are needed to confirm our findings. one of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of mers-cov in the riyadh and al-qassim regions. secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in saudi arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. in addition to this, quality assurance policies are implemented at hesn in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. according to our knowledge, this is one of the few studies that have specifically investigated mers-cov risk factors in the riyadh and al-qassim areas (two major regions in ksa). given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. it must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed mers-cov with those with suspected mers-cov who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. in conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting mers-cov signs with a negative lab result may need retesting. our study covered two main regions in saudi arabia and provides evidence that the mers-cov epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. future studies should aim to confirm such findings in other regions in saudi arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by riyadh regional laboratory under license and are not freely available. however, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia who, middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome (mers), who cov outbreak largest outside kingdom of saudi arabia middle east respiratory syndrome coronavirus in children hospital outbreak of middle east respiratory syndrome coronavirus clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection e pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction epidemiological, demographic, and clinical 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factors: an analysis of globally reported cases of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): impact on saudi arabia 2000-2025) standard-standard populations-seer datasets [internet], acute myeloid leukemia-cancer stat facts acute viral respiratory infections among children in mers-endemic riyadh estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study diagnostic delays in 537 symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia e predictors of 3-and 30-day mortality in 660 mers-cov patients risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel clinical validation of 3 commercial real-time reverse transcriptase polymerase chain reaction assays for the detection of middle east respiratory syndrome coronavirus from upper respiratory tract specimens acknowledgments e authors would like to thank dr. waleed alsalem, dr. ahmed hakawi, and dr. mutaz mohammed from ministry of health, saudi arabia, dr. kamel al-dossari from riyadh regional lab for their help in this research, hatim al-mutairi for data cleaning, dima zailaey for structuring, and dr. munazza jawed from dow university of health sciences, karachi, for proofreading. e authors would also like to thank miss laila mohamed ghoneim from the american university cairo for english-language editing. all authors contributed to the writing of the manuscript and had access to the data. all authors read and approved the final manuscript. key: cord-319635-kh99n7q2 authors: chiang, wei-wei; chuang, ching-kai; chao, mei; chen, wei-june title: cell type-dependent rna recombination frequency in the japanese encephalitis virus date: 2014-07-22 journal: biomed res int doi: 10.1155/2014/471323 sha: doc_id: 319635 cord_uid: kh99n7q2 japanese encephalitis virus (jev) is one of approximately 70 flaviviruses, frequently causing symptoms involving the central nervous system. mutations of its genomic rna frequently occur during viral replication, which is believed to be a force contributing to viral evolution. nevertheless, accumulating evidences show that some jev strains may have actually arisen from rna recombination between genetically different populations of the virus. we have demonstrated that rna recombination in jev occurs unequally in different cell types. in the present study, viral rna fragments transfected into as well as viral rnas synthesized in mosquito cells were shown not to be stable, especially in the early phase of infection possibly via cleavage by exoribonuclease. such cleaved small rna fragments may be further degraded through an rna interference pathway triggered by viral double-stranded rna during replication in mosquito cells, resulting in a lower frequency of rna recombination in mosquito cells compared to that which occurs in mammalian cells. in fact, adjustment of viral rna to an appropriately lower level in mosquito cells prevents overgrowth of the virus and is beneficial for cells to survive the infection. our findings may also account for the slower evolution of arboviruses as reported previously. japanese encephalitis (je) is an important mosquito-borne viral disease, occasionally causing encephalitic symptoms [1] . nowadays, it is extensively distributed in most asian countries and was also recently reported from australia [2] . the je virus (jev) is one of some 70 members of the genus flavivirus belonging to the family flaviviridae [3] , the genome of which contains a linear, single-stranded positive-sense rna (∼11 kb long) that encodes 3 structural proteins including nucleocapsid (c), membrane (prem/m), and envelope (e) proteins, as well as 7 nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5) [4] . due to lack of a proofreading mechanism and an inability to repair errors during rna synthesis, spontaneous mutations frequently occur which contribute to the formation of genetically diversified populations or so-called "quasispecies" in flaviviruses including the jev [5] . maintenance of genetic diversity theoretically reduces the rapid loss of fitness via muller's ratcheting during viral passage from one host to another [6] , which provides benefits to a virus that is adapting to a new niche or selective regimen of its environment [7] . possibly, this feature differentially occurs in different types of host cells [8] . in addition to gene mutations [9] , rna recombination, at least in some cases, can also serve as a factor helping a virus escape from accumulated deleterious effects in a viral population [10] . in other words, rna recombination may serve as an alternate means to generate genetic changes [11] and likely produces a new form of rna comprising genetic information from multiple sources [12] . the viral rna recombination was first reported in the poliovirus, a picornavirus [13] , and subsequently in a variety of viruses that infect humans, animals, plants, and bacteria [14] [15] [16] [17] [18] . therefore, a new virus may be generated through rna recombination between different strains. among arboviruses, at least the western equine encephalitis virus is believed to be a recombinant virus that arose from distant viral progenitors, including an eastern equine encephalitis virus-like virus and a sindbis-like virus [19] . as a result, the ability to form unpredictable recombinant strains or species between virus populations is of considerable concern [20] , particularly the possibility of rna recombination occurring from cocirculated live-attenuated vaccine strains and wild viruses during synthesis of new rnas [21, 22] . flaviviruses naturally comprise multiple genotypes or strains [23, 24] , making them likely to undergo rna recombination. the first rna recombination of the jev was proposed based on a bioinformatics analysis [17] . furthermore, rna recombination was found to occur unequally in mosquito and mammalian cells [25] . herein, we provided evidences of rna recombination of the jev that occurs at a lower frequency in mosquito cells, which may, at least partly, contribute to evolution of the virus [26] . lines. three strains of the jev, including nakayama (the vaccine strain), t1p1-s1 (a small plaque clone from the t1p1 strain) [27] , and cjn-s1 (a small plaque clone from the cjn strain, a kind gift from dr. m. h. ho, academia sinica, taipei, taiwan), were used in this study. of these, further purification via the plaque-picking method to select t1p1-s1 and cjn-s1 strains was implemented as part of the present study [27] . the viruses were propagated in c6/36 mosquito cells and titrated in baby hamster kidney-(bhk-) 21 cells. both cell lines were maintained as previously described [27] . titration. virus titers were determined by means of a plaque assay of bhk-21 cells following descriptions in our previous report [27] . calculation of virus titers was based on the number of formed plaques, expressed as plaque-forming units (pfu)/ml. to detect viral infection in cells, extracted rna was applied to perform rt with the reverse primer at 42 ∘ c for 30 min to generate complementary (c) dna. pcr cycling was then carried out using the forward primer which was subsequently run to amplify a gene fragment with a size of 529 bp under the following conditions: 25 cycles of 95 ∘ c for 30 s, 60 ∘ c for 30 s, and 72 ∘ c for 1 min. the primers used to amplify specific regions are presented in individual sections below. all procedures in this portion of the study followed our previous description [25] . coinfection of jev strains was verified by a method described in our previous report [25] . in brief, extracted viral rnas were applied to perform the rt-pcr with the primer pair, 10-36f (5 -ctgtgtgaactt c t t g g c t t a g t a t c g-3 ) and 850-877r (5 -cagttttcatgagatatcgtgtgtggc-3 ). fragments (868 bp) amplified from jev strains simultaneously infecting bhk-21 or c6/36 cells were subjected to restriction fragment length polymorphism (rflp) with the restriction enzyme rsai to verify coinfection. a pattern showing fragments of 219, 401, and 248 bp represented t1p1-s1 infection, while that showing fragments of 219 and 649 bp represented cjn-s1 infection. those exhibiting all size of fragments indicated that both viral strains had coinfected a single cell. in addition, rflp using specific restriction enzymes as shown in our previous report [25] was used to verify rna recombination between viral strains in a single cell. in some experiments for assay of rna recombination, rflp was carried out by using cells cultured in the presence of an exoribonuclease inhibitor (3 -phosphoadenosine-5phosphate, pap) (sigma-aldrich, st. louis, mo, usa). region-(utr-) i. in order to evaluate rna recombination between genomic rna and a transfected rna sequence, the p(+)t1p1-5 3 -utr-1 plasmid was constructed as described here. viral rna derived from the t1p1 strain of the jev was used as a template to generate dna fragments corresponding to the 5 -or 3 -end of genomicsense rna. to prepare the 5 -end sequence, a primer (5 -ctgccaagcatccagccaagta-3 , complementary to nt 895∼916 of the 5 -end of the t1p1 genome) was used for rt to synthesize the first-strand cdna. subsequently, another primer (5 -taatacgactcactatagagaagtttatctgtgtg-3 ) containing a partial sequence of the t7 polymerase promoter used as a tag (italicized) at the 5 -end (nt 1∼18) of the t1p1 5 -end sequence was used in the pcr to amplify a 934 bp dna fragment. in the meantime, the primer 5 -gtgttcttcctcaccaccagctac-3 (nt 10,946∼10,969 at the 3 -end of the t1p1 genome) was used for rt to generate cdna. another primer (5 -gaaaattatgttgactac-3 , corresponding to the sequence nt 10,320∼10,337) was subsequently used for the pcr under conditions described above to amplify a 650 bp dna fragment. both types of pcr products were separately digested with the restriction enzyme, aatii; the resultant dna fragments were ligated to form subgenomic dna which contained both 5 -end (nt 1∼599) and 3 -end (nt 10,367∼10,969) sequences. subsequently, the subgenomic dnas were cloned into pgem-t (promega, madison, wi, usa) to form a plasmid designated p(+)t1p1-5 3 -utr-i which contained an insert of a 1202 bp fragment. in order to see the stability of viral fragments in host cells, the p(+)5 3 -utr-ii plasmid was constructed. to construct the plasmid, the pt1p1-5 3 -utr was used as a template, and the pcr was performed under conditions described above with the primers 5 -taatacgactca-ctatagagaagtttatctgtgtg-3 (the italics indicate a partial t7 polymerase promoter sequence) and 5 -aagatatcgtgttcttcctcac cacc-3 (the italics indicate an ecorv restriction enzyme site). the pcr products were digested with spei and aatii to delete a fragment from nt 178∼599; the resultant dna fragments were then treated with klenow fragment enzyme (fermentas, hanover, md, usa) and ligated to form subgenomic dna which only contained the 5 -and 3 -utrs of the t1p1 genome. subsequently, the subgenomic dnas were cloned into pgem-t (promega, fitchburg, wi, usa) to form plasmids designated p(+)5 3 -utr-ii. negative (−) sense 5 -end rna sequences and derived dsrna. both (+) and (−) sense 5 -end rna sequences were prepared from the pt1p1-5 3 -utr-ii plasmid. in preparation of the (+) sense 5 -end rna sequence, the plasmid was linearized by ndei and transcribed with t7 rna polymerase using an in vitro transcription system (fermentas). the rna products (599 bp) were extracted with phenol-chloroform, precipitated in ethanol, and then stored in a deep freezer until used for transfection. to prepare the (−) sense 5 -end rna sequence, the plasmid was first linearized, and the 3 -end sequence of the subgenomic dna was deleted with ndei. the resultant linear forms of the plasmid were religated and then redigested with sacii. the products were transcribed with t7 rna polymerase using an in vitro transcription system (fermentas) to generate the (−) sense 5 -end rna sequence which was harvested as done for the (+) sense 5 -end rna sequence. to prepare dsrna, positive-and negative-stranded rna described from pt1p1-5 3 -utr-ii were mixed together, incubated at 95 ∘ c for 5 min and then 4 ∘ c for 10 min. ultimately, 2 l rnase was added to cleave single-stranded rna that failed to anneal in the mixture. the product was used to evaluate degradation of dsrna fragments in host cells. sequence and viral infection in cells. transfection of dsrna or (+) sense 5 -end rna-i prepared from the plasmids (+) pt1p1-5 3 -utr-ii was carried out in bhk-21 and c6/36 cells. at 5 h posttransfection (hpt), cells were infected with the nakayama strain of the jev, at an moi of 5. the detailed procedure followed a previous description, from which efficacy of transfection was demonstrated [25] . sequences derived from (+)5 3 -utr-ii rna were transfected into cells either treated or untreated with an exoribonuclease inhibitor (3 -phosphoadenosine-5 -phosphate, pap) (sigma-aldrich, st. louis, mo, usa) and incubated for 5 h. rna was extracted with the trizol reagent (5 prime, gaithersburg, md, usa), and then dnase (promega) was added to delete interference of genomic dna. rt was subsequently run in a mixture containing 4 g rna, 1 l 100 mm random hexamer primer, and 1 l 10 mm dntp, and double-distilled (dd) h 2 o water was added to bring the volume up to 12 l. this was heated at 65 ∘ c for 5 min, allowed to stand at 4 ∘ c for 2 min, and then 4 l 5x first-strand buffer, 2 l dithiothreitol (dtt), 1 l of an rnase inhibitor (rnase outtm; invitrogen, carlsbad, ca, usa) were added. after incubation at room temperature for 5 min, 1 l of reverse transcriptase m-mlv (invitrogen) was added and allowed to react for 1 h at 37 ∘ c, followed by 15 min at 75 ∘ c. the cdna produced was then used for the subsequent pcr under the conditions described above. the primers used for the pcr included the 5 -utr (5 -agaagtttatctgtgtgaac-3 ) and 3 -utr (5 -agatcctgtgttcttcc-3), which generated pcr products predicted to be 907 bp. to assess integration of dsrna, the same cdna and primer pair described above were used to amplify a fragment 807 bp long. 2.10. assay for rna recombination from transfected as well as infected cells. bhk-21 and c6/36 cells transfected with transcribed rna fragments were then infected by the nakayama strain of the jev. total rna extracted from cells that had been transfected with (+) sense 5 -end rna-1 was run for rt using a primer (850-877r: 5 -tcagttttc-atgagatatcgtgtgtggc-3 ) complementary to the sequence of nt 850∼877. amplification using the forward primer (rvf1: 5 -gcgggatttaatacgactcactat-ag-3 ) which is a partial sequence of the plasmid that serves as a tag and the reverse primer (rvr1/nt 516∼538: 5 -ctgcaatatccgtattgttgac-3 ) produced a specific region comprised of 564 nt. the reverse primer used here was specific for the nakayama strain. as a result, the fragments amplified by this primer pair must represent a strain of genetic recombination. infected by the jev. viral replication was validated by rna accumulation through a real-time rt-pcr with cdnas reverse-transcribed from extracted rna of infected (at an moi of 1) or uninfected c6/36 and bhk-21 cells. the primer pairs ts1-f/ts1r (5 -tgtggcttgcgagcttggcag-3 /5 -acatgtagccgacgtcgatt-3 ) and cjn1-f/cjn1r (5 -tgtggcttgcgagcttggcta-3 /5 -acatgtagccgacgtctatc-3 ) were used to amplify specific regions of the t1p1-s1 and cjn-s1 strains, respectively. levels of 18s rrna designed from the genome of c6/36 or bhk-21 cells were also amplified as an internal control as our previous report [25] . results are expressed as the relative quantities, so fold change was used to represent the amount of viral rna that accumulated at each time point of infection. to monitor synthesis of viral rna including positive and negative strands in a time course in c6/36 cells, viral rna extracted from infected cells (0∼15 hpi) was used to run rt-pcr as the procedures described previously [27] . as above, 18s rrna designed from the genome of c6/36 cells was also amplified as an internal control. the amplified cdna fragment was then identified by running the pcr product on a 2% (w/v) agarose gel. yates' chi-square test was used to assess the frequency of rna recombination in cells coinfected by two virus strains or transfected by viral rna fragments. fragment of cjn-s1 fragment of ts1 (t1p1-s1) smai (120) ts1 figure 1 : the schematic sketch designed to identify rna recombination between viral strains. a fragment (868 bp) comprised of the c/prem junction (nt 10∼877) of viral rna extracted from coinfected bhk-21 or c6/36 cells was amplified, cloned, and then used for an rflp analysis with smai or alw44i. two and one recombinant form(s) were, respectively, identified in selected samples from bhk-21 and c6/36 cells, when they were coinfected with the t1p1-s1 and cjn-s1 strains of the japanese encephalitis virus. stastistical analysis * * < 0.05 > 0.05 * mixed rna was a mixture of rnas separately extracted from t1p1-s1 and cjn-s1 strains of the japanese encephalitis virus, being used as the internal control. * * yates' chi-square test was used to assess the difference of rna recombination in cells coinfected by two virus strains at 5% level of significance. cells. viral rna extracted from single infectious centers (ics) which were randomly selected and picked out from infected bhk-21 or c6/36 cells was subjected to an rsai rflp assay as described in our previous report. the result reveals that different strains of the jev can coinfect a single bhk-21 or c6/36 cell. the c/prem junction comprising 868 nucleotides (nt 10∼877) of viral rna extracted from bhk-21 or c6/36 cells coinfected with the tap1-s1 and cjn-s1 strains was cloned and used for the smai-alw44i rflp analysis (figure 1 ). the recombinant forms of the viral genome were actually identified in bhk-21 and c6/36 cells, when they were coinfected by the 2 strains of the jev. totally, 20 recombination clones (20.4%) were found from 98 clones coinoculated with the 2 strains in bhk-21 cells while being 5 out 38 (13.1%) in c6/36 cells (table 1) . probability of occurring rna recombination was significantly different, compared with the mixed rna control, in bhk-21 cells while being nonsignificant in c6/36 cells (table 1 ). in other words, the frequency of rna recombination is significantly higher in bhk-21 cells than in c6/36 cells. a 564 bp fragment was significantly amplified in bhk-21 and c6/36 cells which were infected by the jev (nakayama strain) following transfection with the (+)5 3 -utr-i rna plasmid, although a light band was also shown in the control group that contained a mixture of rnas extracted from transfected cells.a specific fragment of viral rna (529 bp) was amplified as an internal control in all groups with viral infection. in addition, no fragment presenting an artifact of rna recombination was shown in the control groups of mock treatment (neither infection nor transfection), transfection with only the (+)5 3 -utr rna-i plasmid, or infection with only a single strain. an image-density analysis revealed recombination in bhk-21 cells to be 10.7-fold higher than that of the control group, while it was 7.73-fold higher in c6/36 cells, suggesting that rna recombination may occur in both mammalian and mosquito cells. however, a slightly lower frequency of rna recombination was eventually shown in mosquito cells (figure 2 ). treated with pap, the inhibitor of exoribonuclease, compared to untreated cells. in contrast, no effect of pap on increasing rna recombination was seen in c6/36 cells; only a low level of rna recombination was found in this test (figure 3(a) ). looking at transfected viral fragment (+) rna in c6/36 cells treated with pap, enzymatic cleavage by exoribonuclease did not occur at 3 h after transfection while it evidently decreased preservation of such rna fragment at 6 h after transfection in mosquito cells (figure 3(b) ). viral genomic rna was not affected when treated with pap in both cell types (figure 3(a) ), implying that the transfected viral rna fragment may not be further degraded by exoribonuclease mostly in mammalian cells; which leads to a higher possibility of occurring rna recombination in such cells. strains. when we coinfected t1p1-s1 and cjn-s1 strains of jev into c6/36 cells and treated with pap, only 1 of 30 clones occurred rna recombination while 4 out of 31 clones occurred in the control group (without treatment with pap). the rna recombination rate did not change significantly ( value = 0.370; yates' chi-square test) in coinfected c6/36 cells and even their function of exoribonuclease was inhibited and thus unable to dissolve viral rna ( table 2 ). the result implicated that the low level of viral rna at the early phase of infection may not be fully exoribonuclease-mediated but, as above, is probably contributed by the rnai-dependent effect. the dsrna intermediates are generally formed during virus replication in host cells, however, which may be cleaved in invertebrate cells. through an rt-pcr, a corresponding segment of rna (807 bp) was detected in c6/36 cells immediately after transfection (0 hpt) with a fragment of dsrna derived from (+) or (−) 5 3 -utr rna; however, it had faded by 3 and 6 hpt (figure 4 ). this suggests that transfected dsrnas may have been cleaved and presumably generated short interfering (si)rnas which were not shown on the gel. it suggested that a part of viral rnas may be degraded at the early phase of infection, likely to modulate virus growth, in mosquito cells. yates' chi-square test was used to assess the difference of rna recombination ( value = 0.370). virus during early infection. appropriate accumulation of viral rna in host cells is essential for prosperous production of progeny virions. according to the results, rnas of both the t1p1-s1 and cjn-s1 strains accumulated more slowly in c6/36 cells than bhk-21 cells (figure 5(a) ). specifically, the rna amount of the t1p1-s1 strain remained at the baseline level until 12 hpi (3.81-fold change), compared with an increase of 169.72-fold at 24 hpi in c6/36 cells. in contrast, t1p1-s1 rna, respectively, increased to 3.09-, 28.99-, 429.05-, 4396.07-, and 5487.75-fold, at 3, 6, 9, 12, and 24 hpi in bhk-21 cells. similarly, the rna amount of cjn-s1 also accumulated more slowly in c6/36 cells than in bhk-21 cells. the rna amount remained at the baseline level until 12 hpi (2.36-fold increase) and subsequently increased to 152.32-fold at 24 hpi in c6/36 cells. in