key: cord-263094-5zbzm1b0 authors: dane, hannah; duffy, catherine; guelbenzu, maria; hause, ben; fee, sean; forster, fiona; mcmenamy, michael j.; lemon, ken title: detection of influenza d virus in bovine respiratory disease samples, uk date: 2019-07-07 journal: transbound emerg dis doi: 10.1111/tbed.13273 sha: doc_id: 263094 cord_uid: 5zbzm1b0 influenza d is a newly described virus of cattle, pigs and small ruminants first detected in north america during 2011. cattle have been shown to be the main viral reservoir and mounting evidence indicates that infection with influenza d may contribute to the development of bovine respiratory disease. the virus has been detected across the united states, europe and asia. to date, influenza d has not been reported in the uk. during the winter and spring of 2017/2018, we performed molecular testing of cattle submitted for post‐mortem examination where respiratory disease signs were present. we detected influenza d virus in 8.7% of cases, often as the sole viral agent and always in conjunction with bacterial co‐infection with one or more agents. viral rna was present in both the upper and lower respiratory tract and pathological changes in lung tissues were observed alongside signs of concurrent bacterial infections. sequencing of one uk isolate revealed that it is similar to viruses from the republic of ireland and italy. and antibiotics, deaths attributed to brd have steadily increased since the mid-1990s (loneragan, dargatz, morley, & smith, 2001; snowder et al., 2007) . orthomyxoviridae family. although initially identified during a 2011 outbreak of respiratory disease in north american pigs (hause et al., 2013) , cattle were subsequently shown to be the main reservoir of the virus (collin et al., 2015; ducatez, pelletier, & meyer, 2015; ferguson et al., 2015; hause et al., 2014) . idv has a wide geographic distribution with high seroprevalence in many herds and has been circulating in the united states since at least 2003 (ferguson et al., 2015; luo et al., 2017) . accumulating evidence indicates that idv is associated with brd complex. a recent metagenomic study has shown that idv was among the top 3 viruses associated with brd in dairy calves (ng et al., 2015) . in another study, metagenomics was used to characterize the virome of nasal swab samples collected from beef cattle with acute brd at feedlot operations in mexico and the united states (mitra, cernicchiaro, torres, li, & hause, 2016) . although 21 different viruses were detected, including those normally associated with influenza d is a newly described virus of cattle, pigs and small ruminants first de(table 1 ). the lower respiratory tract of five animals was also positive for idv, with c t values in lung tissue ranging from 19.6 to 32.4. samples from idv positive animals were tested for co-infections with other respiratory pathogens. all animals were negative for brsv, bohv-1, bpiv-3 and bvdv using either real-time pcr or ifat. co-infections with bcov were detected in three of the nine samples. our results demonstrate that idv is circulating in cattle herds in northern ireland. its detection in animals with confirmed respiratory disease at a prevalence of 8.7% is in broad agreement with similar studies performed in the united states, europe and asia (collin et al., 2015; ducatez et al., 2015; ferguson et al., 2015; flynn et al., 2018; zhai et al., 2017) . for example, flynn et al. detected idv at a prevalence of 5.6% in cattle respiratory samples in the republic of ireland (flynn et al., 2018) we thank eoin ryan and orla flynn (department of agriculture, food and marine) and feng li (south dakota state university) for providing advice and reagents. there are no potential conflicts of interest. ken lemon https://orcid.org/0000-0001-9844-1573 cocirculation of two distinct genetic and antigenic lineages of proposed influenza d virus in cattle influenza d virus in cattle influenza d virus infection in mississippi beef cattle influenza d virus in cattle characterization of a novel influenza virus strain in cattle and swine: proposal for a new genus in the orthomyxoviridae family isolation of a novel swine influenza virus from oklahoma in 2011 which is distantly related to human influenza c viruses trends in mortality ratios among cattle in us feedlots serological evidence for high prevalence of influenza d viruses in cattle metagenomic characterization of the virome associated with bovine respiratory disease in feedlot cattle identified novel viruses and suggests an etiologic role for influenza d virus a metagenomics and case-control study to identify viruses associated with bovine respiratory disease pathogenesis, host innate immune response and aerosol transmission of influenza d