contrast, the rna amount of cjn-s1 rna, respectively, increased by 16.64-, 111.43-, and 554.87-fold at 9, 12, and 24 hpi, despite it having been unchanged at 6 hpi (1.35fold change) in bhk-21 cells. the result revealed that progeny rna of the virus is delayed to accumulate in mosquito compared to mammalian cells, especially at the early phase of infection. the stability of viral rna is crucial for the productivity of the progeny virions, which was evaluated after transfection of an rna fragment prepared from (+)5 3 -utr-ii into either bhk-21 or c6/36 cells. results showed that transfected fragments had not significantly degraded even at 3 or 6 hpt in bhk-21 cells, while those in c6/36 cells had more obviously degraded ( figure 5(b) ), implying that different outcomes of rna existed in the 2 cell types especially in the early phase of infection. rna viruses generate new genetic strains with approximately 6 orders of magnitude higher rates of nucleotide substitutions compared to dna viruses [28] . thus, the rate of spontaneous mutations is a critical parameter modeling the genetic structure of viral populations [29] . the primary variation following a mutation may provide for further evolutionary processes, for example, selection and/or recombination [30] . those in turn lead to the generation of viral strains which are more adept and fit in nature. rna recombination is now believed to be a strategy for the evolution of many viruses [12] , for instance, the poliovirus [31] , hepatitis c virus [32] , hepatitis d virus [33] , and norovirus [34] . a variety of flaviviruses including dengue virus and jev were also reported to carry out rna recombination according to bioinformatics inferences [17, 35] and experimental demonstration [25] . currently, 2 possible mechanisms are reported to lead to the occurrence of recombination [36] : a copy-choice mechanism and a breakage and rejoining mechanism. of these, the former apparently occurs more commonly as it has been shown in the poliovirus [37] , coronaviruses [38] , and plant viruses [39] . this mechanism of viral rna recombinations can further be divided into 3 types: precisely homologous, imprecisely (aberrantly) homologous, and nonhomologous [16] . among these, precisely homologous recombination through a template-switching (copy-choice) mechanism is probably most common [40] . as in our previous report, different strains of the jev can coinfect host cells derived from mosquitoes or mammals [25] , which actually generates recombinant forms of the virus [30] . in this study, we infected host cells with nakayama strains of the jev, followed by transfection of the (+)5 3 -utr-i rna fragment. the result was parallel to our previous observation [25] , showing imbalanced rna recombination between bhk-21 and c6/36 cells. looking at rna accumulation of jev in host cells, it takes longer, at least a 24 h difference, in mosquito cells to reach the level of that in mammalian cells. the stability of viral rna was also shown by degradation of transfected single-stranded rna fragments, either positive or negative sense, particularly in mosquito cells. it implicated that viral rna is less stable at least at the early phase of infection by jev in mosquito cells, which may result in delayed growth of the virus. since transfected rna fragments were degraded in both c6/36 cells and bhk-21 cells, rnase cleavage may be actually involved in viral rna degradation to form small rnas [41] . however, degradation of transfected rna fragments in c6/36 cells was partially ameliorated by treatment with pap, suggesting that viral rnas are not completely degraded by the rnase cleavage pathway [42] . perhaps rna interference (rnai) plays an important role in the related events [43] . generally double-stranded replicative-form rna (dsrf-rna) accumulates to provide an immediate signal which activates specific transcription factors such as type-i interferon (ifn) [44] and facilitates the triggering of intracellular figure 5 : viral rna, either t1p1-s1 or cjn-s1, accumulated in c6/36 cells more slowly than in bhk-21 cells. (a) the rna amount of t1p1-s1 remained at the baseline level until 12 h after infection (hpi) (3.81-fold change), which increased to 169.72-fold at 24 hpi in c6/36 cells. in contrast, t1p1-s1 rna, respectively, increased to 3.09-, 28.99-, 429.05-, 4396.07-, and 5487.75-fold, at 3, 6, 9, 12, and 24 hpi in bhk-21 cells. the rna amount of cjn-s1 also accumulated more slowly in c6/36 cells than bhk-21 cells, which remained at the baseline level until 12 hpi (2.36-fold increase) and had increased to 152.32-fold by 24 hpi in c6/36 cells. although the amount of cjn-s1 rna did not evidently increase until 6 hpi (1.35-fold change), it increased to 16.64-, 111.43-, and 554.87-fold at 9, 12, and 24 hpi, respectively, in bhk-21 cells. (b) stability of viral rna was evaluated after a fragment of (+)5 3 -utr-ii rna was transfected into bhk-21 or c6/36 cells. transfected fragments were insignificantly degraded even at 3 or 6 h after transfection in bhk-21 cells while more obvious degradation appeared in c6/36 cells. innate immunity in mammalian cells [45] . on the other hand, dsrnas formed in invertebrate cells are usually cleaved to be sirna that consequently degrades viral rnas [46] , leading to rnai-mediated innate immunity [47] . small rnas ranging from 10 to 24 mer have been identified in c6/36 cells infected by west nile virus [43] . we have also detected normal expression of dicer-2 in c6/36 cells infected by jev for 12 h although it was almost half of inhibition at 6 hpi (data not shown). as a result, dsrf-rna of the jev may have a great potential for viral rna degradation at least in the early phase of infection in mosquito cells. this adjustment of rna amount is believed to be the way for a delay in rna accumulation and thus a lower frequency of rna recombination of the jev. in contrast, dsrnas are recognized as a central component of ifn and therefore are incapable of mediating rnai in mammalian cells [48] . eventually, our results have shown that protection of rna from rnase cleavage increases the efficiency of rna recombination particularly in mammalian cells. rna recombination creates advantageous genotypes by evolutionary jumps [30] , which permits the removal of deleterious genes based on the notion of "muller's ratchet" from the host cell, usually mammalian cells [12] . notably, viral rnas usually accumulated at a lower amount in mosquito cells through rnase cleavage as well as rna-mediated pathways, leading to stagnancy of rna recombination which may brake evolution of the jev and probably most, if not all, arboviruses which are maintained in nature by alternate cycles involving mosquitoes and vertebrates [28] . autoimmunity-related demyelination in infection by japanese encephalitis virus flavivirus encephalitis flaviviridae: the viruses and their replication complete nucleotide sequence of the japanese encephalitis virus genome rna quasispecies theory in virology rapid fitness losses in mammalian rna virus clones due to muller's ratchet genetic diversity in rna virus quasispecies is controlled by host-virus interactions mosquitoes put the brake on arbovirus evolution: experimental evolution reveals slower mutation accumulation in mosquito than vertebrate cells e/ns1 modifications of dengue 2 virus after serial passages in mammalian and/or mosquito cells the advantage of sex in the rna virus 6 genetic evolution of japanese encephalitis virus evolutionary aspects of recombination in rna viruses a genetic map of poliovirus temperaturesensitive mutants heterologous recombination in the double-stranded rna bacteriophage 6 rna recombination in brome mosaic virus, a model plus strand rna virus how rna viruses exchange their genetic material the extent of homologous recombination in members of the genus flavivirus a phylogenetic survey of recombination frequency in plant rna viruses western equine encephalitis virus is a recombinant virus evidence for recombination in natural populations of dengue virus type 1 based on the analysis of complete genome suquences phylogenetic evidence for recombination in dengue virus rna recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease adaptation of two flaviviruses results in differences in genetic heterogeneity and virus adaptability quasispecies of dengue virus experimental evidence that rna recombination occurs in the japanese encephalitis virus genetic diversity and slow rates of evolution in new world alphaviruses mutations in the ns3 gene and 3 -ncr of japanese encephalitis virus isolated from an unconventional ecosystem and implications for natural attenuation of the virus rates of molecular evolution in rna viruses: a quantitative phylogenetic analysis mutation rates among rna viruses why do rna viruses recombine? genetic recombination with poliovirus type 1. studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain evidence of structural genomic region recombination in hepatitis c virus rna recombination of hepatitis delta virus in natural mixed-genotype infection and transfected cultured cells evidence of recombination in the norovirus capsid gene recombination and positive selection identified in complete genome sequences of japanese encephalitis virus rna recombination in animal and plant viruses the mechanism of rna recombination in poliovirus highfrequency rna recombination of murine coronaviruses rna-rna recombination in plant virus replication and evolution efficient in vitro system of homologous recombination in brome mosaic bromovirus accumulation of a 3 -terminal genome fragment in japanese encephalitis virusinfected mammalian and mosquito cells exonucleases and endonucleases involved in polyadenylation-assisted rna decay c6/36 aedes albopictus cells have a dysfunctional antiviral rna interference response how cells respond to interferons effect of double-stranded rna and interferon on the antiviral activity of atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic necrosis virus inhibition of viral gene expression and replication in mosquito cells by dsrna-triggered rna interference mosquito rnai is the major innate immune pathway controlling arbovirus infection and transmission the response of mammalian cells to double-stranded rna the authors thank yi-hsuan chiang, chao-fu yang, and tien-huang chen for their technical assistance in this study. this work was supported by a grant from chang gung memorial hospital (cmrpd 190161∼3) and partly from the ministry of science and technology, executive yuan, taiwan (nsc100-2313-b-182-001-my3). the authors declare that there is no conflict of interests regarding the publication of this paper. wei-wei chiang and ching-kai chuang contributed equally to this work. key: cord-303978-z3888e3g authors: hong, ka lok; sooter, letha j. title: single-stranded dna aptamers against pathogens and toxins: identification and biosensing applications date: 2015-06-23 journal: biomed res int doi: 10.1155/2015/419318 sha: doc_id: 303978 cord_uid: z3888e3g molecular recognition elements (mres) can be short sequences of single-stranded dna, rna, small peptides, or antibody fragments. they can bind to user-defined targets with high affinity and specificity. there has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the gold and szostak laboratories. a large number of target specific nucleic acids mres and their applications are currently in the literature. this review first describes the general methodologies used in identifying single-stranded dna (ssdna) aptamers. it then summarizes advancements in the identification and biosensing application of ssdna aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. lastly, an overview of the basic principles of ssdna aptamer-based biosensors is discussed. target detection in diagnostics and sensors relies on successful molecular recognitions. traditionally, antibodies have been used in biosening applications due to their target specificities and affinities. however, the inherent properties of proteins give rise to many shortcomings of antibodies. in 1990, the gold laboratory first described a process, termed systematic evolution of ligands by exponential enrichment (selex) [1] , which identifies one or few molecular recognition elements (mres) with high affinity and specificity toward their intended targets. mres can be short sequences of single-stranded dna, rna, small peptides, or antibody fragments. all types of mres are capable of binding to user-defined targets with high affinity and specificity, and these targets include proteins, small molecules, viruses, whole bacteria cells, and mammalian cells [2] . in order to identify nucleic acid mres, the selex process generally begins from a very large random library consisting of 10 13 to 10 15 different molecules. an individual nucleic acid mre is composed of two constant regions for primer attachment during polymerase chain reaction (pcr) amplification flanked by 20-80 bases of random region [3] . the target of interest is first incubated with the library under specific ionic and temperature conditions. library molecules that bind to the target are retained and amplified by pcr, while nonbinding library molecules are discarded. negative or counter selections are often performed to increase the specificity of the library or direct the enrichment process away from binding to negative targets. negative targets are often chosen for their structural similarities or the likelihood to coexist in the native environment with the target of interest. in this case, library molecules that bind to negative targets are discarded and those that do not bind are retained and amplified and thus completing one round of in vitro selection ( figure 1 ). it is expected that the library is enriched enough after approximately 12 rounds of selex. one or few nucleic acid mres with high specificity and affinity toward their targets can be identified. both dna and rna mres can conform into three dimensional structures, which include stem-loop, bulges, and/or hairpin regions and give rise to binding pockets for their respective targets [4] . there are reports suggesting that rna mres generally have a higher affinity for their target than their dna counterparts [5] . however, unmodified rna molecules are more susceptible to nuclease degradations than dna. modification on the 2 hydroxyl of rna molecules can increase their stabilities but may have negative impact on their binding affinities [6, 7] . it is also more difficult to amplify rna mres during selection, as reverse transcription to dna must be performed prior to pcr. for these given reasons, there is a bigger hurdle to successfully identify and apply rna mres in molecular detection, and thus this review has chosen to focus on the discussion of ssdna mres in biosening applications. single-stranded dna mres have high affinity and specificity toward their targets that is comparable to antibodies. in addition, ssdna mres have several advantages over antibodies. firstly, ssdna mres are more thermostable and can be reversibly denatured. this reusability is particularly desired for molecular sensing applications. secondly, ssdna mres can be identified for targets that are nonimmunogenic or toxic to cells, as the selex process can be performed completely in vitro and independent of living systems. lastly, identified ssdna mres with known sequences can be chemically synthesized at low cost and without batch to batch variations [8] . different modifications such as thiol or amino functional groups can also be easily incorporated onto the 3 and/or 5 ends of oligonucleotides during synthesis and utilized for immobilization on solid platforms. similarly, labeling molecules such as biotin or fitc can also be covalently attached and serve as reporters in sensing applications. the attractive features of ssdna mres allow researchers to investigate the translational application of biosensors. this review focuses on the recent advancements in the identification and biosensing application of ssdna mres specific for bacteria, viruses, their associated biomolecules, virulence factors, and selected biological and chemical toxins. detection of these targets has been shown to be important in medical diagnosis, food safety, and environmental monitoring. additionally, major principles in mre based biosensors are briefly discussed. recognition elements 2.1. general methodology of selex. the general process of in vitro selection of ssdna mres starts from design and chemical synthesis of ssdna library. ssdna library consists of two predetermined constant regions for primer attachment during pcr amplification flanking a random region. this random region gives raise to the diversity of the library, which can be designated by 4 , where is the number of bases in the random region. longer random regions not only may result in increased library diversity, but also may risk inhibition of pcr amplification due to secondary structure formation. therefore, the overall libraries lengths are usually designed to be less than 150 bases in total length, including a random region of 20 to 80 bases, and are chemically synthesized using phosphoramidite chemistry [3] . the selex process begins by incubating up to 10 15 different ssdna molecules with the target of interest. one of the key steps in the selex process is the separation of bound mres from unbound mres. the separation process is often achieved by target immobilization. immobilization options include nitrocellulose membranes that can be used to adsorb protein targets [9] and histidine tags on recombinant proteins that can be with a metal affinity chromatography column [10] . however, ssdna molecules may nonspecifically adsorb to immobilizing substrates. a round of negative selection is typically performed prior to the start of the first round of positive selection to reduce the nonspecific adsorption between the library and immobilizing substrates. magnetic beads have also been used to immobilize a wide range of targets [11] [12] [13] [14] . the terminal primary amine or a surface lysine on a protein can be used to conjugate onto carboxylic acid coated magnetic beads via edc/nhs reactions. small molecule targets or target analogs with available functional groups can also be biotinylated and immobilized on streptavidin coated magnetic beads based on the strong affinity between biotin and streptavidin [14, 15] . magnets can then be used for the separation of bound and unbound molecules. however, this technique runs the risk of selecting mres bound to magnetic beads and/or streptavidin. sooter and coworkers successfully showed that competitive elution with free target can effectively isolate ssdna mres specific for the target of interest and not for the immobilizing substrates or analog molecules [14] [15] [16] . amplification of the ssdna library is also crucial to the success of the in vitro selection process. pcr conditions have to be determined and optimized before the selection process. after the retrieval of target bound ssdna molecules for each round of selection, a small-scale pcr can be carried out to determine the cycles of pcr needed to successfully amplify the library. large-scale pcr can subsequently be performed based on the determined number of reaction, and thus decreasing the chance of overamplification and the generation of undesired pcr amplicons. it is necessary to obtain ssdna from double-stranded pcr product prior to the subsequent rounds of selection. several techniques have been shown to effectively isolate the single-stranded binding element from double-stranded dna, such as asymmetric pcr, biotin-streptavidin separation, lambda exonuclease digestions, and size separation on denaturing urea polyacrylamide gel electrophoresis. asymmetric pcr uses a different ratio of forward and reverse primer in the reaction mixture to generate both dsdna and ssdna allowing the two types of dna molecules to be visualized and separated using agarose gel electrophoresis. the ssdna is then excised and purified [17] . biotin-streptavidin separation uses a biotin-tagged primer in the pcr amplification process to generate biotinylated dsdna. the dsdna can then be captured by streptavidin coated beads. the unbound strand of dna can be retrieved using sodium hydroxide [18] . lambda exonuclease can selectively digest a phosphorylated strand of the dsdna in 5 to 3 direction. pcr reactions carried out with a phosphorylated reverse primer can be selectively digested by lambda exonuclease, leaving only the forward strand [19] . modified primers can be used to create size differences between the forward and reverse strands and be detected by using urea denaturing page, and subsequently ssdna can be excised and purified [20] . the general process of selex has been modified over the past two decades. these modifications mostly focus on increasing the efficiency in separating bound and unbound mres, increasing specificity of the selected mres, eliminating the need for immobilizing target molecules, selecting against live whole cells, and decreasing the overall labor intensiveness of the selex process. selected modified selex methods pertinent to this review are briefly discussed. negative or counter selection is incorporated into the normal selex process by introducing negative targets that have structural similarity to the target of interest or are likely to coexist in the target's environment. this modification is to increase the overall specificity of the library during selection and thus identify mres that are highly specific to the target. williams and coworkers identified ssdna mre target for herbicide, atrazine, with 2.1-fold higher binding affinity to atrazine than to a closely related herbicide, simazine, by introducing multiple negative selection rounds and increasing stringency during the selection [14] . this stringent negative selection scheme was utilized to obtain two other ssdna mres that bind to their respective targets with high affinity and specificity [15, 16] . capillary electrophoresis can separate molecules based upon their charges. target bound and unbound dna molecules migrate at different rates due to differences in their overall charges, and therefore different species can be separated and collected at different time points. mendonsa and bowser were the first to use capillary electrophoresis to identify a ssdna mre specific for human ige. due to its high efficiency in separating different molecules, mres can generally be identified in 4 to 6 rounds of capillary electrophoresis based selex (ce-selex) [21] . ce-selex can also select mres bound to free targets in solution and without the need of immobilization. a variant of ce-selex utilizes nonequilibrium capillary electrophoresis of equilibrium mixtures (neceem) to achieve separation (non-selex) has also been developed. in non-selex, repetitive rounds of selection are performed without pcr amplification. berezovski and coworkers were the first to use non-selex to identify a high affinity mre ( : 0.3 nm) specific for hras protein [22] . park and coworkers developed an immobilization-free selex method based on -stacking interaction between dna and graphene oxide (go-selex). in go-selex, ssdna library is adsorbed on graphene oxide and then incubated with the target. in the presence of the target, a portion of the ssdna library is released from graphene oxide and bind preferentially to the target, while unbound ssdna remains adsorbed and can be separated by centrifugation [23] . this method was used to isolate ssdna mres specific for bovine viral diarrhea virus type 1 [24] . a highthroughput modification of go-selex was also developed by nguyen and coworkers to identify flexible ssdna mres that are specific for multiple pesticides with affinities in the nanomolar range [25] . nutiu and li developed a different target immobilization-free selex method using a ssdna library containing a 15-base constant region, sandwiched by two random regions, and finally encompassed by two constant primer hybridization regions at both 3 and 5 ends [26] . the 15 bases constant region can hybridize with biotinylated complementary strand and can be captured by streptavidin coated beads. binding of the ssdna library to target molecules induces conformational changes, thus releasing the binding-strand from the complementary strand. this method has been adapted to screen for ssdna mres specific for multiple pesticides [27, 28] . flumag-selex was developed by stoltenburg and coworkers by immobilizing targets on magnetic beads, using fluorescently labeled forward primer during pcr amplification [29] . magnetic separation of bound and unbound mres is performed similarly to traditional magnetic bead based selex. however, the overall binding capacity of the library can be monitored precisely with the presence of fluorescence tag. the selection process can then be terminated when the overall library binding affinity toward the target reaches a plateau. a similar technique has been incorporated in single microbead selex described by tok and fischer. in their work, only 2 cycles of selex were performed to identify multiple ssdna mres specific for botulinum neurotoxin with low micro-to nanomolar values [30] . the usage of fluorescence tag in the library is further investigated by lauridsen and coworkers by performing a one-step selection against alpha-bungarotoxin [31] . microfluidic chips are also being investigated to facilitate the selex process (m-selex). microfluidic chips are capable of manipulating a very small amount of immobilized target on magnetic beads, thus achieving a more efficient separation of bound mres [32] . qian and coworkers were able to identify ssdna mres specific for botulinum neurotoxin type a with low nanomolar binding affinity after only one round of selection [32, 33] . recently, mres with nanomolar binding affinity specific for whole influenza a/h1n1 virus were selected using m-selex [34] . complex targets such as live mammalian and bacteria whole cells have become popular targets for selection. these types of selection are called cell-selex or whole cell-selex. early works mostly focused on identifying mres specific for tumor cells [35] [36] [37] [38] . the general methodology of cell-selex is very similar to traditional selex, but fluorescence-activated cell sorting (facs) can be utilized to achieve a very high level of separation of mre bound and unbound cell targets. multiple pathogenic bacteria genera, such as salmonella, pseudomonas, staphylococcus, listeria, and escherichia have been chosen as a selection target. the selection and biosening application of ssdna mres targeting bacteria, viruses, and associated biomolecules are discussed in the following section. singlestranded dna mres targeting bacteria can be classified into two general categories, (1) targeting whole cells with known or unknown molecular targets and (2) targeting predefined bacteria cell surface targets or bacteria spores (table 1) . multiple virulent strains of the gram-negative bacteria, escherichia coli, have been chosen as targets for the selection of specific ssdna mres due to their enterotoxigenic effects and the potential of contaminating food and water [39] . peng et al. enriched ssdna mre library specific for e. coli k88 whole bacteria [40] . they also developed a sandwich detection system, in which biotinylated antibodies targeting the k88 strain were immobilized on magnetic beads as the capturing element and the 5 fitc labeled ssdna library from round 13 selection served as the reporter in a fluorescent assay. a lower limit of detection (lod) of 1100 cfu/ml was achieved in pure culture. artificial contaminated fecal samples were also tested with a lod of 2200 cfu per gram. however, no individual ssdna mre was able to achieve the same degree of binding affinity as the whole library and ssdna mre with high affinity and specificity against k88 fimbriae protein was selected after 11 rounds [41] . a fluorescence binding assay was used to obtain the affinity of the selected mre candidates. the reported equilibrium dissociation constant ( ) for the best candidate mre was 25â±4 nm. kim et al. performed 10 rounds of selection against a fecal strain of e. coli along with multiple negative selections against other species of bacteria. they identified four candidate sequences with high affinity for the target strain. all four candidates were highly selective against negative target bacteria. however, they all showed cross-binding activity with other strains of e. coli. this suggested that the selected candidates potentially bound to common antigens expressed in multiple strains of e. coli [42] . savory et al. identified ssdna mre with high specificity and affinity ( = 110 nm) for an uropathogenic strain of e. coli. quantitative pcr was used to monitor the selex process in order to minimize the number of rounds of selex required. after 5 rounds of selex, a selected ssdna mre containing a guaninequadruplex sequence motif showed low cross-binding activities toward other strains of e. coli [43] . in addition to selecting whole e. coli bacteria as targets, outer membrane protein from e. coli 8739 (crook's strain) and lipopolysaccharide from o111:b4 strains were also chosen as targets for selection. a fluorescence resonance energy transfer (fret) assay was developed to detect e. coli 8379 with a lod of 30 cfu/ml [44] . the ssdna mre targeting lipopolysaccharide showed antibacterial effects on both o111:b4 and k12 strains [45] . however, values were not reported in either study. several ssdna mres have been selected against species of foodborne bacteria including salmonella, listeria, and vibrio. dwivedi et al. identified ssdna mre specific for whole cell salmonella enterica serovar typhimurium with a reported of 1.73 â± 0.54 m after eight rounds of selection [46] . two rounds of negative selection against a mixture of nontarget bacteria were also performed to enhance the selectivity of the library. a detection application was developed using immobilized biotinylated mres on streptavidin coated magnetic beads as the capturing elements and was coupled with quantitative pcr. the reported lod of this assay was between 100 to 1000 cfu in a 290 l sample volume. duan et al. performed a similar selection on the same organism with nine rounds of target selection and two rounds of negative selection against mixtures of nontarget bacteria [47] . the best candidate ssdna mre had a value of 6.33 â± 0.58 nm and high specificity based upon flow cytometry analysis. a fluorescence bioassay achieved a lod of 25 cfu/ml. another similar study performed by moon et al. showed relatively high affinities and specificities of selected candidate sequences after ten rounds of target and six rounds of negative selections. however, no values were reported in the study [48] . outer membrane proteins of salmonella enterica serovar typhimurium were chosen as selection target by joshi et al. in that study seven rounds of selection were performed with three rounds of negative selection against e. coli outer membrane proteins and lipopolysaccharides. a magnetic bead based quantitative real-time pcr assay was developed using immobilized ssdna mre as the capturing element. food and environmental samples were tested to demonstrate the translational usage of the assay. a lod of less than 10 cfu per gram of artificially contaminated bovine feces was reported. additionally, 10 to 100 of cfu were detected in 9 ml of artificially contaminated whole carcass chicken rinse sample solution in a pull-down assay [49] . two recent studies identified ssdna mres specific for two serovars of salmonella, typhimurium, and enteritidis [50, 51] . park et al. truncated out the random region (29 to 30 mer) of selected candidates and identified three ssdna mres with values in micromolar range toward their respective serovars after ten rounds of mixed target and counter target selection. poly-d-lysine was conjugated to the selected mres and achieved an approximately 20-to 100-fold enhancement in their binding affinities [51] . kolovskaya et al. also performed a similar selection on the two serovars of salmonella [50] . after twelve rounds of selection, two ssdna mres with values range in nanomolar were identified (enteritidis: = 7nm; typhimurium: = 25nm). to identify an mre with high affinity ( = 47 â± 3 nm) and specificity toward paratyphi a. lod of 1000 cfu/ml was achieved using chemiluminescence assay based on selfassembled single-walled carbon nanotubes and dnazymeslabeled mre as detection elements [52] . mre with high specificity toward salmonella o8 was identified by liu et al. after eleven rounds of positive and two rounds of negative selection. the selected mre had a reported of 32.04 nm. a preliminary fluorescent in situ labeling assay was developed with the mre. however, no lod was reported [53] . consumption of uncooked or undercooked seafood contaminated by vibrio bacteria can lead to food poisoning [54] . two different species, vibrio parahaemolyticus and vibrio alginolyticus were chosen as selection targets. nine rounds of cell-selex using flow cytometry were carried out to identify ssdna mre with high affinity and specificity for vibrio parahaemolyticus ( = 16.88 â± 1.92 nm) [55, 56] . tang et al. performed 15 rounds of cell-selex on inactivated vibrio alginolyticus. negative selection was performed every third positive target round to improve the library specificity. the study did not characterize affinities and specificities of candidate ssdna mres from the last round of selection. instead, the whole library was characterized to have a value of 27.5 â± 9.2 nm and was highly specific toward the target. the enriched library was able to detect 100 cfu/ml of the bacteria based on a pcr amplification assay [56] . listeria monocytogenes is a foodborne gram-positive bacterium that can cause serious illnesses and even death. fda and european union both have zero tolerance of l. monocytogenes in ready-to-eat food products. suh et al. conducted two studies to identify ssdna mres specific for l. monocytogenes [57, 58] . in their earlier study, mre with a micromolar value was identified after six rounds of positive and two rounds of negative selections. the mre showed low cross-binding to negative target bacteria but had similar binding affinity for other members of the listeria genus. a magnetic bead based capture assay coupled with quantitative pcr was developed. the assay was able to detect less than 60 cfu in 500 l of binding buffer containing a mixture of non-listeria bacteria [58] . in their later study, the affinities of selected candidate mres were improved with reported values of in the nanomolar range and were specific for the target bacteria at different growth phases [57] . duan et al. performed similar whole cell in vitro selection on l. monocytogenes. the selected mre had high affinity ( = 48.74 â± 3.11 nm) and was highly specific toward the target. a fluorescent cross-binding assay showed significantly lower binding activities toward negative bacteria targets and other bacteria species in the listeria genus. a sandwich fluorescent bioassay was developed and demonstrated a lod of 75 cfu/ml [59] . most recently, liu et al. performed eight rounds of selection to identify ssdna mres specific for l. monocytogenes. the best candidate mre reported to have a value of 60.01 nm and had high specificity. a fluorescent based detection assay was developed to enable the observation of binding between the mre and target bacteria using fluorescent microscope, but the lod was not reported [60] . ohk et al. selected ssdna mre specific for internalin a of l. monocytogenes. internalin a is a major invasion protein expressed on the cell surface of l. monocytogenes [61] . a highly specific sandwich style fiber optic biosensor was developed by using the selected mre and antibody. a reported lod of 1000 cfu/ml was achieved. the sensor also successfully detected the bacteria in artificially contaminated ready-to-eat meat products. however, affinity data was not reported in the study [62] . shigella dysenteriae is a gram-negative bacterium that causes severe epidemic diarrhea in many countries [63] . duan et al. used cell-selex methodology to identify ssdna mre specific for s. dysenteriae [47, 55, 59, 64] . the best candidate mre had a reported value of 23.47 â± 2.48 nm and low cross-binding activities toward negative bacteria targets. a fluorescent based detection assay demonstrated a lod of 50 cfu/ml [64] . campylobacter jejuni is a highly infectious gram-negative bacterium that is one of the leading causes of acute diarrheal sickness worldwide [65] . bruno et al. performed an in vitro selection by extracting surface proteins of c. jejuni and immobilizing them on magnetic beads. no values of were reported in the study. however, a fluorescent assay based on magnetic beads and quantum dot was developed to detect the bacteria in different food matrices. the assay showed low cross-binding activities with other species of bacteria but was not able to distinguish between bacteria in the campylobacter genus. the reported lods were 2.5 cfu and 10 to 250 cfu in buffer solution and in different food matrices, respectively [66] . ce-selex was employed by stratis-cullum et al. to identify mres specific for c. jejuni. killed bacteria were used as target in their study. a qualitative capillary electrophoresis immunoassay was developed with a lod of 6.3 ã� 10 6 cells/ml [67] . dwivedi et al. performed cell-selex on live c. jejuni. a total of ten positive rounds and two negative rounds were carried out to identify ssdna mres with high affinity and specificity toward the target bacteria ( = 292.8 â± 53.1 nm) [68] . bacteria that are associated with common infectious diseases, such as streptococcus, staphylococcus, and pseudomonas, are also popular targets for in vitro selection. identification of mres targeting infectious bacteria could be potentially used to facilitate diagnosis and thus decreasing the time between culture collections to specific antibiotic treatment. savory et al. performed cell-selex on proteus mirabilis, a common cause of catheter associated urinary tract infections in long-term catheterized patients. mres specific for two different strains of p. mirabilis with low nanomolar range values were identified after 6 rounds of in vitro selection. additionally, an in silico maturation (ism) process was performed to increase the specificity of the selected mre. it was reported that a 36% higher specificity was achieved after the ism process [69] . this same technique was again employed to select mre specific for streptococcus mutants, the main causative pathogen of dental caries. the affinity of the identified mre was improved up to 16-fold and the specificity was increased 12-fold after ism. a gold colloids based colorimetric flow-through assay was developed and demonstrated the detection s. mutants in the range of 10 5 -10 8 cfu/ml [70] . streptococcus pyogenes (group a streptococcus) is often the causative pathogen of a wide range of infectious diseases, such as streptococcal pharyngitis and streptococcal toxic shock syndromes [71] . different m-types of live s. pyogenes were chosen for selection by hamula et al. after 20 rounds of target selection, the two best candidate mres yielded high affinity for group a streptococcus ( = 9-10 nm). it was noteworthy that the candidate mres showed good specificities, even though the authors did not perform any negative selections [72] . staphylococcus aureus is a gram-positive bacteria associated with numerous of infections in human [73] . cao et al. selected a panel of ssdna mres specific for s. aureus after several rounds of target and counter target selection. the reported values of individual candidate mres were in the nanomolar range with high specificity. the study showed that the combination of the panel of mres yielded a better sensitivity in recognizing s. aureus than any single mre [74] . change et al. selected two ssdna mres with high affinities and specific toward s. aureus ( = 35 and 129 nm). the reported values of improved to 3.03 and 9.9 nm, respectively, after thiol-modification and conjugation to gold nanoparticles. subsequently, the mre conjugated gold nanoparticles were utilized to capture target bacteria and a resonance light-scattering signal demonstrated the detection of single s. aureus cell in 1.5 hours [75] . pseudomonas aeruginosa is a gram-negative bacterium that is commonly associated with nosocomial infections [76, 77] . wang et al. performed 15 rounds of positive and 2 rounds of counter target selection to identify ssdna mres with values in the low nanomolar range. the selected mre showed negligible binding to counter bacteria cell targets. a fluorescence in situ hybridization (fish) assay was developed to show rapid detection of p. aeruginosa. however, the detection ranges were not reported [78] . mycobacterium tuberculosis is the etiologic pathogen for tuberculosis [79] . chen et al. reported ssdna mre with an apparent association constant ( ) between 10 5 -10 6 m and was highly specific. the authors reported an antibacterial effect of the selected mre with both in vitro and in vivo models [80] . highly infectious bacteria and bacteria spores have been considered as potential biological warfare agents, and it is important to detect these biological threats rapidly [81] . bruno and kiel 1999 performed an in vitro selection of ssdna mres targeting bacillus anthracis spores, the causative agent of anthrax. autoclaved anthrax spores were used in the selection. mre-magnetic bead electrochemiluminescence sandwich assay was developed with a reported detection range of 10-10 6 spores [82] . ikanovic et al. performed a selection of ssdna mres specific for bacillus thuringiensis spores, a closely related species to b. anthracis. in this study, the methodology was adopted from bruno and kiel 1999. a fluorescent assay based on cadmium selenide quantum dots was developed with a reported detection limit at about 1000 cfu/ml [83] . bruno and carrillo 2012 revisited the selection of bacillus spores. in this later study, anthrose sugar on anthrax spores was chosen as target for selection. mre beacon based on fluorescent signals was developed and generated strong signal at spore concentrations greater than 30,000 spores/ml. the authors also compared the mre sequences pattern to previous studies and identified similarities in sequences composed of t/g rich bases. it was also reported that mres specific for whole spores did not generate fluorescent signals in the presence of anthrose sugar, suggesting that the selected spore specific mres possibly bound to a different epitope and warranting further research [84] . francisella tularensis is an encapsulated, gram-negative coccobacillus that is highly infectious. reports show as few as 25 organisms in aerosol can cause diseases [85] . vivekananda and kiel performed ten rounds of selection on francisella tularensis subspecies japonica bacterial antigen. a cocktail of 25 ssdna mres was reported to have high specificity toward the target bacteria. mre modified enzyme linked immunosorbent assay was developed, and demonstrated binding to the target and other subspecies of f. tularensis but not to other species of bacteria and chicken lysozyme or chicken albumin. in addition, the assay was able to achieve better sensitivity then traditional elisa using anti-tularemia antiserum and anti-tularemia polycolonal antibodies. the reported lod is 1700 bacteria/ml [86] . peptidoglycan is a macromolecule universally expressed on bacteria outer cell wall [87] . ferreira et al. identified two ssdna mres with sub-to low micromolar values that can bind specifically to both gram-positive and gram-negative bacteria. neither mre bounded to human fibroblasts or candida albicans and could potentially be used as generic detection elements for bacteria [88] . lipopolysaccharide (lps or endotoxin) is expressed in the outer membrane of gram-negative bacteria and can illicit strong immune response upon entering into mammalian cells. [89, 90] kim et al. used nonequilibrium capillary electrophoresis of equilibrium mixtures (neceem) based non-selex to identify multiple ssdna mres with high affinities toward lipopolysaccharide in only three rounds of selection. selected mres also demonstrated very low cross-binding activities to bovine serum albumin and other intracellular molecules, such as dna, rna, glucose, and sucrose, in an electrochemical assay. this assay resulted in a target detection range of 0.01 to 1 ng/ml [91] . there is a wealth of literature describing ssdna mres targeting various virus life cycle regulator proteins with the purpose of therapeutic application. in contrast, there is a lesser amount of research on ssdna mres for virus biosensing application ( table 2 ). for the focus of this review, those mres with therapeutic applications are listed in the following table without further detail discussions (table 3) . in recent years, there has been an increase in the interest in the application of ssdna mres for virus detection. [10] . go-selex was utilized to identify ssdna mres specific for bovine viral diarrhea virus. after five rounds of positive and negative selections, three best candidate mres had reported values of 4.08 ã� 10 4 , 4.22 ã� 10 4 , and 5.2 ã� 10 4 tcid 50 /ml, respectively, by spr kinetics analysis. all candidate mres showed very high specificity toward the target. a sandwich spr detection assay was developed wherein a biotinylated mre was immobilized on streptavidin coated gold chip as the capturing mre, and a second different mre with thiol modification was conjugated to gold nanoparticle as the reporting mre. a lod of 800 copies of virus/ml was reported with this assay [24] . hepatitis c virus (hcv) envelope surface glycoprotein e2 was chosen as target for selection by chen et al. e2 protein was expressed on a murine colon carcinoma cell line, ct26 cells, and used as a target for positive selection. the native ct26 cells were used as counter target. after 13 rounds of selection, the best candidate mre showed high affinity and specificity toward e2-positive cells. an elisa virus capture assay was developed by using biotinylated mre as reporter and demonstrated the detection of hcv in clinical human serum samples. in addition, the mre, termed ze2 also displayed therapeutic effect by inhibiting e2 protein binding to cd81 and blocking hcv infection of human hepatocytes in vitro [100] . dengue virus is a member of family flaviviridae, genus flavivirus. it is a mosquito-borne rna virus that can cause gangue fever, dengue hemorrhagic fever, and dengue shock syndrome [101] . gandham (table 4) . staphylococcus aureus can secrete a group of thermostable enterotoxins that have been shown to contaminate food. reports suggest that these toxins are a common cause of foodborne illnesses [104] . there are many types and subtypes of staphylococcus enterotoxins. bruno and kiel first selected ssdna mres that bind to enterotoxin b by using magnetic bead immobilized target. an electrochemiluminescence assay was developed to demonstrate a detection limit of less than 10 pg of enterotoxin b. however, no kinetic data or crossing-binding profiles were presented in the study [105] . degrasse recently identified ssdna mre specific for enterotoxin b after fourteen rounds of mixed target and negative targets selection. mre based precipitation assay was used to analyze the selectivity of candidate mres in cellfree culture supernatant from multiple strains of s. aureus. the high selectivity of candidate mres was confirmed by capturing only the target toxin in the precipitation assay. however, no quantitative binding data was presented in the study [11] . enterotoxin subtype c1 was chosen as a target for selection by huang et al. after twelve rounds of selection, the best candidate mre demonstrated high affinity for enterotoxin c1 ( = 65.14 â± 11.64 nm). crossbinding experiments showed that the selected mre had high specificity and low cross-binding activities on staphylococcus enterotoxin a, enterotoxin b, and other protein molecules. a graphene oxide based fluorescence detection assay was