virus in cattle bovine respiratory disease in feedlot cattle: phenotypic, environmental, and genetic correlations with growth, carcass, and longissimus muscle palatability traits influenza d virus in animal species in guangdong province, southern china key: cord-316617-8cqxz3wi authors: ward, michael p. title: sars‐cov‐2, where to now? date: 2020-06-19 journal: transbound emerg dis doi: 10.1111/tbed.13654 sha: doc_id: 316617 cord_uid: 8cqxz3wi nan these gaps. to reiterate a statement made in our previous issue, transboundary and emerging diseases is committed to publishing high-quality research on sars-cov-2 (ward, li, & tian, 2020) . yu et al. (2020) in this issue describe the epidemiological and clinical characteristics of 333 confirmed covid-19 cases in shanghai, during the early phase of the pandemic. estimates of some key epidemiologic parameters-such as incubation period-are presented, and risk factors are described. importantly, the role of travel to wuhan features prominently. it is also an example of the successful control of covid-19 within a megacity. bauer et al. (2020) present a novel approach to choosing an animal model for sars-cov-2 studies. in the case of a newly discovered virus such as sars-cov-2, the laborious process of matching the available virus strain with an animal model can be substantially improved by assessing genome-wide functionalities. this has some profound implications for the development of diagnostics, vaccines and treatments, all of which are dependent on effective animal models. a key gap in our knowledge of sars-cov-2 transmission is the role of animals, and despite a consensus that this virus arose from an animal reservoir, a fundamental and intriguing question is which species? deng et al. (2020) present the results of a sars-cov-2 serological survey in 35 animal species in china, including the dog of a covid-19 patient and an additional two in-contact dogs. despite testing 1,914 sera using a double antigen sandwich elisa, sars-cov-2-specific antibodies were not detected. as stressed by li (2020) in a letter to the editors, pets such as cats can be infected when the infection pressure is sufficiently high (such as in wuhan, china during the epidemic); however, accumulating evidence suggests that pets such as cats are unlikely to be a reservoir of sars-cov-2. in addition, diagnostic methods used on animals require validation. tests available for the detection of sars-cov-2 are comprehensively described in this issue of transboundary and emerging diseases (li & ren, 2020) . in addition to the publication of new knowledge about sars-cov-2 in this issue of transboundary and emerging diseases, new ideas are also presented. roe (2020a) has suggested an explanation for the neurological symptoms (acute cerebrovascular disease) noted in covid-19 patients, as well as apparent virus reactivation in patients: by analogy with the known pathogenesis of sars and nipah viruses, selective infection of neurons enables a virus to evade attacks from the host's immune system. if demonstrated to be the case, this has public health implications that will remain well after the current pandemic of acute covid-19 is over. in addition, in this issue the mechanism of thrombosis in covid-19 patients is explored (roe, 2020b) . in recent times, we have faced many global health crises-such as covid-19, african swine fever and avian influenza-to the extent that overlapping emergencies have occurred. stoffel et al. (2020) argue in a letter to the editors in the previous issue of transboundary and emerging diseases that to address such a situation we need a there was evidence of human-to-human transmission as early as at least mid-december . in january, the aetiology was identified as a new coronavirus (subsequently named sars-cov-2). thus, disease emergence was followed by discovery of a cause. the general consensus is that sars-cov-2 is of animal origin-a zoonosis-but despite various hypotheses, the species has not been identified. the process of disease emergence and then pathogen discovery (illustrated in the following figure, upper panel) is characteristic of separate human and animal health systems, a lack of coordination between these sectors, and a response which is reactive to clinical disease occurrence. rather than disease emergence (especially in humans in the case of a zoonosis) leading to pathogen discovery, the opposite is what we need to strive for. discovery of zoonotic agents in animal populations, a thorough risk assessment driven by knowledge of the hazard and the likelihood of spillover, and then integrated monitoring and surveillance of animal and human populations can shift the world into a paradigm of discovery that prevents emergence and spread (as