developed and achieved a reported lod of 6 ng/ml in artificially contaminated buffer-diluted milk samples [106] . bruno and kiel 2002 selected ssdna mre against cholera toxin. an enzyme linked colorimetric assay showed a detection limit of less than 10 ng of cholera toxin and electrochemiluminescence assay shows a detection limit of less than 40 ng. however, affinity, crossing-binding data, and mre sequences were not presented in the study [105] . toxigenic strains of clostridium difficile can produce toxins a and toxin b, which are the contributing factor of c. difficile induced diarrhea. rapid diagnosis of the condition is crucial in facilitating patient recovery and disease control [107] . some strains of c. difficile also secret a binary toxin that can inhibit actin polymerization [108] . ochsner et al. selected several slow off-rate modified ssdna mres (somamer) specific for toxins a, b, and binary toxin. several dna libraries with modifications, such as 5-benzylaminocarbonyl-du (bndu), 5-naphthylmethylaminocarbonyl-du (napdu), 5-tryptaminocarbonyl-du (trpdu), 5-phenylethyl-1-aminocarbonyl (pedu), 5-tyrosylaminocarbonyl-du (tyrdu), or 5-(2-naphthylmethyl) aminocarbonyl (2napdu) were used in selections. truncated recombinant toxins were used as targets. equilibrium dissociation constants of selected somamers were in pico to nanomolar range. the affinities for native toxins were slightly lower but were remain in the low nanomolar range for majority of the candidate sequences. pull-down capture, dot blots, and antibody sandwich assays were developed with a reported lod of 300 pg/ml. selected somamers were able to detect all three toxins in cellfree culture supernatants of toxigenic c. difficile [109] . ochsner et al. performed another in vitro selection on c. difficile binary toxin with sandwich selex. the advantage of sandwich selex is to select somamer pairs that target different epitopes of the target protein. the reported values of selected somamers ranged from 0.02 to 2.8 nm. a somamer sandwich assay was developed with a reported lod in the low picomolar range. the authors claimed that these studies showed the high potential for the development of sensitive and specific diagnostic kits [110] . hong et al. performed twelve rounds of positive in vitro selection against c. difficile toxin b and eleven rounds of negative selection. spr binding study determined the selected ssdna mre had a value of 47.3 â± 13.7 nm. fluorescence plate based cross-binding assay showed the selection ssdna mre was two to five times more selective on toxin b than negative targets. a proof-of-concept modified elisa using ssdna as the toxin capturing element was developed and able to detect toxin b at 50 nm concentration in human fecal matter [111] . tuberculosis (tb) remains to be a challenging disease in developing countries. recent discovery of multidrug resistant strains of mycobacterium tuberculosis has further increased public concerns, however, current diagnostic techniques for tb are either time-consuming or insensitive [112] [113] [114] . rotherham et al. performed a selection on cfp-10.esat6 heterodimer, a specific biomarker for tb infections. after six rounds of selection, spr binding studies showed that candidate ssdna mres had affinities in the nanomolar range. one of the candidate mre was tested in an enzyme linked oligonucleotide assay (elona). the authors reported that the assay had 100% sensitivity and 68.75% specificity in clinical sputum samples using youden's index. however, the time needed for assay completion and crossing-binding activities to other antigens were major limitations of the assay [9] . tang et al. performed a selection on the same cfp-10.esat6 heterodimer. after seventeen rounds of selection, values of candidate mres were in the low nanomolar range. two ssdna mres (ce24, ce15) were used in an elona assay. the reported sensitivity and specificity of ce24 mre based elona were 100% and 94.1%, respectively. ce15 mre based elona had a lower sensitivity of 89.6%, but the specificity was the same. assays were tested both pulmonary and extrapulmonary with serum samples from tb patients [115] . mpt64 is a secreted protein of m. tuberculosis and can be used as biomarker for active tb infections [116] . qin et al. performed twelve rounds of selection on mpt64. affinities of truncated candidate ssdna mres, containing only a 35-base central random region, were qualitatively observed using streptavidin-horse radish peroxidase (hrp) binding to protein-bound biotin-tagged mres. a colorimetric sandwich assay using two different mres was developed to detect the presence of mpt64 in culture filtrates. the sandwich assay achieved sensitivity and specificity of 86.3% and 88.5%, respectively [117] . protective antigen (pa) is a secreted virulence factor of bacillus anthracis that binds to anthrax toxin receptors on mammalian cells and subsequently causes cell dysfunction and death [118] . cella et al. utilized ce-selex to identify ssdna mre targeting pa with high affinity and specificity. after six rounds of ce-selex, the best candidate had alpha toxin filter --[155] a reported of 112 nm. an electrochemical biosensor was developed by immobilizing 5 amino modified mre on 1-pyrenebutanoic acid succinimidyl ester (pase) modified single wall carbon nanotubes (swnt). the sensor showed low cross-binding activity toward human and bovine serum albumin at 100 nm concentration. the sensor surface could be regenerated using 1 l of 6 m guanidine hydrochloride for 15 minutes followed by a wash with 10 mm phosphate buffer. a reported lod of 1 nm was achieved [119] . choi et al. performed an in vitro selection on pa. after eight rounds of selection, four candidate sequences had high affinities for pa ( in low nanomolar range), and two of the four candidates had low cross-binding activities toward bovine serum albumin and bovine serum [120] . botulinum neurotoxins (bont) are produced by clostridium botulinum. in addition to its medical uses, it can also cause serious foodborne illness and may potentially be used as a biological weapon [121] . tok and fischer used a novel single microbead selex to perform selection of ssdna mres specific to aldehyde-inactivated bont type a toxin (bont/a-toxoid) and bont type a heavy chain peptide (bont/a hc-peptiod). targets were immobilized onto ni-nta agarose or amine-functionalized polystyrene tentagel beads. a single target-immobilized microbead was incubated with the ssdna library and retrieved for pcr amplification of bound ssdna molecules. after only two rounds of selection, five candidate sequences specific for bont/a hc-peptiod had values ranging from 1.09 m to 4.20 m. three candidate sequences specific for bont/atoxoid had values ranging from 3 nm to 51 nm. the authors reported that all mres specific to bont/a hc-peptoid were able to competitively inhibit the binding between the toxin peptide and anti-bont antibody and potentially be used as a therapeutic agent [30] . lou et al. utilized a novel microfluidic device to facilitate the partitioning of a small volume of target coated magnetic beads (m-selex). the library achieved a very high overall affinity ( = 33 â± 8 nm) against bont/a light chain after only one round of selection. four candidate sequences had a range of values between 34 and 86 nm. the authors claimed that their m-selex could be readily adapted to any bead immobilized targets or whole cell target [33] . bruno et al. immobilized bont/a light chain on magnetic beads and performed 10 rounds of selection. the best candidate mre was fluorescently tagged and used as a reporter for target detection. the reported lod of 1 ng/ml was achieved in buffer. however, the mre reporter also bound to structurally similar targets, bont type b, type e holotoxins, and heavy or light chain components, in a soil dilution. the author compared their mre sequence to previous ssdna mre specific for bont and found consensus short sequence segments. this suggested that the binding between bonts and mres may be conserved within these consensus segments [122] . microcystin is a hepatotoxin produced by cyanobacteria. three different analogs of microcystin were used in the study performed by nakamura et al. microrocystin lr, containing a leucine substituent, was immobilized and used for twelve rounds of target selection. however, surface plasmon resonance binding data indicated a higher binding level between the selected mre and microcystin yr, an analog containing a tyrosine substituent. there was also significant binding to microcystin rr, an analog containing an arginine substituent. the reported binding affinity ( ) was low, at approximately 10 3 m â��1 . this early work did not demonstrate the high affinity and specificity properties of mres; however, it did show the possibility of using mres as a binding molecule in a label-free detection system [123] . cylindrospermopsin (cyn) is another water soluble and heat stable alkaloid secreted by a large group of fresh water cyanobacteria. it has a variety of toxic effects in human bodies upon exposure to cylindrospermopsin usually through drinking water or food [124] . elshafey et al. recently selected ssdna mre with high affinity and specificity toward cyn, with a reported of 88.78 nm. circular dichroism measurements showed that mre had a conformational change upon binding to cyn. this property was exploited in a labelfree impedimetric biosensor. the reported lod of the sensor was 100 pm with a linear range of 80 nm. it also showed negligible responses toward coexistent cyanobacterial toxins of microcystin-lr and anatoxin-a. cyn was recoverable in a spike test with tap water [125] . saxitoxin is a small neurotoxin produced by few dinoflagellates and certain cyanobacteria that affect marine organisms [248] . handy et al. were the first to select ssdna mre against target saxitoxin. in their study, saxitoxin was conjugated to keyhole limpet hemocyanin (klh) via a spacer compound, 2,2 -(ethylenedioxy)bis(ethylamine), or jeffamine and then the protein-toxin conjugate immobilized on magnetic beads. ten rounds of selection were performed, and negative selection against klh-bead was carried out from round four to the round ten, in order to decrease nonspecific binding to klh and beads. one candidate sequence was analyzed by spr and demonstrated a concentration-dependent and selective binding to saxitoxins. however, the of the selected mre was not presented in the study [145] . okadaic acid (oa) is a phycotoxin produced by dinophysis and prorocentrum algae. it can accumulate in shellfish due to its lipophilic and heat-stable nature. human consumption of oa can lead to a variety of gastrointestinal symptoms [249] . eissa et al. identified ssdna mre with high affinity and specificity toward oa after eighteen rounds of mixed target and negative target selection. the candidate mre with the highest affinity ( = 77 nm) was chosen for circular dichroism analysis. a conformational change in the mre was observed upon binding of oa. a label-free electrochemical impedimetric biosensor was developed with this mre and achieved a lod of 70 pg/ml. it demonstrated no crossbinding activity toward structurally similar toxins, including dinophysis toxins-1 and -2 and microcystin-lr [12] . ochratoxin a (ota) is a mycotoxin produced by members of the aspergillus and penicillium genera. it is a nephrotoxin and has potential carcinogenic effects in humans. it has been shown as a contaminant in many food products, such as grains and wine [250] . however, the current detection method for ota is both expensive and time-consuming [251] . cruz-aguado and penner identified ssdna mre specific for ota after thirteen rounds of selection. the best candidate mre reported had a value of 200 nm. it did not bind nonspecifically to warfarin, n-acetyl-l-phenylalanine, or ochratoxin b in a fluorescent based cross-binding assay [146] . subsequently, the authors developed a detection system based on a fluorescence polarization displacement assay. the author reported that the assay was sensitive to ota but not to warfarin and n-acetyl-l-phenylalanine, with a lod of 5 nm. however, the detection assay did not test ochratoxin b (otb) binding activity or sensitivity in food sample [147] . barthelmebs rounds of selection. after binding and cross-binding analysis, the best candidate had a value of 96 nm with minimal binding to otb and phenylalanine. it was incorporated into an elisa and elaa assays for the detection of ota spiked in pretreated wine samples. different elaa and elisa assays were compared, and a direct competitive elaa had the lowest detection limit of 1 ng/ml with the shortest analysis time of 125 minutes [148] . mckeague et al. performed fifteen rounds of in vitro selection to identify ssdna mres specific for ota. two candidate mres had reported values of 110 â± 50 nm (designated b08) and 290 â± 150 nm (designated a08). a08 ssdna mre was utilized in a labelfree fluorescence detection assay and achieved a lod of 9 nm. it also had low cross-binding activity on otb and warfarin. the authors reported a truncated version of a09 also had similar specificity and binding affinity profiles [149] . fumonisins are heat-stable mycotoxins present in most corn and are produced by fungi, fusarium verticillioides and fusarium proliferatum. fumonisin b 1 (fb 1 ) is a nephrotoxin and potential carcinogen in humans. as the toxin cannot be inactivated by cooking in high temperature, it is crucial to monitor its level during food production [252] . mckeague et al. performed eight rounds of selection to identify ssdna mre with high binding affinity toward fb 1 . unmodified magnetic beads (immobilization substrate), l-homocysteine, l-cysteine, and l-methionine l-glutamic acid were used as negative targets in the selection. six candidate mres were identified, and the best candidate mre had a reported of 100 nm. however, the authors did not test the specificity of the selected mre on other mycotoxins [13] . zearalenone (zen; f-2 toxin) is a nonsteroidal estrogenic mycotoxin produced by many fungus species in the fusarium genus. it has been shown to be present in many grains worldwide, such as oats, wheat, rice, and their derived food products [253] . chen et al. performed fourteen rounds of selection, and the best candidate mre had reported of 41 â± 5 nm and high specificity. cross-binding assays showed insignificant binding to other mycotoxins, -zearalenol, aflatoxin b1, aflatoxin b2, fumonisin b 1 , and fumonisin b 2 . circular dichroism measurement showed a conformational change of the mre after binding of zearalenone. a detection assay using mre immobilized magnetic beads and the bluegreen florescence property of zearalenone was developed. a lod of 0.785 nm was achieved in pretreated beer samples [150] . t-2 toxin (t-2) is a trichothecene mycotoxins produced by many species in the fusarium genus and is harmful to humans. it is a very stable small molecule biological toxin that is resistant to high temperature and is present in variety of grains, such as oats, barley, and wheat. currently, it can only be detected by labor intensive and costly instruments and it is thus difficult to monitor its level in food [254] . chen et al. recently utilized ten rounds of go-selex to identify ssdna mre specific for t-2 with high affinity and specificity. fluorescent binding and cross-binding assay showed that the of the best candidate mre was in the nanomolar range, with insignificant cross-binding activities on fb 1 , zen, ota, and aflatoxin b1. there was a conformational change upon mre-t-2 binding. the authors also developed a fluorescent assay to detect spiked t-2 level in beer. a lod of 0.4 m was achieved [151] . aflatoxins are highly toxic natural compounds produced by many species of filamentous fungi and can contaminate agricultural products. the ld 50 can be as low as 0.5 mg/kg, and acute toxicity is even higher than many chemical toxins, such as cyanide or arsenic [255, 256] . ma et al. performed an in vitro selection on a subtype of aflatoxins, aflatoxins b1 (afb1). after ten rounds of target and negative target selection, the best candidate mre had a reported of 11.39 â± 1.27 nm and with minimal cross-binding activities on aflatoxins b2, g1, g2, ota, and fb 1 . a fluorescent assay similar to the authors' previous study on zen and t-2 specific mre was developed to detect spiked levels of afb1 in methanol-extracted peanut oil. the assay achieved a lod of 35 ng/l [152] . malhotra et al. perform two selections (selex1 and selex2) using slightly different methodologies to identify ssdna mres specific for both afb1 and aflatoxins m1 (afm1). in selex1, lambda exonuclease was used to generate ssdna from amplified dsdna. afm1 coated magnetic beads were used as a positive target from round 1 to round 10, and afb1 coated magnetic beads were used as positive target at round 11 (last round) only. free targets were used to competitively elute ssdna that bound to toxin coated beads in round 10 and round 11. in selex 2, each round started from preincubation with counter targets (uncoated beads, afb1 beads) followed by incubation with afm1 beads. snap cooling was used to obtain ssdna from dsdna. in selex 2, only eight rounds were carried out. multiple candidate mres were analyzed and their values were in the nanoto low micromolar range. one mre with the best affinity ( = 35.6 â± 2.9 nm), designated afas3, was used in developing a colorimetric assay based on mre immobilized gold nanoparticles. this assay had a detection range of 250 to 500 nm of afm1 and only minor interaction with afb1. however, there were no reported cross-binding data on other mycotoxins [153] . two studies identified ssdna mres specific for biological toxins with therapeutic intentions. alpha-bungarotoxin is a toxic substance in krait snake venom and can bind irreversibly to acetylcholine receptors and eventually lead to death in victims [257, 258] . lauridsen et al. performed a rapid one-step selex and identified ssdna mre with relatively high binding affinity toward alpha-bungarotoxin ( = 7.58 m). the authors claimed that rapid selection technique could potentially be used with a chemically modified nucleic acid library and generate mres suitable for diagnostic and therapeutic purposes [31] . vivekananda et al. selected ssdna mre specific for alpha-toxin of staphylococcus aureus. several candidate sequences showed cell rescuing effects when coadministrated with alpha toxin in multiple in vitro neutralization assays. the authors claimed that it was possible to generate mres against alpha-toxin for the treatment of s. aureus infections [155] . hong et al. also performed an in vitro selection against s. aureus alpha toxin. twelve rounds of positive and eleven rounds of negative rounds of negative selection were performed to identify the candidate ssdna mre. the reported determined by spr single cycle kinetics was 93.7 â± 7 nm. acetamiprid immobilization free 4.98 m -[27] fluorescence plate based cross-binding assay showed the ssdna mre was approximately two to five times more selective on the alpha toxin than negative targets. a proofof-concept modified elisa using the selected ssdna mre had a reported sensitive target detection at 200 nm in human serum [154] . toxins. the detection of chemical toxins is important in both food safety and environmental monitoring. environmental and food contamination by various kinds of chemical toxins have been reported and even at low concentrations can still be detrimental to human health. currently, the majority of small chemical toxins can only be detected by labor intensive and costly laboratory equipment such as liquid and/or gas chromatography coupled with mass spectrometry. in order to address these current limitations, there has been an increase in the identification and biosensing applications of mres with high affinity and specificity to capture and detect chemical toxins. however, the in vitro selection of ssdna mres targeting small molecule chemical toxins has several inherent challenges, such as difficulties in efficient separation between bound and unbound dna molecules, limited chemical motifs on target surfaces for sufficient binding, lack of chemical functional groups for target immobilization, and candidate mres that may not have sufficient specificities to distinguish molecules with very similar chemical structures if selection schemes are not carefully designed. for these reasons, there are a limited number of ssdna mres specific for chemical toxins currently in the literature (table 5) however, due to the small molecular weight of e2, there were no observable binding events by spr. an electrochemical platform measured under swv was eventually utilized to detect e2 with a lod of 0.1 nm in buffer solutions [156] . alsager et al. selected a 75-mer ssdna mre specific for e2 with a of 50 nm after eighteen rounds of selection. the 5 amino-modified mre was covalently conjugated to carboxylated nanoparticles and dynamic light scattering/resistive pulse sensing was used to observe size contraction in particle size upon e2 binding. a detection range of 5 nm to 100 nm was achieved with this detection platform. progesterone, testosterone, bis (4-hydroxyphenyl) methane (bpf), and bisphenol-a (bpa) were also tested for the specificity of the selected mre. the assay showed minimal binding to both bpa and bpf; however, the mre was not able to distinguish the other two steroids [157] . bisphenol a (bpa) is an estrogen mimicking chemical that has been classified as an endocrine-disrupting compound. it is used in the manufacture of polycarbonate plastic products, such as plastic bottles and containers. it has been shown to be released into food after heating and can accumulate in human [259] . jo et al. selected ssdna mre specific for bisphenol a with high affinity and specificity. the reported was 8.3 nm with only minimal binding to structurally related chemical molecules, including 6f biophenol a, bisphenol b, and 4, 4 -bisphenol. a cy-3 labeled mre pair was immobilized on sol-gel biochip and a sandwich detection assay was developed with nanomolar range sensitivity. however, the authors acknowledged the assay system can only detect a limited range of bpa concentrations [158] . polychlorinated biphenyls (pcb) are a group of chlorinated hydrocarbons that are used in varies of industrial settings. pcbs are highly toxic and are reported to be an environmental contaminant affecting water bodies and food sources [260] . mehta et al. identified pcb binding ssdna mres with nanomolar range affinity. in their study, two pcb compounds with hydroxyl functional group were immobilized on magnetic beads and used as target for selection. after nine rounds of selection, three candidate sequences were chosen for characterization. two of the three candidate sequences (9.1 and 9.3) showed comparable binding affinities to both immobilized targets. in subsequent crossingbinding analysis, candidate 9.1 showed broad substrate binding affinity to other pcb compounds, while candidate 9.2 showed a high specificity for the two pcbs with hydroxyl functional groups. the study did not test specificity on other hydrocarbons that are structurally similar to pcb [160] . xu et al. immobilized a primary amine modified pcb compound (pcb77-nh 2 ) on epoxy-activated sepharose agarose as the target for in vitro selection. after 11 rounds of selection, four candidate sequences were characterized to have affinity in the low micromolar range. cross-binding assays showed only minimal binding toward other hydrocarbons and agarose substrate. a fluorescent based detection assay was developed using the fluorescence quenching property of gold nanoparticle. upon binding to target, the fluorescent signal was released. a detection range of 0.1-100 ng/ml was achieved. this assay detected other pcb compounds with different sensitivities [159] . the current detection method for herbicides and pesticides environmental contaminants in the environment relies on using time-consuming and labor intensive laboratory based equipment. mres have been investigated as binding elements in rapid, field deployable detection systems. atrazine is a widely used herbicide worldwide [261] . sanchez utilized ce-selex to identify ssdna mre specific for atrazine with a of 890 nm. however, the mre did not show specificity in binding between atrazine and structurally closely related simazine at concentration below approximately 2 m in a fluorescence polarization detection assay [161] . williams et al. also performed an in vitro selection of ssdna mre specific for atrazine. a derivative of atrazine, desethyl-atrazine was first biotinylated and then immobilized on streptavidin coated magnetic beads. the selection scheme was designed with increasing selection stringency, by incorporating negative selections on streptavidin magnetic beads, simazine, metabolites of atrazine, and other commonly used pesticides. competition selection was also performed to ensure the library bound only to free atrazine in solution, but not to desethyl atrazine. as a result, ssdna mre with subnanomolar affinity and high specificity was identified after twelve rounds of selection. a magnetic bead based capture assay coupled with capillary electrophoresis was developed to detect atrazine in artificially contaminated river water samples. the assay was able to detect atrazine in the nanomolar range [14] . similar in vitro selection methodology was also employed by williams et al. to identify mres specific for a commonly used organophosphate pesticide, malathion. in their second selection, the selected mre had high nanomolar range affinity, and minimal binding to metabolites of malathion and other herbicides. however, the author noted that the cross-binding activity was high on bovine serum albumin possibility due to the large, globular characteristics of the protein [15] . william et al. subsequently performed another selection on an herbicide, bromacil. this study further validated the methodology the authors employed to identify mres with high affinity and low cross-binding activities on structurally similar compounds and compounds that were likely to coexist in the environment. the authors noted that these properties were particularly important for incorporating ssdna mres as sensing elements in biosensors [16] . as noted above, not every chemical toxin can be readily immobilized for portioning during selection. in order to circumvent this limitation, wang et al. utilized an immobilization free in vitro selex developed by li and coworkers to select ssdna mres specific for four different organophosphorus pesticides, phorate, profenofos, isocarbophos, and omethoate [26, 28] . after twelve rounds of selection, two candidate sequences reported values in the low micromolar range for all four targets. cross-binding assays showed good specificities for the selected two mres, with only minimal observed binding to eight other different pesticides [28] . the same group of researchers later developed a fluorescence polarization assay using the selected mres to detect phorate, profenofos, isocarbophos, and omethoate at a lod of 19.2, 13.4, 17.2, and 23.4 nm, respectively [247] . he et al. employed immobilization-free selex to identify ssdna mre specific for pesticide, acetamiprid. after eighteen rounds of selection, the best candidate mre was reported to have a of 4.96 m. specificity of the selected mre was tested and cross-binding data showed no significant change in fluorescent signals in the presence of three other pesticides, imidacloprid, nitenpyram, and chlorpyrifos. the authors noted that the affinity of the selected mre was lower than typical antibodies [27] . go-selex was used to identify three ssdna mres specific to three different pesticides: tebuconazole, mefenacet, and inabenfide [25] . the reported values of were in the range of 10 to 100 nm. high specificity of each identified mre was also determined by isothermal titration calorimetric and gold nanoparticle colorimetric assays. a simple, rapid detection method using gold nanoparticles was developed with lod ranges from 100 to 400 nm. in recent years, a large number of researches have taken place in applying ssdna mres for the use in biosensors. major detection methods can be categorized into three classes: (1) electrical/electrochemical, (2) optical, and (3) mass sensitive. the following section highlights the basic principles of the general classes of detection methods that have been utilized influenza h5n1 surface plasmon resonance -0.128 hau poultry [183] widely in the development of ssdna mre based biosensors. the relative portability of different detection methods is also briefly discussed. recent literatures describing the detection of pathogens and toxins using ssdna mres biosensors are summarized in tables 6, 7 , and 8. the principle of electrochemical detection is based on measuring changes in electrical properties of the sensing platform. in this method, ssdna mre is usually immobilized on a gold electrode via thiol-gold linkage. a redox label, such as methylene blue, can be used to detect binding between mre and the target [262] . in a "signal on" system, the redox label is away from the electrode surface, and the binding of target causes a conformational change in the mre and brings the redox label into close proximity with the electrode, thus causing a measurable change in electrical properties ( figure 2) . a "signal off " system behaves similarly, but the binding of target causes the redox label to move [192] aflatoxin b1 fluorescence dipstick -0.3 ng/g corn [193] aflatoxin m1 electrochemical redox current magnetic nanoparticles 8 ng/l milk [194] ochratoxin a colorimetric -20 nm [195] ochratoxin a electrochemical impedance graphene oxide, gold nanoparticles 0.74 pm red wine [196] ochratoxin a fluorescence -1 ng/ml beer [197] ochratoxin a electrochemical redox current gold nanoparticles 0.75 pm red wine [198] ochratoxin a electrochemiluminescence loop-mediated isothermal amplification 10 fm red wine [199] ochratoxin a fluorescence -2 pg/ml wheat [200] ochratoxin a localized surface plasmon resonance -1 n m c o r n p o w d e r [201] ochratoxin a rt-qpcr -1 fg/ml red wine [202] ochratoxin a fluorescence -0.2 ng/ml red wine [203] ochratoxin ochratoxin a fluorescence -0.01 ng/ml maize flour [232] away from the electrode. this system can also be modified as a "label-free" system, in which the redox molecule is intercalated in a hairpin structure of a mre in a target unbound state, and binding of the target causes the release of the redox molecule ( figure 2 ). in addition to measuring redox current, the changes in impedance upon binding of the target can also be measured. in this case, no labeling of mre is required and the conformational changes in mre upon target binding cause a measurable change in impedance that can be recorded by voltammetry [212] . nanomaterials can also be incorporated into electrochemical sensor to enhance signals. single-walled carbon nanotube field effect transistors (swcnt-fet) can be used to build electrochemical biosensors (figure 3 ). in this system, mres are immobilized on swcnts and swcnts are sandwiched between a source and a drain electrode. when the immobilized mres bind to the target, there is a measurable change in the conductance of the system [240] . gold nanoparticles (aunp) are also widely used as signal enhancers. aunps can be coated on electrodes and greatly increase the surface area. as a result, more mres can be immobilized on the electrode, thus enhancing the system's sensitivity. aunps can also be coated with a second mre and reporting probes in a sandwich assay (figure 3 ). in this case, the target first binds to a primary capturing mre, followed by the binding a secondary reporting mre along with a redox molecule, which can generate an enhanced signal for sensitive detection [263] . in general, electrical/electrochemical detection systems are relatively smaller and more easily adapted into portable, chip-based platforms. this allows the ssdna mres biosensors to be used for on-site target detection [211, 212] . 3.2. optical. optical detection methods can be classified into three major categories: (1) fluorescence detection, which require specialized instruments to measure fluorescent signals; (2) colorimetric detection, which color changes can be observed by the naked eye or measured in terms of optical density; (3) absorbance assay can enhance detection signals, and subsequently be measured by instruments as well. fluorescence. the principle of fluorescence detection is based on the generation or quenching of fluorescence signals upon target binding. various fluorescence molecules and quantum dots can be linked to ssdna mres. conformational changes induced by target binding can alter the fluorescence signal generated by the fluorophore and therefore can be measured (figure 4 ) [264] . quenching molecules can also be linked to the other end of the ssdna mre. in this system, the quencher completely blocks the fluorescence signal from the fluorophore and target binding can move the quencher away from the fluorophore and have "signal on" detection ( figure 4 ) [265] . the same principle can also be applied for a "signal off " system. carbon nanotubes and graphene can also be used as quenchers, where fluorescent labeled ssdna mres is adsorbed on the carbon quenchers via -stacking interactions. fluorescence resonance energy transfer (fret) can also be utilized as measurements when the distance of the two fluorescence molecules linked to mres is changed upon target binding. fluorescence based detection systems are frequently developed because of their high sensitivity and the ease of fluorescently labeling ssdna mres. traditionally, the complete detection system requires large, costly components, including lasers, filters, and detectors. a recent study reported a portable ssdna mre based biosensor utilizing an optic fiber for sensitive detection of bpa [233] . gold nanoparticles (aunp) have been widely used in various colorimetric assays. aunps aggregate in salt solution and appear in purple color. when they are dispersed, they are in red color. this special absorbance property of aunps allows observation of target binding by naked eye. mres in salt solution can bind to aunps, dispersing the aunps. when targets are introduced into the system, mre preferably bind to the targets, therefore causing aunps to aggregate, and a red to purple color change is observed ( figure 5 ) [266] . alternatively, ssdna mres can be used to link aunps that are functionalized with probe strands. in this case, the initial state of the mre/aunps solution is aggregated purple. upon target binding, the linked aunps are released, and a purple to red color change is observed ( figure 5 ) [267] . furthermore, aunps can be used in a sandwich colorimetric assay, in which the secondary reporting mre linked aunp can grow in size when the detection system is placed in a growth solution containing haucl 4 , thus enhancing the detection limit [268] . colorimetric detection systems are attractive for onsite target sensing due to the ease of observation with the naked eye. these systems are often developed into hand-held laminar flow devices [163, 204, 226] . nucleic acid mres have been used in modified enzyme linked immunoassays, usually substituting for either the capturing or the reporter antibodies. in a direct oligonucleotide enzyme link assay, often the protein target is adsorbed on plate and biotinylated mres bind to the target and then followed by the addition of streptavidin-horse radish peroxidase (hrp) conjugate and enzyme substrate for signal development [148] . in a sandwich assay, biotinylated mres can be immobilized on streptavidin plate and then followed by the addition of the protein target, hrp linked antibody, and enzyme substrate [98] . this detection method is mostly limited to clinical laboratory settings and detection of protein targets for which antibodies have been isolated ( figure 6 ). mass sensitive detection is a class of label-free detection system that can be subdivided into four surface plasmon resonance (spr) sensors measure a change in the refractive index and resonance angle when a mass change occurs upon target binding. mres are often biotin-tagged and immobilized on streptavidin coated gold chip. when targets in solution pass through the flow cell, the binding between targets and immobilized mres cause a change in mass on the sensor chip surface and is subsequently translated into a change in the refractive index. this change in resonance is proportional to the amount of target bound to the immobilized mres and therefore providing a realtime detection of the target in solution (figure 7 ) [183] . commercially available spr systems are typically large and limited to bench top use. one study reported a portable spr biosensor based on ssdna mres for the detection of h5n1 influenza virus [183] . a quartz crystal microbalance (qcm) is an acoustic wave resonator based on the piezoelectric property of quartz crystal. nucleic acid mres can be immobilized on goldcoated quartz. the binding between target and mre increases the mass on the surface of the crystal and leads to a detectable decrease in the resonance frequency of the crystal (figure 8 ) [181] . the detection principle of surface acoustic wave (saw) based biosensor is similar to qcm. nucleic acid mres have been utilized to fabricate a special type of love-wave sensor that uses shear horizontal waves to enhance the surface sensitivity and achieve ultrasensitive detection of the target [269] . micromechanical cantilevers have been investigated for mre based biosensors. the major advantage of this type of sensor is that it can be readily scale up and perform parallel analysis for multiple analytes with low background interference [270] . when the target binds to the mre on the surface of the cantilever, a nanometer scale deflection in the cantilever can be detected optically ( over the last two decades, there has been a continuous increase in the research of molecular recognition elements. single-stranded dna mres have several advantages over antibodies, in terms of stability, reusability, and production cost. however, ssdna mres are not without limitations. the binding affinity of mres is highly dependent on their three-dimensional structure and is influenced by factors including the ionic condition, temperature, and ph of the binding condition [4] . challenges remain in eliminating cross-binding activities to other molecules in native environments. these limitations hinder the use of mre for detection in many real world complex samples, such as biological fluids and food matrices. a carefully designed selection scheme can greatly improve the specificity of the identified mre, which can better distinguish closely related molecules at low concentrations. using modified bases in pcr amplification or performing base modifications after selection can also help improving resistance to nucleases in many biological fluids, such as human serum [271] . as the field of ssdna mre based biosensors continues to grow, improvements in selex methodology will be necessary to more rapidly isolate mres with the desired affinity and specificity. improvements will also be necessary to allow mres against more targets and a wider variety of targets to be isolated. the development of mre based sensors is becoming an increasingly diverse field. scientist and engineers from many disciplines must work together in order to create the optimal end product. portable ssdna mre based biosensors may be utilized in a variety of settings, such as food safety, environmental monitoring, and health care. the many attractive features of ssdna mres prompt researchers to continue to investigate and optimize their applications in biosensing. the commercialization of these devices should continue to increase in the future. systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t4 dna polymerase aptamers: an emerging class of molecules that rival antibodies in diagnostics design, synthesis, and amplification of dna pools for in vitro selection structure, recognition and adaptive binding in rna aptamer complexes biophysical characterization of dna and rna aptamer interactions with hen egg lysozyme modified-rna aptamer-based sensor for competitive impedimetric assay of neomycin b selection of 2 -fluoromodified rna aptamers for alleviation of cocaine and mk-801 inhibition of the nicotinic acetylcholine receptor aptamers as functional nucleic acids: in vitro selection and biotechnological applications selection and application of ssdna aptamers to detect active tb from sputum samples novel system for detecting sars coronavirus nucleocapsid protein using an ssdna aptamer a single-stranded dna aptamer that selectively binds to staphylococcus aureus enterotoxin b selection and identification of dna aptamers against okadaic acid for biosensing application screening and initial binding assessment of fumonisin b 1 aptamers in vitro selection of a single-stranded dna molecular recognition element against atrazine in vitro selection of a single-stranded dna molecular recognition element for the pesticide malathion in vitro selection of a single-stranded dna molecular recognition element specific for bromacil generation of singlestranded dna by the polymerase chain reaction and its application to direct sequencing of the hla-dqa locus direct solid phase sequencing of genomic and plasmid dna using magnetic beads as solid support efficient preparation of singlestranded dna for in vitro selection use of pcr primers containing a 3 -terminal ribose residue to prevent cross-contamination of amplified sequences in vitro selection of high-affinity dna ligands for human ige using capillary electrophoresis nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers immobilization-free screening of aptamers assisted by graphene oxide an ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening multiple go-selex for efficient screening of flexible aptamers in vitro selection of structure-switching signaling aptamers isolation and identification of the dna aptamer target to acetamiprid selection of dna aptamers that bind to four organophosphorus pesticides flumag-selex as an advantageous method for dna aptamer selection single microbead selex for efficient ssdna aptamer generation against botulinum neurotoxin rapid one-step selection method for generating nucleic acid aptamers: development of a dna aptamer against alpha-bungarotoxin generation of highly specific aptamers via micromagnetic selection micromagnetic selection of aptamers in microfluidic channels influenza a virus-specific aptamers screened by using an integrated microfluidic system neutralizing aptamers from whole-cell selex inhibit the ret receptor tyrosine kinase a tenascin-c aptamer identified by tumor cell selex: systematic evolution of ligands by exponential enrichment aptamers evolved from live cells as effective molecular probes for cancer study selection of aptamers for molecular recognition and characterization of cancer cells presence of faecal coliforms, escherichia coli and diarrheagenic e. coli pathotypes in ready-to-eat salads, from an area where crops are irrigated with untreated sewage water rapid fluorescent detection of escherichia coli k88 based on dna aptamer library as direct and specific reporter combined with immunomagnetic separation aptamer selection for the detection of escherichia coli k88 isolation and characterization of dna aptamers against escherichia coli using a bacterial cell-systematic evolution of ligands by exponential enrichment approach selection of dna aptamers against uropathogenic escherichia coli nsm59 by quantitative pcr controlled cell-selex a novel screening method for competitive fret-aptamers applied to e. coli assay development in vitro antibacterial effects of antilipopolysaccharide dna aptamer-c1qrs complexes selection of dna aptamers for capture and detection of salmonella typhimurium using a whole-cell selex approach in conjunction with cell sorting selection and characterization of aptamers against salmonella typhimurium using wholebacterium systemic evolution of ligands by exponential enrichment (selex) identification of salmonella typhimurium-specific dna aptamers developed using whole-cell selex and facs analysis selection, characterization, and application of dna aptamers for the capture and detection of salmonella enterica serovars development of bacteriostatic dna aptamers for salmonella development of ssdna aptamers for the sensitive detection of salmonella typhimurium and salmonella enteritidis highly specific and cost-efficient detection of salmonella paratyphi a combining aptamers with single-walled carbon nanotubes screening and preliminary application of a dna aptamer for rapid detection of salmonella o8 infectious diseases associated with molluscan shellfish consumption selection and identification of a dna aptamer targeted to vibrio parahemolyticus selection of aptamers against inactive vibrio alginolyticus and application in a qualitative detection assay selection and characterization of dna aptamers specific for listeria species nucleic acid aptamers for capture and detection of listeria spp selection, identification and application of a dna aptamer against listeria monocytogenes in vitro selection of dna aptamers and fluorescence-based recognition for rapid detection listeria monocytogenes internalins: a complex family of leucine-rich repeat-containing proteins in listeria monocytogenes antibody-aptamer functionalized fibre-optic biosensor for specific detection of listeria monocytogenes from food global burden of shigella infections: implications for vaccine development and implementation of control strategies in vitro selection of a dna aptamer targeted against shigella dysenteriae risk factors for sporadic campylobacter infection in the united states: a case-control study in foodnet sites plastic-adherent dna aptamer-magnetic bead and quantum dot sandwich assay for campylobacter detection evaluation of relative aptamer binding to campylobacter jejuni bacteria using affinity probe capillary electrophoresis selection and characterization of dna aptamers with binding selectivity to campylobacter jejuni using whole-cell selex in silico maturation of binding-specificity of dna aptamers against proteus mirabilis simultaneous improvement of specificity and affinity of aptamers against streptococcus mutans by in silico maturation for biosensor development invasive group a streptococcal disease: epidemiology, pathogenesis and management dna aptamers binding to multiple prevalent m-types of streptococcus pyogenes staphylococcus aureus infections combining use of a panel of ssdna aptamers in the detection of staphylococcus aureus rapid single cell detection of staphylococcus aureus by aptamer-conjugated gold nanoparticles pouring salt on a wound: pseudomonas aeruginosa virulence factors alter na+ and clflux in the lung opportunistic infections in lung disease: pseudomonas infections in cystic fibrosis utility of aptamer-fluorescence in situ hybridization for rapid detection of pseudomonas aeruginosa treatment of tuberculosis aptamer from whole-bacterium selex as new therapeutic reagent against virulent mycobacterium tuberculosis bioterrorismrelated inhalational anthrax: the first 10 cases reported in the united states in vitro selection of dna aptamers to anthrax spores with electrochemiluminescence detection fluorescence assay based on aptamer-quantum dot binding to bacillus thuringiensis spores development of aptamer beacons for rapid presumptive detection of bacillus spores nature of protective immunity to francisella tularensis anti-francisella tularensis dna aptamers detect tularemia antigen from different subspecies by aptamer-linked immobilized sorbent assay from the regulation of peptidoglycan synthesis to bacterial growth and morphology selection of peptidoglycanspecific aptamers for bacterial cells identification the lipooligosaccharides of pathogenic gram-negative bacteria receptor-dependent mechanisms of cell stimulation by bacterial endotoxin harnessing aptamers for