illustrated in the following figure, lower panel) . a key enabler of such a shift in our thinking and approach to disease emergence and spread is a one health workforce capable of undertaking integrated monitoring, surveillance, risk assessment and response activities. specifically in terms of one health surveillance, barriers to operationalization include legal issues, hurdles to data sharing, unclear responsibilities and structural barriers between human and animal authorities that prevent integrated action (stärk et al., 2015) . in addition, as identified by stärk et al. (2015) , 'policy makers in the health sector often perceive one health as a veterinary-driven initiative that is not particularly relevant to their priority problems'. there is a lack of funding for the development of a sustainable one health workforce, perhaps because of the scarcity of a strong business case (costs versus benefits) for one health surveillance. integration (versus specialization) in heath training at an undergraduate level, to promote the inter-relatedness of medical, veterinary and environmental sciences, is also a need (stärk et al., 2015) . the covid-19 pandemic could be a catalyst for such a seismic shift in how we approach emerging infectious diseases and one health. the emergence of infectious diseases can be driven by interconnected economic, social and environmental changes. we have many tools available for identification, prioritization and investigation of emerging infectious diseases impacting human and animal healthincluding environmental and horizon scanning, surveillance, disease prioritization, risk assessment and disease modelling (brookes, hernandez-jover, black, & ward, 2015) . eids are complex, multifactorial health problems for which a single tool is not sufficient; however, integration of the tools already available into a systematic approach for the development of tactical and strategic plans for emerging risk preparedness is lacking (brookes et al., 2015) . we can be sure, even when the current covid-19 pandemic is resolved, that the need for surveillance, response and prevention of transboundary and emerging diseases will remain. an integrated approach to health-regardless of specific pathogens, domains or nations-is a critical need. issues of politics, trade and commerce must be recognized, but can no longer be used as excuses for inaction. the author confirms that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. no ethical approval was required for this editorial. supporting pandemic response using genomics and bioinformatics: a case study on the emergent sars-cov-2 outbreak preparedness for emerging infectious diseases: pathways from anticipation to action serological survey of sars-cov-2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals recent progress on the diagnosis of 2019 novel coronavirus cats under the shadow of the sars-cov-2 pandemic explanation for covid-19 infection neurological damage and reactivations high covid-19 virus replication rates, the creation of antigen-antibody immune complexes, and indirect hemagglutination resulting in thrombosis one health surveillance: more than a buzz word? preventive veterinary medicine the ongoing crises in china illustrate that the assessment of epidemics in isolation is no longer sufficient a novel coronavirus outbreak of global health concern novel coronavirus 2019, an emerging public health emergency epidemiological and clinical characteristics of 333 confirmed cases with coronavirus disease key: cord-280328-16fpiuc2 authors: villanueva‐cabezas, jp; rajkhowa, arjun; campbell, angus j.d. title: one health needs a vision beyond zoonoses date: 2020-08-12 journal: transbound emerg dis doi: 10.1111/tbed.13782 sha: doc_id: 280328 cord_uid: 16fpiuc2 the unprecedented pandemic events that currently affect animals and humans have fueled calls for one health action. we argue that the one health framework must be accompanied by ‘rich outcomes’ to avoid a reductionist one health focus on zoonotic pathogens, that forgoes the benefits of the framework. we propose that the united nation’s sustainable development goals provide an adequate multidimensional set of targets that can help researchers and policymakers contextualise emerging diseases, and guide one health long‐term solutions that are equitable, efficacious, and sustainable. approach is crucial because these diseases' effects transcend their original and current host species, affecting the wellbeing of animals, people, and the environment in multiple ways. therefore, calls to adopt oh approaches in dealing with these pandemics (amuasi et al., 2020) must transcend reductionist, pathogencentric approaches and focus on holistic outcomes, embracing the intricate interactions