electrochemical detection of endotoxin noroviruses: the leading cause of gastroenteritis worldwide ultrasensitive norovirus detection using dna aptasensor technology selection, characterization and application of nucleic acid aptamers for the capture and detection of human norovirus strains selection of a dna aptamer against norovirus capsid protein vp1 understanding the symptoms of the common cold and influenza selection and characterization of dna aptamers for use in detection of avian influenza virus h5n1 selection of dna aptamers that bind to influenza a viruses with high affinity and broad subtype specificity characterization of monoclonal antibody against sars coronavirus nucleocapsid antigen and development of an antigen capture elisa cs-selex generates high-affinity ssdna aptamers as molecular probes for hepatitis c virus envelope glycoprotein e2 dengue virus life cycle: viral and host factors modulating infectivity thioaptamers targeting dengue virus type-2 envelope protein domain iii capillary electrophoresis-selex selection of aptamers with affinity for hiv-1 reverse transcriptase foodborne illness acquired in the united states-major pathogens use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods selection and characterization of dna aptamers against staphylococcus aureus enterotoxin c1 clinical recognition and diagnosis of clostridium difficile infection production of a complete binary toxin (actin-specific adp-ribosyltransferase) by clostridium difficile cd196 detection of clostridium difficile toxins a, b and binary toxin with slow off-rate modified aptamers systematic selection of modified aptamer pairs for diagnostic sandwich assays in vitro selection of single-stranded dna molecular recognition elements against clostridium difficile toxin b and sensitive detection in human fecal matter multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review cfp10 and esat6 aptamers as effective mycobacterial antigen diagnostic reagents clinical evaluation of mpt-64 and mpt-59, two proteins secreted from mycobacterium tuberculosis, for skin test reagents the selection and application of ssdna aptamers against mpt64 protein in mycobacterium tuberculosis anthrax toxin nano aptasensor for protective antigen toxin of anthrax screening and characterization of high-affinity ssdna aptamers against anthrax protective antigen botulinum toxin: bioweapon & magic drug an aptamer beacon responsive to botulinum toxins usage of a dna aptamer as a ligand targeting microcystin freshwater cyanobacteria (blue-green algae) and human health in vitro selection, characterization, and biosensing application of high-affinity cylindrospermopsin-targeting aptamers dna aptamers selected against the hiv-1 rnase h display in vitro antiviral activity novel aptamer inhibitors of human immunodeficiency virus reverse transcriptase high-affinity ssdna inhibitors of the reverse transcriptase of type 1 human immunodeficiency virus dna aptamers to human immunodeficiency virus reverse transcriptase selected by a primer-free selex method: characterization and comparison with other aptamers dna aptamers derived from hiv-1 rnase h inhibitors are strong anti-integrase agents structure-activity of tetrad-forming oligonucleotides as a potent anti-hiv therapeutic drug dna aptamers selected against the hiv-1 trans-activation-responsive rna element form rna-dna kissing complexes in vitro selection of dna aptamers against the hiv-1 tar rna hairpin inhibition of hepatitis c virus (hcv) rna polymerase by dna aptamers: mechanism of inhibition of in vitro rna synthesis and effect on hcvinfected cells an aptamer targets hbv core protein and suppresses hbv replication in hepg2.2.15 cells differential inhibitory activities and stabilisation of dna aptamers against the sars coronavirus helicase a dna aptamer prevents influenza infection by blocking the receptor binding region of the viral hemagglutinin potent inhibition of human influenza h5n1 virus by oligonucleotides derived by selex neutralizing dna aptamers against swine influenza h3n2 viruses dna aptamers against the receptor binding region of hemagglutinin prevent avian influenza viral infection single-stranded dna aptamer that specifically binds to the influenza virus ns1 protein suppresses interferon antagonism isolation of ssdna aptamers that inhibit rabies virus use of cell-selex to generate dna aptamers as molecular probes of hpv-associated cervical cancer cells one-step selection of vaccinia virus-binding dna aptamers by monolex first report of the use of a saxitoxin-protein conjugate to develop a dna aptamer to a small molecule toxin determination of ochratoxin a with a dna aptamer fluorescence polarization based displacement assay for the determination of small molecules with aptamers enzyme-linked aptamer assays (elaas), based on a competition format for a rapid and sensitive detection of ochratoxin a in wine selection and characterization of a novel dna aptamer for label-free fluorescence biosensing of ochratoxin a selection and identification of ssdna aptamers recognizing zearalenone screening and identification of dna aptamers against t-2 toxin assisted by graphene oxide selection, identification, and application of aflatoxin b1 aptamer selection of aptamers for aflatoxin m1 and their characterization in vitro selection of single-stranded dna molecular recognition elements against s. aureus alpha toxin and sensitive detection in human serum dna aptamers as a novel approach to neutralize staphylococcus aureus -toxin electrochemical detection of 17beta-estradiol using dna aptamer immobilized gold electrode chip small molecule detection in solution via the size contraction response of aptamer functionalized nanoparticles development of single-stranded dna aptamers for specific bisphenol a detection selection of dna aptamers against polychlorinated biphenyls as potential biorecognition elements for environmental analysis selection and characterization of pcb-binding dna aptamers dna aptamer development for detection of atrazine and protective antigen toxin using fluorescence polarization, microbiology a simple aptamer biosensor for salmonellae enteritidis based on fluorescence-switch signaling graphene oxide lateral flow biosensor for dna extraction-free detection of salmonella based on aptamer mediated strand displacement amplification a visual detection method for salmonella typhimurium based on aptamer recognition and nanogold labeling an aptamer-based electrochemical biosensor for the detection of salmonella aptamer-based impedimetric sensor for bacterial typing gold nanoparticle-based enzymelinked antibody-aptamer sandwich assay for detection of salmonella typhimurium a dual-color flow cytometry protocol for the simultaneous detection of vibrio parahaemolyticus and salmonella typhimurium using aptamer conjugated quantum dots as labels dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticlesbased bioassay for the simultaneous detection of salmonella typhimurium and staphylococcus aureus label-free detection of staphylococcus aureus in skin using real-time potentiometric biosensors based on carbon nanotubes and aptamers graphene-based potentiometric biosensor biomed research international for the immediate detection of living bacteria a sensitive gold nanoparticlebased colorimetric aptasensor for staphylococcus aureus a new aptamer/swnts ide-spqc sensor for rapid and specific detection of group a streptococcus aptamer cocktails: enhancement of sensing signals compared to single use of aptamers for detection of bacteria aptasensors for rapid detection of escherichia coli o157: h7 and salmonella typhimurium a rapid and sensitive aptamer-based electrochemical biosensor for direct detection of escherichia coli o111 realtime potentiometric detection of bacteria in complex samples potential of fluorophore labeled aptamers for pseudomonas aeruginosa detection in drinking water simultaneous aptasensor for multiplex pathogenic bacteria detection based on multicolor upconversion nanoparticles labels a pdms/paper/ glass hybrid microfluidic biochip integrated with aptamerfunctionalized graphene oxide nano-biosensors for one-step multiplexed pathogen detection hydrogel based qcm aptasensor for detection of avian influenza virus aptamer-based viability impedimetric sensor for viruses a spr aptasensor for detection of avian influenza virus h5n1 an aptamerfunctionalized gold nanoparticle biosensor for the detection of prion protein aptamer biosensor for sensitive detection of toxin a of clostridium difficile using gold nanoparticles synthesized by bacillus stearothermophilus target-induced aptamer release strategy based on electrochemical detection of staphylococcal enterotoxin b using gnps-zro2-chits film attomole sensitivity of staphylococcal enterotoxin b detection using an aptamer-modified surface-enhanced raman scattering probe aptasensor for staphylococcus enterotoxin b detection using high snr piezoresistive microcantilevers evanescent wave dna-aptamer biosensor based on long period gratings for the specific recognition of e. coli outer membrane proteins a label-free dna aptamer-based impedance biosensor for the detection of e. coliouter membrane proteins aptamer-based electrochemical biosensor for botulinum neurotoxin development of an ultrasensitive aptasensor for the detection of aflatoxin b1 an aptamerbased dipstick assay for the rapid and simple detection of aflatoxin b1 label-free detection of aflatoxin m1 with electrochemical fe 3 o 4 /polyaniline-based aptasensor aptamer-based colorimetric biosensing of ochratoxin a using unmodified gold nanoparticles indicator amplified impedimetric aptasensor based on gold nanoparticles covalently bound graphene sheet for the picomolar detection of ochratoxin a a simple and sensitive approach for ochratoxin a detection using a label-free fluorescent aptasensor ultrasensitive electrochemical aptasensor for ochratoxin a based on two-level cascaded signal amplification strategy ultrasensitive electrochemiluminescent aptasensor for ochratoxin a detection with the loop-mediated isothermal amplification a fluorescent aptasensor based on dna-scaffolded silver-nanocluster for ochratoxin a detection a regeneratable, label-free, localized surface plasmon resonance (lspr) aptasensor for the detection of ochratoxin a femtogram ultrasensitive aptasensor for the detection of ochratoxin a fluorescent sensing ochratoxin a with single fluorophore-labeled aptamer signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin a highly sensitive ochratoxin a impedimetric aptasensor based on the immobilization of azido-aptamer onto electrografted binary film via click chemistry electrochemical grafting of long spacer arms of hexamethyldiamine on a screen printed carbon electrode surface: application in target induced ochratoxin a electrochemical aptasensor design of pegaptamer two piece macromolecules as convenient and integrated sensing platform: application to the label free detection of small size molecules electrochemical dna aptamer-based biosensor for ota detection, using superparamagnetic nanoparticles development of an automated flow-based electrochemical aptasensor for on-line detection of ochratoxin a an aptamer-based chromatographic strip assay for sensitive toxin semi-quantitative detection fluorescent strip sensor for rapid determination of toxins an electrochemical competitive biosensor for ochratoxin a based on a dna biotinylated aptamer fabricated aptamer-based electrochemical 'signal-off ' sensor of ochratoxin a an electrochemical biosensor based on hairpin-dna aptamer probe and restriction endonuclease for ochratoxin a detection ultrasensitive one-step rapid detection of ochratoxin a by the folding-based electrochemical aptasensor aptamerfunctionalized magnetic nanoparticle-based bioassay for the detection of ochratoxin a using upconversion nanoparticles as labels electrochemical aptasensor for the determination of ochratoxin a at the au electrode modified with ag nanoparticles decorated with macrocyclic ligand development of an electrochemical method for ochratoxin a detection based on aptamer and loop-mediated isothermal amplification a simple and rapid biosensor for ochratoxin a based on a structure-switching signaling aptamer a signal-on fluorescent aptasensor based on tb3+ and structure-switching aptamer for label-free detection of ochratoxin a in wheat analytical performances of a dna-ligand system using timeresolved fluorescence for the determination of ochratoxin a in wheat impedimetric dna aptasensor for sensitive detection of ochratoxin a in food gold nanoparticle-based fluorescence resonance energy transfer aptasensor for ochratoxin a detection simply amplified electrochemical aptasensor of ochratoxin a based on exonuclease-catalyzed target recycling double-probe signal enhancing strategy for toxin aptasensing based on rolling circle amplification pvp-coated graphene oxide for selective determination of ochratoxin a via quenching fluorescence of free aptamer electrochemiluminescent aptamer biosensor for the determination of ochratoxin a at a gold-nanoparticles-modified gold electrode using n-(aminobutyl)-n-ethylisoluminol as a luminescent label aptamer-dnazyme hairpins for biosensing of ochratoxin a rapid high-throughput analysis of ochratoxin a by the selfassembly of dnazyme-aptamer conjugates in wine single-walled carbon nanotubes based quenching of free fam-aptamer for selective determination of ochratoxin a polyaniline langmuir-blodgett film based aptasensor for ochratoxin a detection an aptamer-based fluorescence assay for ochratoxin a a portable optic fiber aptasensor for sensitive, specific and rapid detection of bisphenol-a in water samples resonance light scattering determination of trace bisphenol a with signal amplification by aptamer-nanogold catalysis an electrochemical aptasensor based on gold nanoparticles dotted graphene modified glassy carbon electrode for label-free detection of bisphenol a in milk samples functionalized aptamers as nano-bioprobes for ultrasensitive detection of bisphenol-a ultrasensitive one-step rapid visual detection of bisphenol a in water samples by label-free aptasensor one-step signal amplified lateral flow strip biosensor for ultrasensitive and on-site detection of bisphenol a (bpa) in aqueous samples a highly sensitive and selective resonance rayleigh scattering method for bisphenol a detection based on the aptamer-nanogold catalysis of the haucl 4 -vitamin c particle reaction aptamer sandwich-based carbon nanotube sensors for single-carbon-atomic-resolution detection of non-polar small molecular species asymmetric plasmonic aptasensor for sensitive detection of bisphenol a a femtomolar level and highly selective 17 -estradiol photoelectrochemical aptasensor applied in environmental water samples analysis label-free aptamer-based electrochemical impedance biosensor for 17 -estradiol aptamer-based optical biosensor for rapid and sensitive detection of 17 -estradiol in water samples aptamer-based colorimetric sensing of acetamiprid in soil samples: sensitivity, selectivity and mechanism a highly selective electrochemical impedance spectroscopy-based aptasensor for sensitive detection of acetamiprid organophosphorus pesticides detection using broad-specific single-stranded dna based fluorescence polarization aptamer assay non-traditional vectors for paralytic shellfish poisoning determination of okadaic acid content of dinoflagellate cells: a comparison of the hplcfluorescent method and two monoclonal antibody elisa test kits ochratoxin a from a toxicological perspective analytical methods for determination of mycotoxins: a review us food and drug administration's monitoring and surveillance programs for mycotoxins, pesticides and contaminants in food review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin t-2 toxin, a trichothecene mycotoxin: review of toxicity, metabolism, and analytical methods mycotoxins aflatoxin contamination in foods and foodstuffs in tokyo: 1986-1990 a controlled clinical trial of a novel antivenom in patients envenomed by bungarus multicinctus three-finger -neurotoxins and the nicotinic acetylcholine receptor, forty years on parent bisphenol a accumulation in the human maternal-fetal-placental unit levels and congener distributions of pcdds, pcdfs and dioxin-like pcbs in environmental and human samples: a review impacts of atrazine in aquatic ecosystems labelfree electronic detection of thrombin in blood serum by using an aptamer-based sensor impedimetric aptasensor with femtomolar sensitivity based on the enlargement of surfacecharged gold nanoparticles affinity analysis of dna aptamer-peptide interactions using gold nanoparticles aptamer biosensor for protein detection using gold nanoparticles unmodified gold nanoparticles as a colorimetric probe for potassium dna aptamers fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticles aptamerfunctionalized au nanoparticles for the amplified optical detection of thrombin a love-wave biosensor using nucleic acids as ligands nanomechanical microcantilever operated in vibration modes with use of rna aptamer as receptor molecules for label-free detection of hcv helicase improving the stability of aptamers by chemical modification this work was supported by the national science foundation cooperative agreements (nsf-1003907 and nsf-0554328), department of defense cooperative agreement (w911nf-09-2-0044), and west virginia university. the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord-300944-c57impca authors: huang, xiaolan; cheng, qiang; du, zhihua title: a genome-wide analysis of rna pseudoknots that stimulate efficient −1 ribosomal frameshifting or readthrough in animal viruses date: 2013-11-04 journal: biomed res int doi: 10.1155/2013/984028 sha: doc_id: 300944 cord_uid: c57impca programmed −1 ribosomal frameshifting (prf) and stop codon readthrough are two translational recoding mechanisms utilized by some rna viruses to express their structural and enzymatic proteins at a defined ratio. efficient recoding usually requires an rna pseudoknot located several nucleotides downstream from the recoding site. to assess the strategic importance of the recoding pseudoknots, we have carried out a large scale genome-wide analysis in which we used an in-house developed program to detect all possible h-type pseudoknots within the genomic mrnas of 81 animal viruses. pseudoknots are detected downstream from ~85% of the recoding sites, including many previously unknown pseudoknots. ~78% of the recoding pseudoknots are the most stable pseudoknot within the viral genomes. however, they are not as strong as some designed pseudoknots that exhibit roadblocking effect on the translating ribosome. strong roadblocking pseudoknots are not detected within the viral genomes. these results indicate that the decoding pseudoknots have evolved to possess optimal stability for efficient recoding. we also found that the sequence at the gag-pol frameshift junction of hiv1 harbors potential elaborated pseudoknots encompassing the frameshift site. a novel mechanism is proposed for possible involvement of the elaborated pseudoknots in the hiv1 prf event. during the translation process, ribosomes are capable of performing some nonstandard decoding events which provided that appropriate signals are present in the mrna being translated. these unusual events are referred to as "recoding" [1, 2] . two of the major recoding mechanisms are programmed −1 ribosomal frameshifting (prf) and stop codon readthrough. these mechanisms are utilized by retroviruses and some other rna viruses to express their structural and enzymatic proteins at a defined ratio [1, [3] [4] [5] . both −1 frameshifting and stop codon readthrough are site specific and occur at a defined frequency much higher than the background error rates of maintaining the reading frames. the discovery of the −1 prf mechanism was made by atkins and coworkers [6] , and the utilization of this recoding mechanism by viruses was described as a strategy by which rous sarcoma virus (rsv) expresses its gag-pol polyprotein from the overlapping gag and pol open reading frames from a single translation initiation codon of the 5 gag reading frame [7] . in −1 frameshifting, only a defined percentage of the translating ribosomes shifts to the −1 reading frame and translates the downstream gene. this percentage is referred to as the frameshifting efficiency, which dictates the molar ratio of viral structural and enzymatic proteins, encoded by the gag and pol gene, respectively. for efficient −1 frameshifting to happen, two cis-acting elements programmed in the overlapping region of the mrna are often required to signal the translating ribosomes to shift backward by one nucleotide. the first element is a heptanucleotide stretch termed "slippery sequence" with a typical composition of x xxy yyz (xxx and yyy: a stretch of three identical nucleotides; the triplets indicate the 0 reading frame). although the slippery sequence is the site of action where the ribosomes actually shift to the 2 biomed research international s1 5 l1 s2-5 l3 s2 l2 s2-3 3 s1-5 s1-3 figure 1 : schematic diagrams of the sequence elements for forming an h-type rna pseudoknot. abbreviations used are: s1, stem1; s2, stem2; s1-5 and s1-3 , the 5 and 3 strands of stem1; s2-5 and s2-3 , the 5 and 3 strands of stem2; l1, loop1; l2, loop2; l3, loop3. (a): linear sequential arrangement of the pseudoknot-forming sequence elements. residues involved in the formation of s1 and s2 are represented as black and gray squares, respectively. residues in the single-stranded loop region are represented as unfilled circles. (b): schematic representations of folded pseudoknots. left: with a nonzero l3 sequence; right: with the absence of l3, s1 and s2 can stack coaxially to form a quasicontinuous double helix. l1 and l2 locate on the same side of the double helix, with l1 crossing the major groove of s2 and l2 crossing the minor groove of s1. −1 frame, this element alone is not sufficient to cause efficient shifting. a secondary signal called "stimulator" is usually required in the form of an rna structure downstream from the frameshift site, separated by a spacer region (typically 6-9 nucleotides in length). in some cases, the stimulator is a conventional stem-loop structure, but most often it is a pseudoknot, which is a structural motif of rna formed when a stretch of nucleotides within a loop region in a secondary structure basepairs with residues outside that loop [8] [9] [10] (figure 1 ). the indispensable role of a downstream pseudoknot in efficient −1 frameshifting has been established in a large number of rna viruses including members from the retroviridae family, coronaviridae family (such as sars cov), totiviridae family, and luteoviridae family [5, [11] [12] [13] [14] [15] [16] [17] [18] . although a 3 rna structure is utilized as a frameshift stimulator in most cases, absence of such a structure has also been reported in efficient frameshifting such as in the case of semliki forest virus [19] . the involvement of rna pseudoknots in stop codon readthrough has also been established. in the gag-pol junction region of moloney murine leukemia virus (mo-mulv), a pseudoknot located several nucleotides 3 to the uag termination codon of the gag gene was found to be required [20, 21] . strong similarities between the sequences in the gagpol region of mulv and the other viruses of the readthrough retrovirus group imply that the other readthrough retroviruses may use a similar pseudoknot structure to stimulate the stop codon readthrough as well [22, 23] . the vast majority of the established frameshift-or readthrough-stimulating pseudoknots belong to the socalled h (hairpin)-type pseudoknots, in which a stretch of nucleotides within a hairpin loop basepairs with a complementary region outside of the hairpin (see figure 1 for the secondary structure and terminology of an h-type pseudoknot). all h-type pseudoknots contain two helical stems, s1 and s2, and two nonequivalent loops, l1 and l2. some h-type pseudoknots also contain a third loop, l3. if