within a system and confronting the problems that beset it (zinsstag, schelling, waltner-toews, whittaker, & tanner, 2015) . without this approach, the oh response to covid-19 and asf will likely continue to result in scattered actions confined to the immediate need for disease control. multidimensional targets such as the sustainable development goals ("transforming our world: the 2030 agenda for sustainable development," 2015) can provide a set of 'rich outcomes' for systems-based oh strategies to address these pandemics. the sdgs offer a way to systematically understand the pandemic effect on the interrelationships between the human, animal, and environmental elements of the oh framework. understanding the interactions and overlaps between the sdgs can help policymakers and researchers prioritise and identify points of leverage for oh actions, making these more efficient and sustainable, minimising antagonistic outcomes, and generating explicitly defined, maximal benefits. much has already been written about the antagonism between the human health (sdg 3) and socioeconomic effects (sdg 8) of covid-19 responses (hodgins & saad, 2020) . the sdgs may help to contextualise the human, animal and environmental effects of diseases and mitigation efforts more widely. for example, laborde et al. (2020) suggest that covid-19 will increase extreme poverty (sdg 1) globally by 20% and increase agricultural labour availability caused by job losses in the urban service sector. although the latter may boost rural agricultural production (sdg 2), it may simultaneously depress incomes (sdg 1 & 8). covid-19 has also augmented recognition of the oh implications of trading wildlife (sdgs 12 and 15), but a systems oh perspective is needed to achieve effective and sustainable changes to this activity. in the absence of support for alternative livelihoods for those engaged in the exotic species trade, banning wildlife markets may unintentionally increase illicit trade, hamper conservation efforts, and undermine disease surveillance and reporting (eskew & carlson, 2020) . these consequences should be acknowledged in response strategies and their intended outcomes. the emergence of the covid-19 pandemic is necessarily intertwined with increasing human pressures on the environment (sdgs 12, 14, 15) and climate change (sdg 13) (who, 2020). in turn, climate change may expand the distribution of asf reservoirs and soft tick vectors (costard et al., 2009 ). in areas where pig this article is protected by copyright. all rights reserved farming supports food security (sdg 2), and underpins peri-urban and urban sustainability (sdg 11) (costard et al., 2009) , smallholders respond to asf outbreaks by selling or consuming infected pigs (chenais et al., 2017) . these practices may result in counter-intuitive shorter-term nutritional, economic, schooling and/or healthcare benefits (sdgs 2, 4, 3) . however, the large fluctuations in food supply, prices, and incomes caused by these practices may create groups of poor urban consumers who obtain unconventional foods from unregulated sources through preference or necessity (blecha, 2015) , with direct oh implications for foodborne illness, household nutrition, and disease emergence (sdgs 2, 3, 10) . a pathogen-centric oh approach that only advocates biosecurity interventions to control asf may overlook how such actions magnify socioeconomic and gender inequality (sdgs 5, 8, 10) by disproportionately reducing smallholder incomes to the benefit of livestock traders in the absence of good market linkages (ouma et al., 2018) . thus, more holistic asf management that promotes semi-intensive urban pig rearing and more efficient value chain operation can support urban income generation (sdg 1, 2, 8, 11, 12) , reduce zoonoses such as cysticercosis (sdg 3), and reduce trading of free-ranging pigs and other wildlife species (sdgs 12, 14, 15 ). the merit of the oh framework is that it helps not only to identify the emergence and spread of diseases between humans, animals and the environment, but also conceptualises the synergistic and antagonistic effects of disease outbreaks and mitigation efforts on these domains. figure 1 is a simple example of such an oh approach, which may serve as a model for others interested in this framework. when the advantages of this holistic, systems-based view are appreciated, other system-based instruments such as the sdgs can be integrated to help researchers and policymakers delineate outcomes and pathways to them. in turn, the oh paradigm-called for but still under-implemented-can help promote long-term solutions that are equitable, efficacious, and sustainable. this article is protected by copyright. all rights reserved if after covid-19 the community resumes the trade of wildlife, positive sdg effects associated with the trading ban will be lost (c  b) to improved human population wellbeing (c  c). if pig production is promoted as an alternative livelihood in the region, the value chain must be strengthened at all levels (d) to create resilient systems that warrant simultaneous community animal, and environmental wellbeing. if in contrast, the systems lack resilience and asf outbreaks result in pathogen-centric approaches (for example, culling of animals with minimum or no compensation), the positive sdgs effects associated with the pig value chain are severely undermined (e  d). if wildlife trading resumes due to asf, there is increasing negative sdgs outcomes on the environment (e  b), and increased risk of new emerging zoonoses and adverse sdg outcomes on people (b  a). calling for a covid-19 one health research coalition regulating backyard slaughter: strategies and gaps in municipal livestock ordinances quantitative assessment of social and economic impact of african swine fever outbreaks in northern uganda african swine fever: how can global spread be prevented? overcoming challenges for designing and implementing the one health approach: a systematic review of the literature overselling wildlife trade bans will not bolster conservation or pandemic preparedness. the lancet planetary health will the higher-income country blueprint for covid-19 work in lowand lower middle-income countries? global health: science and practice poverty and food insecurity could grow dramatically as covid-19 spreads early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia african swine fever control and market integration in ugandan peri-urban smallholder pig value chains: an ex-ante impact assessment of interventions and their interaction the ongoing crises in china illustrate that the assessment of epidemics in isolation is no longer sufficient transforming our world: the 2030 agenda for sustainable development, a/res/70/1 stat q&a: climate change and covid-19 one health: the theory and practice of integrated health approaches the authors have nothing to disclose.role of funding source:no funding was received to write or publish this manuscript. the authors do not have data to share. the authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. no ethical approval was required as this is a letter to the editor with no original research data. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved accepted article tbed_13782_f1.tiff key: cord-347475-ttmactz0 authors: mesquita, j. r.; hakze‐van der honing, r.; almeida, a.; lourenço, m.; van der poel, w. h. m.; nascimento, m. s. j. title: outbreak of porcine epidemic diarrhea virus in portugal, 2015 date: 2015-09-07 journal: transbound emerg dis doi: 10.1111/tbed.12409 sha: doc_id: 347475 cord_uid: ttmactz0 an outbreak of porcine epidemic diarrhea virus (pedv) in the south of portugal in january 2015 and the spread of pedv northwards in the territory are described. comparative analysis of the amplified sequences showed a very high (99.0%) identity with the pedv variant most recently reported in the united states and also show complete (100%) identity to the strains recently reported in germany, supporting the hypothesis that a unique strain is currently circulating in europe. the origin of this pedv variant still needs to be elucidated and further studies in the remaining european countries may contribute to the knowledge. porcine epidemic diarrhea (ped) is an acute and highly con-tagious enteric disease of pigs. typical clinical symptoms of ped include watery diarrhea, vomiting, dehydration, and porcine epidemic diarrhea (ped) is an acute and highly con-tagious enteric disease of pigs. typical clinical symptoms of ped include watery diarrhea, vomiting, dehydration, and porcine epidemic diarrhea (ped) is an acute and highly con-tagious enteric disease of pigs. typical clinical symptoms of ped include watery diarrhea, vomiting, dehydration, and porcine epidemic diarrhea virus (pedv; family coronaviridae, subfamily coronavirinae, genus alphacoronavirus) is a highly contagious virus responsible for enteric disease in swine characterized by an acute onset of symptoms including severe watery diarrhea, vomiting, dehydration, and high mortality in suckling piglets (ictv, 2012; song and park, 2012) . upon its first description in 1971 in the united kingdom, this disease was initially termed "epidemic viral diarrhea" due to the quick spread across europe (wood, 1977; song and park, 2012) . since then pedv has caused substantial economic losses, predominantly in asia, but in may 2013 a new pedv variant has emerged and rapidly spread in the us (stevenson et al., 2013) . from this time to early