l3 is absent, s1 and s2 can form a quasicontinuous double helix, with loops l1 and l2 crossing the major groove and minor groove of stem s2 and stem s1, respectively ( figure 1 ). the structures of many −1 frameshift stimulating pseudoknots have been determined by nmr or x-ray crystallography, including those at the gag-pro junctions from the mouse mammary tumor virus (mmtv) (24) (25) (26) and the simian retrovirus-1 (srv-1) [24, 25] , the p1-p2 junctions from several plant luteoviruses: the beet western yellows virus (bwyv) [26] [27] [28] , the pea enation mosaic virus (pemv-1) [29] , the potato leaf roll virus (plrv) [30] , and the sugar cane yellow leaf virus (scylv) [31] . in the mmtv pseudoknot, l3 consists of an unpaired adenosine that is intercalated between the two stems s1 and s2 thereby inducing a bent conformation of the pseudoknot. in the srv-1 pseudoknot, l3 is absent, and the two stems s1 and s2 stack coaxially to form a quasicontinuous helix. the luteoviral pseudoknots are small and compact (with only 4-5 basepairs in s1 and 3 basepairs in s2). extensive s1-l2 and s2-l1 interactions are present in all of the luteoviral pseudoknots. overall, the available structures do not share common structural feature(s) other than the fact that they all adopt the general pseudoknotted topology. these results indicate that the frameshift stimulating ability of a pseudoknot is not dependent on its specific or unique structural feature(s). several lines of evidence suggest that the thermodynamic stability and mechanical strength of a frameshift stimulating pseudoknot to resist unwinding by the helicase activity of the ribosome may correlate more strongly with frameshift stimulation. it is known that some mrna structures, especially pseudoknots, can cause the translating ribosome to pause upstream to such structures [32] [33] [34] . in a low resolution (∼16å) structure, obtained by cryoelectron microscopy, of mammalian 80s ribosome in complex with the infectious bronchitis virus (ibv) pp1a/pp1b pseudoknot frameshift signal, as well as eef2 occupying the a-site and a trna occupying the p-site [35] , it is found that the paused pseudoknot is stuck at the entrance of the mrna tunnel of the ribosome, and the d-helix of the p-site trna bends heavily toward the 3 direction (compared to a structure with nonframeshift stimulating stem-loop rna). the opposing forces placed by translocation and the wedged pseudoknot may create local tension on the mrna, which can be relaxed by frameshifting. the superior ability of pseudoknots to pause ribosomes is presumably due to the unique pseudoknotted topology. the presence of stem2 greatly limits the rotational freedom of stem1, making the pseudoknots harder to unwind by the translating ribosomes than simple stem-loop structures with comparable thermodynamic stability [36] . single molecule studies using optical tweezers to pull pseudoknots apart [37] [38] [39] showed that the mechanical force required to unfold pseudoknots is much larger than the gibbs free-energy difference (δ ) between folded and unfolded pseudoknots. the requirement for extra energy input may explain why the pseudoknot is more resistant to unfolding by optical tweezers (presumably by ribosomes as well). the mechanical strength of pseudoknots was further correlated with the frameshift stimulating ability of the pseudoknots [37, 38] . by extrapolating the data, it was proposed that pseudoknots with certain mechanical strength would be able to stimulate −1 frameshifting of 100% efficiency, and pseudoknots with even higher mechanical strength would stall the ribosomes completely like a roadblock (with or without frameshifting), leading to translation termination [38] . a more recent study confirmed the ribosomal roadblocking effect of strong pseudoknots [40] . by investigating a number of designed pseudoknots with varied numbers of basepairs in the stems, it was shown that the strongest pseudoknots (as predicted) only induced limited frameshifting, as judged by the amount of full-length frameshifted products. however, analysis of the pulse labeled proteins revealed that a significant fraction of the ribosomes did shift frames but failed to pass the pseudoknot structures to continue translation; the strength of the pseudoknots correlated not only with the fraction of frameshifted ribosomes but also the roadblocking effect. based on these observations, it was proposed that the optimal frameshifting efficiency would be produced when a balance of the two effects is achieved. according to this hypothesis, the naturally occurring frameshift stimulating pseudoknots should have optimal mechanical strength to cause the right amount of translating ribosomes to shift frames but should not be too strong in order to ensure that the ribosomes are not stalled permanently. it is also implied that strong pseudoknots that exhibit a roadblocking effect should not be present in the coding regions of mrnas. given these recent progresses on the mechanisms of frameshifting, it would be interesting to assess the uniqueness of the frameshift stimulating pseudoknots in the viral genomic rnas. how many other potential pseudoknots are present in the viral mrnas? how do the other pseudoknots compare to the frameshift stimulating pseudoknots in terms of thermodynamic stability and mechanical strength? are there strong roadblocking pseudoknots in the viral genomic mrnas? to address these questions, we have developed a computer program capable of identifying potential h-type pseudoknots in any given mrna sequence and ranking the identified pseudoknots according to the relative strength of their helical stems. using the program, we have analyzed the full-length genomic mrnas of 81 animal viruses that are known or expected to use −1 frameshifting or readthrough as a decoding mechanism for protein expression. a computer program has been developed to identify all putative h-type pseudoknots within any given rna sequence (bioinformation, in press). figure 1 (a) shows a linear presentation of the sequence elements of a typical h-type pseudoknot, which requires that both helical stems (s1 and s2) can form simultaneously. if a given rna sequence contains two pairs of complementary stretches (s1-5 complementary to s1-3 and s2-5 complementary to s2-3 ) g-u is considered a legitimate basepair separated by two or three connecting unpaired regions (l1, l2, and optionally l3) with a sequential arrangement as shown in figure 1 (a), then an h-type pseudoknot can potentially form within this sequence. the computer program tests all possible combinations of stem and loop lengths within certain ranges to see whether the pseudoknotforming criteria can be met. the ranges for the lengths of the stems and loops can be set by the user. the default ranges are as follows: s1 has 5 to 20 base pairs; s2 has 5 to 20 base pairs; l1 has 1 to 10 nucleotides; l2 has 3 to 50 nucleotides, and l3 has 0 to 10 nucleotides. in order to compare the relative thermodynamic stability and mechanical strength of the identified pseudoknots within a given mrna sequence, we implemented free energy (δ ∘ 37 ) calculation for the two helical stems s1 and s2. in calculating the free energy, the turner's nearest-neighbor parameters are used [41] . if l3 = 0, the two stems are taken as a continuous helical stem for the calculation but only half of the value is given to the s1-s2 stack to account for the quasicontinuous nature of the stacked stems. if an l3 is present, the free energy is calculated as a sum of the energies of the two individual stems. although this simplified free energy calculation should only be viewed as semi quantitative, it provides a reasonable estimation of the relative stability of the detected pseudoknots, which are ranked according to the calculated free energies. the calculated free energy value is also used as a criterion to discard those pseudoknots with less stable stems. by default, only those pseudoknots with a free energy value lower than −18 kcal/mol are kept for further analysis. the output file of the program contains information about whether pseudoknots are found and how many are found; the detected pseudoknots are then listed in the order of calculated free energy of the stems. for each of the detected pseudoknots, the following information is given: lengths of s1, s2, l1, l2, and l3; free energy value of the stems; size and location of the pseudoknot. a schematic diagram is then drawn showing the actual pseudoknot forming sequence and base pairing of the two stems; a sequence of 20 nucleotides immediately 5 -to the pseudoknot is also shown because frameshift or readthrough pseudoknots usually appear several nucleotides downstream of the frameshift or readthrough sites (see supplementary file 1, for an example output file in supplementary materials available online at http://dx.doi.org/10.1155/2013/984028). in our study, a total of 81 full-length genomic rna sequences of animal viruses were analyzed for the presence of potential h-type pseudoknots. to facilitate the analysis of such a large number of full-length sequences, in house-developed computer program is used, which is capable of identifying h-type pseudoknots efficiently and reliably. in brief, the program identifies pseudoknots by scanning through the input rna sequence and testing every possible combination of stem and loop (s1, s2, l1, l2, and l3) lengths within the predefined ranges to see whether two helical stems can form simultaneously in a linear sequential topology as shown in figure 1 (a). this approach ensures that no potential pseudoknots with stem and loop lengths that fall within the predefined ranges would escape from being detected. in our pseudoknot search, we set the default ranges for stem and loop lengths as follows: stem1 (s1) and stem2 (s2) both have from 5 to 20 base pairs, loop1 (l1) has from 1 to 10 nucleotides, loop2 (l2) has from 3 to 50 nucleotides, and loop3 (l3) has from 0 (l3 is absent) to 10 nucleotides. these default ranges are very generous because most established htype pseudoknots have stem and loop lengths that fall within these ranges. to evaluate the relative strength of the identified pseudoknots in a given viral genomic rna sequence, the free energy of the two stems for each of the identified pseudoknots was calculated, based on the turner's nearest-neighbor parameters. the pseudoknots were ranked according to the calculated free energies. for a bioinformatic investigation, this calculated free energy represents the best way to evaluate the relative stabilities of the putative h-type pseudoknots within a given viral genome. the free energy value is also used as a criterion for the search; only those pseudoknots with a free energy value lower than −18 kcal/mol are kept for further analysis. in a typical search, tens of potential pseudoknots were identified within the full-lengths genomic viral mrnas. for example, in the full-length genomic rna of simian retroviruses type-1 (srv-1, accession number m11841) that has 8173 nucleotides, 50 potential pseudoknots were identified using the default stem and loop ranges for pseudoknot formation. some of these potential pseudoknots have overlapped pseudoknot-forming sequences; that is, two or more potential pseudoknots are mutually excluded and cannot exit at the same time. after eliminating overlapped pseudoknots with higher free energy, the number of potential pseudoknots in the srv-1 genomic mrna decreases to 31. of course, it is possible that some of the detected pseudoknots may not really exist. among the 31 detected pseudoknots, the established −1 frameshift stimulating pseudoknot at the gag-pro junction [25, 43] is identified as the most stable pseudoknot as biomed research international 5 judged by the lowest calculated free energy of −33.7 kcal/mol (table 1) . while pseudoknots were detected shortly downstream from the frame-shift or read-through sites in most of the viral sequences using the default ranges of stem and loop lengths, the default search did miss some known cases, such as the frameshift stimulator pseudoknot in human coronavirus 229e that has a 164 nt l2. for such cases, the ranges of stem and loop lengths were increased accordingly for another round of search. at the end, possible pseudoknots were identified shortly downstream from the frame-shift or read-through sites in 69 full-length viral genomic mrna sequences (85% of the 81 sequences). table 1 lists information related to the detected frameshift-or readthroughstimulating pseudoknots. this table does not include those viral mrnas in which no pseudoknot was identified downstream from the slippery sequence. in table 1 , the viruses are grouped into different families and listed in the particular orders as in the ictv (international committee on taxonomy of viruses) 2011 master species list (msl) version 2. as documented in recode v2.0: database of translational recoding events [42] , a large number of viruses are known or expected to use a pseudoknot as the stimulator rna structure for −1 frameshifting or readthrough. all but one (human astrovirus) of these documented pseudoknots are identified by our pseudoknot searching program. the putative frameshift stimulator pseudoknot in human astrovirus as shown in the recode database has three mismatched pairs in a row within a five-basepair stem2, which explains why it is not detected by our program. interestingly, the program identifies many potential frameshift stimulating pseudoknots in viruses whose frameshift stimulators are indicated as simple stem-loop structure (or absence of structure) in the recode database. below, we briefly describe the results. the arteriviridae family. −1 frameshift stimulating pseudoknots identified in this family of viruses are pretty much identical to those shown in the recode database (see table 1 for summarized information of the pseudoknots and figure 2 for schematic drawings of representative pseudoknots from various virus families). the involvement of the lv and ldv pseudoknots in efficient frameshifting was established [44, 45] . the pseudoknots in eav, lv, ldv, and prrsv are comparable to each other in terms of the lengths of the spacer region, s1, s2, l1, and l3, and the calculated stem free energies. the lengths of l2 are more varied. the length of l2 in eav (68 nt) is substantially longer than those in other viruses of this family. interestingly, we find that this l2 sequence harbours a potential pseudoknot with 31 nt (figure 2 ). this pseudoknot seems very credible because it is very similar to the structurally well characterized t2 bacteriophage gene 32 mrna autoregulatory pseudoknot [46] and the srv-1 gagpro frameshift stimulating pseudoknot [24, 25] . moreover, this potential pseudoknot, when placed in an "up-side-down" orientation, stacks just right on top of the stem1 of the frameshifting pseudoknot. stacking of the four stem regions of the two pseudoknots creates a quasi-continuous double helix of 28 basepairs in length. the slippery sequence in eav (g uua aac) is somewhat deviated from the consensus slippery sequence of x xxy yyz found in most other viruses. whether the elaborate arrangement of pseudoknots in eav plays any role in regulating frameshifting efficiency, and if it does, whether it is related to the "atypical" slippery sequence, await further investigations. all of the frameshift stimulating pseudoknots in eav, lv, ldv, and prrsv rank first among all of the potential pseudoknots identified within the fulllength genomic rnas. the other virus of the arteriviridae family, simian hemorrhagic fever virus (shfv), also has a potential frameshift stimulating pseudoknot, which seems to be different from those pseudoknots in eav, lv, ldv, and prrsv. the potential frameshift stimulating pseudoknot in shfv ranks 78th among all of the potential pseudoknots identified within the full-length genomic rnas. the first ranked potential pseudoknot has a calculated stem free energy of −31.7 kcal/mol. the coronaviridae family. prf stimulating pseudoknots identified in this family of viruses at the orf1a and orf1b overlapping region are basically the same as those shown in the recode database and a number of previous studies [18, 22, [47] [48] [49] [50] . most of these pseudoknots have comparable stems and loops (see figure 2 for the ibv pseudoknot as a representative). all but one of these pseudoknots rank first in terms of calculated stem free energy among all of the potential pseudoknots identified within the full-length genomic rnas. the hcv229e frameshifting pseudoknot has a long (164 nt) l2. it was found that a short stretch of nucleotides at the 3 -end of l2 participated in the formation of an extra helical stem required for efficient frameshifting in hcv229e [49] . the extra stem has the potential to stack on stem s2 of the pseudoknot. the established frameshift stimulator pseudoknot in sars also has an elaborated three stemmed structure [17, 18] . the 5 -end sequence of l2 has the potential to form a stem-loop structure. the extra stem has the potential to stack on stem s1 of the pseudoknot. the frameshift stimulator pseudoknot in pedv is different from those pseudoknots in other viruses of this family. most noticeably, the length of s1 is much shorter (5 bp versus 11-14 bp). correspondingly, the pseudoknots are less stable. it ranks beyond 100th among all the potential pseudoknots within the gemones. the 1st ranked putative pseudoknot in this virus has a calculated free energy of −38.7 kcal/mol. the astroviridae family. there are five astroviruses (human, ovine, mink, turkey and chicken astroviruses) in the recode database. all viruses use the same slippery sequence a aaa aac. according to the recode database, human astrovirus and chicken astrovirus use a pseudoknot and a stem-loop as the frameshift stimulator, respectively, while there is no information on the other three viruses. however, we detected potential pseudoknots 6-8 nt downstream from the slippery sequences in ovine, turkey and chicken astroviruses. the detected pseudoknots have comparable stem and loop lengths, as well as stem free energies. they rank 2nd, 10th, ss or rt: slippery sequence or readthrough of stop codon; sp: length of the spacer sequence between the slippery sequence/stop codon and the downstream pseudoknot; s1, s2, l1, l2, and l3: lengths of the sequence elements of the pseudoknot, stem1, stem2, loop1, loop2, and loop3. see figure 1 for the sequence elements of a typical pseudoknot. calculated free energy of the stem regions of the pseudoknot is listed in the column "δ " in minus kcal/mol. "rank" indicates the relative ranking (according to the calculated free energy of the stems) of the frameshift/readthrough stimulating pseudoknots among all possible pseudoknots detected within the full-length genomic rnas. "cpk1" indicates whether the pseudoknot belongs to the cpk1 family. and 16th among all the potential pseudoknots within the gemones. the flaviviridae family. pseudoknots detected in this family are identical to those documented in the recode database. two slippery sequences are used by these viruses: c ccu uuu and u ccu uuu. the pseudoknots are very similar to each other in terms of the lengths of the stems & loops and stability. in all but one of the viruses, the putative frameshift stimulating pseudoknot rank 1st among all potential pseudoknots within the genome. the frameshift stimulating pseudoknot in murray valley encephalitis virus ranks 2nd. the 1st ranked pseudoknot has a calculated stem free energy of −38.9 kcal/mol, which is slightly lower than the frameshift stimulating pseudoknot. the retroviridae family. viruses in the retroviridae family belong to several genera: alpha-, beta-, gamma-, delta-, or epsilon-retroviruses and lentivirus. these retroviruses utilize three different mechanisms to express their gag, pro and pol genes from a single gag-pro-pol translational unit: (1) inframe readthrough of the gag termination codon (gammaand epsilon-retroviruses); (2) single frameshift event at the gag-pol junction to express the pol gene (alpharetrovirus and lentivirus); (3) double frameshift events at the gag-pro and pro-pol junctions to express the pro and pol genes (beta-and delta-retroviruses). the two alpharetroviruses avian leukosis virus (alv) and rous sarcoma virus (rsv) have very similar sequences at the gag-pol frameshift junction and both have a pseudoknot. the pseudoknot in rsv as a frameshift stimulator has been established [51] . the alv and rsv pseudoknots contain a very long l3 (52 nt) which is much longer than those in most other known frameshift stimulator pseudoknots. due to the unusual length of l3 (not within the default range), the alv and rsv frameshift stimulator pseudoknots initially were not detected. they were detected after we increased the upper limit for l3 to 60 nt. both pseudoknots ranked 1st among all possible pseudoknots within the viral genomes. the rsv and alv pseudoknots as shown in table 1 leave only one nucleotide in the spacer, which is too short to position the slippery sequence at the active site of the ribosome while leaving the pseudoknot at the entrance of the mrna tunnel based on model building studies [13, 52] . however, this problem can be solved easily by breaking an appropriate number of base-pairs within stem1 adjacent to the spacer. many betaretroviruses were investigated (jsrv to miap in table 1 ). all of the viruses in this group rely on double frameshifting mechanism to express their pro and pol genes. htlv-i (gag-pro) table 1 shows the detected pseudoknots associated with gagpro frameshifting (potential pro-pol frameshift stimulator pseudoknots are not listed in table 1 ). most of these pseudoknots are identical to those previously reported and shown in the recode database. these pseudoknots are comparable to each other in terms of the lengths of the stems and loops. they are all very compact pseudoknots with less than 35 nt. l3 is absent in all but one (mmtv) of the pseudoknots. all pseudoknots ranked 1st among all possible pseudoknots within the viral genomes (using the default ranges of stem and loop lengths in the search). no pseudoknot was detected downstream from the propol frameshift site in any of the betaretroviruses when the default ranges for stem and loop lengths were used in the search. however, when we increased the upper limit of l1 to 60 nt and performed another round of search, potential frameshift stimulator pseudoknots were detected in all viruses (for an example pseudoknot, see figure 2 . see biomed research international 9 supplementary figure 1 for all of the potential pseudoknots). these pseudoknots are much bigger than the compact gag-pro frameshift stimulator pseudoknots. they all have a relatively large l1 (ranging from 36 to 52 nt) and l2 (ranging from 15 to 52 nt); the total numbers of basepairs in the two stems are also larger (ranging from 14 to 20 bp). the functional importance of the gag-pro frameshift pseudoknots in the betaretroviruses has been well established [24, 25, [53] [54] [55] . in contrast, the utilization of a pseudoknot as the frameshift stimulator for pro-pol frameshifting has not been established. four deltaretroviruses (blv to stlv in table 1 ) were investigated. these viruses also utilize double frameshift mechanisms to express their pro and pol genes. the secondary rna structures downstream of the gag-pro frameshift sites in this group of retroviruses were generally believed to be simple stem-loops [22, 56, 57] . using the default ranges for stems and loops, potential pseudoknots were detected downstream from the gag-pro frameshift site in all viruses but blv (not shown). however, credibility of the detected pseudoknots is questionable due to the lack of appropriate spacer between the slippery sequence and the pseudoknot. in view of the fact that the previously reported stem-loop structures seem to be conserved in all four viruses, we increased the upper limit for l2 to 85 nt and performed another search. interestingly, potential pseudoknots with decent stability were detected downstream from the gag-pro frameshift site in all four viruses (shown in table 1 ); moreover, the detected pseudoknots are all formed by basepairing of a stretch of nucleotides in the loop of the previously reported stem-loop structure with a complimentary sequence 61-83 nt downstream. very stable pseudoknots were also detected downstream from the pro-pol frameshift site in all four deltaretroviruses (for an example pseudoknot, see figure 2 . see supplementary figure 1 for all of the potential pseudoknots). similar to the srv group, the putative pro-pol frameshift stimulator pseudoknots are bigger than the gag-pro pseudoknots. the pro-pol pseudoknots in htlv-i and stlv-i are the most stable pseudoknots among all of the detected pseudoknots in this study (figure 2) , with a calculated free energy of −54.2 kcal/mol. gammaretroviruses and epsilonretroviruses utilize the in-frame read-through decoding mechanism. for the two epsilonretroviruses walleye dermal sarcoma virus and snakehead retrovirus, no pseudoknot was detected downstream from the gag reading frame stop codon. the most stable pseudoknots detected in these two viruses have calculated free energy values of −33.0 and −40.7 kcal/mol respectively. for the gammaretroviruses (indicated by "rt" in the "ss or rt" column in table 1 ), conserved pseudoknots were detected downstream from the gag reading frame stop codon in all viruses. these pseudoknots are the same as previously proposed [22] and shown in the recode database. the indispensable role of the mo-mulv pseudoknot in readthrough suppression had been established by two independent studies [20, 21] . all of these putative pseudoknots ranked 1st or 2nd among all possible pseudoknots within the viral genome. the lentiviruses investigated include several non-primate lentiviruses (biv to plv-14 in table 1 ) and three primate lentiviruses (hiv-1, hiv-2 and siv). potential pseudoknots were detected downstream from the gag-pol frameshift site in all non-primate lentiviruses. these frameshift stimulator pseudoknots are largely the same as previously reported and documented in the recode database [22, [58] [59] [60] . all but two of these pseudoknots ranked 1st among all possible pseudoknots within the viral genome. there are three primate (simian or human) lentiviruses: simian immunodeficiency viruses (siv) and human immunodeficiency viruses type-1 and type-2 (hiv-1 and hiv-2) in the recode database. the database gives only one representative sequence for each of these viruses. these are the particular sequences we investigate in this study. no potential pseudoknot was detected downstream from the gagpol frameshift site in siv. in hiv-1 (strain hxb2), the sequence downstream from the gag-pol frameshift site harbours two potential pseudoknots that are mutually excluded (figure 3 ). one of the potential pseudoknots is preceded by a normal length spacer, while the other potential pseudoknot follows the slippery sequence by 0 or 1 nucleotides (depending on whether a g-u basepair is formed in s1). the two pseudoknots rank 16th and 10th, respectively, among all potential pseudoknots within the genomic mrna (only the 10th ranked pseudoknot is listed in table 1 ). the 1st ranked potential pseudoknot has a calculated free energy of −29.6 kcal/mol. intriguingly, we detected another potential pseudoknot that involves the slippery sequence u uuu uua (boxed in figure 3 ). the two potential pseudoknots in the ga-pol junction region have the potential to stack their stems together to form a quasicontinuous double helix with 22 basepairs. interestingly, the two potential pseudoknots are very similar to the established srv-1 gag-pro frameshift stimulator pseudoknot. they are all compact pseudoknots belonging to the previously proposed cpk-1 (standing for common pseudoknot motif 1) type [46, 61] (more details in section 4). in hiv-2, a compact pseudoknot was detected immediately downstream from the gag-pol frameshift site (figure 3) . this potential pseudoknot ranks 5th among all potential pseudoknots within the genomic mrna. the 1st ranked potential pseudoknot has a calculated free energy of −30.5 kcal/mol. the togaviridae family. twenty viruses in this family (all belonging to the alphavirus genus) were investigated. for three of these viruses (midv, nduv, and sesv), the recode database predicts a pseudoknot structure downstream from the frameshift site. for the other viruses, the recode database either predicts a simple stem-loop structure downstream from the frameshift site or makes no prediction. using our pseudoknot search method, we detected putative frameshift stimulator pseudoknots in twelve viruses, including midv, nduv and sesv (table 1 and figure 4 ). half of these pseudoknots ranked 1st among all possible pseudoknots within the viral genomes. in those viruses in which no frameshift stimulator pseudoknot is detected or the detected we have used a robust in house-developed computer program to detect potential pseudoknots within the full-length genomic mrnas of a large number of viruses. in many of these viruses, a frameshift or readthrough stimulator pseudoknot was verified or predicted previously (as documented in the recode database). all but one of these pseudoknots were detected by our program. the missed case is in human astrovirus, in which the predicted framshift stimulator pseudoknot by the recode database is very weak. importantly, our approach of pseudoknot detection was not restricted to a limited sequence window downstream from the known frameshift or readthrough recoding sites. instead, the program detects all possible pseudoknots within the fulllength viral genomic mrnas. the effectiveness and reliability of our approach are proven by the fact that almost all of the previously documented frameshift or readthrough stimulator pseudoknots are detected. interestingly, we also detected quite a number of putative frameshift stimulator pseudoknots that were not known before. overall, potential pseudoknots were detected downstream from most (∼90%) of the established or putative frameshift or readthrough sites (the gag-pro and pro-pol sites in the same virus are counted as two different sites). some of these detected pseudoknots may not actually exist. however, the high percentage of possible pseudoknots detected downstream from the strategically important frameshift or readthrough sites still overwhelmingly proves that pseudoknots are the most common stimulators for efficient −1 ribosomal frameshifting and readthrough. since all possible pseudoknots within the full-length viral genomic mrnas are detected in a blind search, the results from this study provide a new way to assess the significance and uniqueness of the frameshift or readthrough stimulator pseudoknots in an unbiased manner. as shown in table 1 , in ∼78% of the viruses, the pseudoknot detected downstream from the frameshift or readthrough site rank 1st or 2nd among all possible pseudoknots within the genome. the pseudoknot with the lowest free energy (−48.7 kcal/mol) in table 1 is found in lelystad virus. the detected pseudoknot downstream from the pro-pol framshift site in htlv-i/stlv-i ( figure 2 , not listed in table 1 ) has an even lower free energy of −54.2 kcal/mol. this pseudoknot is the most stable pseudoknot among all the possible pseudoknots (regardless of locations of the pseudoknots within the viral genomes) detected in this study. in comparison, the artificial strong pseudoknots that can act as ribosomal roadblocks described in a previous study [40] has a calculated free energy of −73.9 kcal/mol (22 bp in s1 and 6 bp in s2). apparently, the frameshift or readthrough stimulator pseudoknots in a lot of viruses have evolved to become the most stable pseudoknot within the viral genomic mrnas; but at the same time they are not too strong. these pseudoknots seem to have optimal stability to stimulate the right amount of frameshifting and readthrough and subsequently be unfolded by the translating ribosomes. in other viruses in which the detected frameshift or readthrough stimulator pseudoknot ranks lower or no pseudoknot is detected downstream from the frameshift site, the most stable potential pseudoknots all have a calculated free energy value higher than the putative pro-pol framshift stimulator pseudoknot in htlv-i/stlv-i. these results clearly show that the viral genomic mrnas do not contain ultra-stable "roadblocking" pseudoknots that would significantly stall ribosomes and might induce no-go decay of the mrnas [40, 62] . it was noticed previously that many naturally occurring pseudoknots (not limited to frameshift and readthrough stimulating pseudoknots) belonged to a structurally related pseudoknot family known as cpk-1, standing for common pseudoknot motif 1 [46, 61] . a typical cpk-1 pseudoknot has a s2 of 6-7 base pairs and a very short l1 of 1-2 nucleotides; l3 is absent therefore the two helical stems s1 and s2 can stack to form a quasicontinuous helix. an inspection of the detected frameshift or readthrough stimulator pseudoknots reveals that more than 40% of these pseudoknots conform to the cpk-1 family ( table 1 ). the alternative tandem pseudoknots (figure 3 . not listed in table 1 ) in hiv1 and the elaborated pseudoknot in eav (figure 2 ) also conform to the cpk-1 family. interestingly, it was found that the founding member of the cpk-1 family, a pseudoknot in gene 32 mrna of bacteriophage t2 whose natural biological function is translational autoregulation, was unable to serve as a frameshift stimulator [63] . most likely, the common features defined by the cpk-1 motif may primarily serve a structural role for maintaining a stable and compact pseudoknotted scaffold upon which diverse biological functions can build on, presumably mainly by the more variable parts of the pseudoknots, including s1 & l2, and possible interactions between them. consistent with this theory, it was found in several different systems that frameshifting efficiency was generally more sensitive to mutations introduced to s1 & l2, and the junction, while mutations to s2 & l1 showed less effect on frameshifting efficiency, as long as integrity and stability of the pseudoknot-forming interaction were maintained [25, [63] [64] [65] [66] . this theory provides a very good explanation for the frequent utilization of cpk-1 type pseudoknots in various viruses. it is clear that while a large number of detected pseudoknots conform to the cpk-1 family, the lengths and compositions of s1 and l2 of these pseudoknots show a fair degree of variations, especially when viruses from different groups are compared. moreover, additional features could be added to the "basic" cpk-1 pseudoknot fold (such as seen in the eav, hcv229e and sars pseudoknots) which would make the pseudoknots even more versatile in fine-tuning the frameshifting. although cpk-1 type pseudoknots occur most frequently among the detected pseudoknots, other types of pseudoknots are also observed. these pseudoknots sample a wide range of stem and loop sizes, as well as the presence or absence of an intervening sequence (l3). given the wide range of sequences studied, it is not particularly surprising to observe all these variations associated with the detected pseudoknots. since different viruses (especially viruses that are remote in evolution) may have different requirements for certain level of frameshifting efficiency, variations in the frameshifting pseudoknots, as well as the slippery sequences and spacers, may be necessary for the fine-tuning of the frameshifting efficiency to meet the specific need of different viruses. the frameshift stimulating secondary structure downstream of the hiv-1 group m (which includes the strain hxb2 investigated in this study) gag-pol frameshift site was originally proposed to be a simple stem-loop [67] , which was shown to be important for wild-type level frameshifting in vivo (in mammalian cells) [68] . it was also shown that a sequence downstream from the originally proposed stemloop also contributed to frameshifting, either modelled as an intramolecular triplex [69] or an extended bulged stemloop [70] . in hiv-1 strain mvp5180 from subgroup o, a very classic h-type pseudoknot locating 8nt downstream from the gag-pol frameshift site was shown to be required for stimulating a higher frameshifting efficiency than that in group m [71] . our study detected potential pseudoknots in the gagpol junction of both hiv-1 and hiv-2. in hiv-1 (hxb2), two mutually excluded potential pseudoknots were detected downstream from the slippery sequence (figure 3) , one with no spacer and the other with a normal length spacer. intriguingly, another pseudoknot that contains the slippery sequence is detected. this pseudoknot can stack on top of the pseudoknot immediately downstream from the slippery sequence. given such an elaborated arrangement of tandem pseudoknots (both belonging to the cpk-1 family) and another mutually excluded pseudoknot at the hiv-1 gag-pol frameshift junction, we asked ourselves this question: can we come up with a reasonable hypothesis about the prf mechanisms in this case that would explain the possible involvement of these pseudoknots? the answer is: yes we can. let us assume that the tandem pseudoknots are present at the gag-pol frameshift junction (due to its lower free energy compared to the alternative pseudoknot). these pseudoknots can significantly slow down the translating ribosome when the pseudoknots are being unwound by the ribosome. when the ribosome scans through the unwound mrna sequence and is approaching the slippery sequence, the top pseudoknot is fully unwound and the stem1 of the bottom pseudoknot should also be disrupted. stem2 of the bottom pseudoknot remains intact. the six basepairs of this stem2 are actually the same as the first six basepairs in stem1 of the mutually excluded pseudoknot ( figure 3 ). now that the tandem pseudoknots are unwound, the alternative pseudoknot can form rapidly (because a large portion of its stem1 is already in place). the newly formed pseudoknot, with an optimal spacer from the slippery sequence, jams the entrance of the mrna tunnel of the ribosome. this novel mechanism of prf elegantly explains the results from our bioinformatic study and is consistent with current paradigm of prf mechanism. equilibrium of relevant alternative rna structures has been shown to play a functional role in the regulation of read-through efficiency in murine leukaemia virus, suggesting a general involvement of equilibrium-based mechanism in translational recoding [72] . we plan to carry out a large scale analysis on the several thousand sequences for different strains of hiv1 viruses to assess the degree of conservation on the putative prf signals. several conclusions can be drawn from our study. at first, the viral genomic mrnas do not contain strong roadblocking pseudoknots that would terminal translation. second, the frameshift or readthrough stimulating pseudoknots in most viruses are among the most stable pseudoknots within the viral genomic mrnas. the stabilities of these pseudoknots have been fine-tuned during evolution to be optimal for the decoding events. third, pseudoknots of the cpk-1 family occur most frequently. the favorable cpk-1 scaffold can accommodate significant variations (especially in the stem1 and loop2 regions) which are presumably important for the fine-tuning of framshift or readthrough stimulating ability of the pseudoknots. fourth, some hiv1 viruses may utilize a novel mechanism that involves three pseudoknots to regulate the frameshift efficiency at the gag-pol junction. results from this study also prove the usefulness of our pseudoknotdetecting program. since this is a general-purpose program that can identify all possible pseudoknots in a long rna sequence, we expect that the program will find its application in some other related studies such as identifying potential cases of pseudoknot-dependent −1 prf in cellular genes. bovine torovirus (breda virus) (brv, nc 007447), and equine torovirus (berne virus) (bev, x52374) rous sarcoma virus (rsv, af033808); jaagsiekte sheep retrovirus nc 001550); simian retroviruses type-1 (srv-1, m11841) and type-2 (srv-2, af126467); mouse mammary tumor virus (mmtv, m15122); squirrel monkey retrovirus (smrv-h, nc 001514); human endogenous retrovirus k10 (herv-k10, m14123); three intracisternal a particle (iap) genetic elements from chinese hamster (chiap34, m73970) human t-cell leukemia virus type-i (htlv-i, af033817) and type-ii (htlv-ii, m10060); simian t-cell leukemia virus type-i (stlv-i walleye dermal sarcoma virus af033822 feline leukemia virus (felv, af052723); gibbon ape leukemia virus moloney murine sarcoma virus (msv, af033813) moloney murine leukemia virus friend murine leukemia virus (f-mulv akv murine leukemia virus (akv-mulv, j01998); bovine leukemia virus jembrana disease virus (jdv, u21603); ovine lentivirus south african ovine maedi visna virus (sa-omvv nc 001463); feline immunodeficiency virus (fiv, m25381); equine infectious anaemia virus (eiav, af033820) simian immunodeficiency virus (siv, m66437, nc 004455). togaviridae family: aura virus barmah forest virus (bfv, nc 001786) eastern equine encephalitis virus (eeev, nc 003899), fort morgan virus getah virus mayaro virus (mayv, nc 003417) o'nyong-nyong virus salmon pancreas disease virus seal louse virus semliki forest virus (sfv, nc 003215) venezuelan equine encephalitis virus (veev, nc 001449); western equine encephalitis virus whataroa virus (whav, af339479) recoding: reprogrammed genetic decoding recoding: translational bifurcations in gene expression ribosome gymnastics-degree of difficulty 9.5, style 10.0 recoding: dynamic reprogramming of translation viral rna pseudoknots: versatile motifs in gene expression and replication normal trnas promote ribosomal frameshifting expression of the rous sarcoma virus pol gene by ribosomal frameshifting a new principle of rna folding based on pseudoknotting pseudoknots: a new motif in the rna game structural and functional aspects of rna pseudoknots ribosomal frameshifting on viral rnas programmed translational frameshifting structure, stability and function of rna pseudoknots involved in stimulating ribosomal frameshifting programmed ribosomal frameshifting in decoding the sars-cov genome regulation of programmed ribosomal frameshifting by cotranslational refolding rna hairpins programmed -1 ribosomal frameshifting in the sars coronavirus a three-stemmed mrna pseudoknot in the sars coronavirus frameshift signal an atypical rna pseudoknot stimulator and an upstream attenuation signal for -1 ribosomal frameshifting of sars coronavirus frameshifting in alphaviruses: a diversity of 3' stimulatory structures evidence that a downstream pseudoknot is required for translational read-through of the moloney murine leukemia virus gag stop codon bipartite signal for read-through suppression in murine leukemia virus mrna: an eight-nucleotide purine-rich sequence immediately downstream of the gag termination codon followed by an rna pseudoknot rna pseudoknots: translational frameshifting and readthrough on viral rnas pseudoknot-dependent read-through of retroviral gag termination codons: importance of sequences in the spacer and loop 2 base-pairings within the rna pseudoknot associated with the simian retrovirus-1 gag-pro frameshift site solution structure of the pseudoknot of srv-1 rna, involved in ribosomal frameshifting metal ions and flexibility in a viral rna pseudoknot at atomic resolution minor groove rna triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot specific mutations in a viral rna pseudoknot drastically change ribosomal frameshifting efficiency solution structure of a luteoviral p1-p2 frameshifting mrna pseudoknot crystal structure of a luteoviral rna pseudoknot and model for a minimal ribosomal frameshifting motif a loop 2 cytidinestem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated -1 ribosomal frameshifting ribosomal pausing at a frameshifter rna pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency ribosomal pausing during translation of an rna pseudoknot ribosomal movement impeded at a pseudoknot required for frameshifting a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting torsional restraint: a new twist on frameshifting pseudoknots correlation between mechanical strength of messenger rna pseudoknots and ribosomal frameshifting triplex structures in an rna pseudoknot enhance mechanical stability and increase efficiency of -1 ribosomal frameshifting characterization of the mechanical unfolding of rna pseudoknots mrna pseudoknot structures can act as ribosomal roadblocks predicting thermodynamic properties of rna recode-2: new design, new search tools, and many more genes analysis of the role of the pseudoknot component in the srv-1 gag-pro ribosomal frameshift signal: loop lengths and stability of the stem regions lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (ldv) structure of the autoregulatory pseudoknot within the gene 32 messenger rna of bacteriophages t2 and t6: a model for a possible family of structurally related rna pseudoknots the primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus mhv-a59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase an "elaborated" pseudoknot is required for high frequency frameshifting during translation of hcv 229e polymerase mrna complete sequence (20 kilobases) of the polyproteinencoding gene 1 of transmissible gastroenteritis virus secondary structure and mutational analysis of the ribosomal frameshift signal of rous sarcoma virus the 9-a solution: how mrna pseudoknots promote efficient programmed -1 ribosomal frameshifting structural and functional studies of retroviral rna pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting a characteristic bent conformation of rna pseudoknots promotes-1. frameshifting during translation of retroviral rna the structure of an rna pseudoknot that causes efficient frameshifting in mouse mammary tumor virus two cis-acting signals control ribosomal frameshift between human t-cell leukemia virus type ii gag and pro genes the sequences of and distance between two cis-acting signals determine the efficiency of ribosomal frameshifting in human immunodeficiency virus type 1 and human t-cell leukemia virus type ii in vivo rna pseudoknots downstream of the frameshift sites of retroviruses identification and analysis of the gag-pol ribosomal frameshift site of feline immunodeficiency virus the stimulatory rna of the visna-maedi retrovirus ribosomal frameshifting signal is an unusual pseudoknot with an interstem element an nmr and mutational study of the pseudoknot within the gene 32 mrna of bacteriophage t2: insights into a family of structurally related rna pseudoknots identification of functional, endogenous programmed-1 ribosomal frameshift signals in the genome of saccharomyces cerevisiae comparative studies of frameshifting and nonframeshifting rna pseudoknots: a mutational and nmr investigation of pseudoknots derived from the bacteriophage t2 gene 32 mrna and the retroviral gag-pro frameshift site analysis of the role of the pseudoknot component in the srv-1 gag-pro ribosomal frameshift signal: loop lengths and stability of the stem regions evidence for an rna pseudoknot loop-helix interaction essential for efficient -1 ribosomal frameshifting the role of rna pseudoknot stem 1 length in the promotion of efficient -1 ribosomal frameshifting characterization of ribosomal frameshifting in hiv-1 gag-pol expression human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mrna secondary structure: demonstration by expression in vivo the frameshift signal of hiv-1 involves a potential intramolecular triplex rna structure characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1 the frameshift stimulatory signal of human immunodeficiency virus type 1 group o is a pseudoknot an equilibrium-dependent retroviral mrna switch regulates translational recoding this work was supported by the start-up fund and a seed grant from southern illinois university carbondale to zhihua du and in part by the national science foundation under grant no. iis-1218712 to qiang cheng. 