june 2014, pedv outbreaks had been reported in 33 states of the united states causing approximately 7 million piglet deaths, and resulting in severe economic losses to the swine industry (us department of agriculture, 2015). in early 2014, a novel variant of pedv (oh 851) was identified in ohio, containing insertions and deletions in the s gene (s indel), causing mild clinical signs and lower mortality rates in suckling piglets (wang et al., 2014) . more recently, an epidemic of severe watery diarrhea in southern germany with typical clinical signs of pedv was reported and found to be caused by a novel pedv, closely related to the us strain oh851, causing concern regarding on the possible circulation of a novel more virulent strain (hanke et al., 2015) . in the present work we describe an outbreak of severe watery diarrhea in swine caused by pedv in portugal, early 2015, and the spread northwards in the country. in january 2015 a pig farm in the south region of portugal reported diarrhea in all animals 2 days after introducing new animals from a different producer. diarrhea lasted for 1 week and a high mortality in piglets was observed. from january to april 2015 another 43 pig farms (4 farms from the south and 39 farms from the center of portugal) have reported similar epidemic diarrheas. a total of 84 fecal samples were collected from all 44 farms and submitted to analysis. stools were diluted (10% phosphate buffered saline), and viral nucleic acid was extracted from centrifuged stool suspensions using high pure rna isolation kit (roche applied sciences, mannheim, germany) and nucleospin rna virus (machery nagel, haerdt, france). nucleic acids were tested for the presence of pedv using a commercial real-time rt-pcr kit according to the manufacturer's instructions that targets the nucleocapsid protein gene (n gene) (viroreal â kit pedv; ingenetix, vienna, austria). a total of four samples positive for pedv rna by realtime rt-pcr (two from a farm in the south and two from a farm in the center of portugal) were further tested by conventional pcr using primers s1-univ-f (5 0 -tac tta caa ctc cac tg ttt -3 0 ) and s1-univ-r (5 0 -cca ttg ata gta gtg tca ga -3 0 ) that amplify a 440 bp region of the s protein. for the cdna synthesis, 0.2 ll of the s1univ-r primer (10 lm), 1 ll of dntp (10 mm), 6.8 ll of h 2 o and 5 ll of rna were incubated for 5 min at 65°c and placed on ice before being added 1 ll of superscript â ii reverse transcriptase (200 u/ll) (invitrogen), 1 ll of rnasin (40 u/ll) (promega), 1 ll 0.1 m dithiothreitol and 4 ll first strand buffer (59) (invitrogen). the rt mix was incubated for 60 min at 50°c. for the pcr, 2 ll of cdna were added to 2 ll of clontech buffer (109), 0.4 ll 509 dntp, 0.4 ll 509 polymerase mix, 1 ll of both s1-univ-f (10 lm) and s1-univ-r(10 lm) primers and 13.2 ll of h20. the pcr reactions were carried out under the following program: 5 min at 94°c followed by 40 cycles of 30 s at 94°c, 30 s at 54°c, 30 s at 68°c and a final extension of 10 min at 68°c. after electrophoresis appropriately sized bands (440 bp) were excised and purified using gel dna recovery kit (zymo reserch, ca, usa), and sequenced in both directions. sequence editing and multiple alignments were performed using bionumerics version 6.6 (applied maths, kortrijk, belgium). the pedv positive samples selected for genetic characterization were also screened for rotavirus group a (viroreal â rotavirus (a); ingenetix, vienna, austria) and transmissible gastroenteritis virus (tgev) (viroreal â tgev; ingenetix) accoding to the manufacturers instructions. in this study we describe an outbreak of severe watery diarrhea in swine in portugal, early 2015, with a rapid spread northwards in the territory. (pedv portugal 2015) . phylogenetic analysis was performed using mega version 6.0 software (tamura et al., 2013) . farms from the center. from the total 84 studied diarrheic stools, pedv was found in 55. four of these samples (two were from a farm in the south and two were from a farm in the center of portugal) were tested by conventional rt-pcr and amplified products (440 bp) were subjected to sequencing in order to obtain information about their genetic relatedness with pedv reference strains (fig. 1) . comparative analyses of these amplicons showed that all amplified sequences were identical (100%), showing that a single strain was responsible for this outbreak. analysis also showed that the amplified sequences share a very high (99.0%) identity with the new pedv variant oh851 (genbank accession no. kj399978) of the united states that affects sows (wang et al., 2014) . interestingly, the amplified sequences showed to be identical (100%) to the strains recently reported in germany pedv/ger/l00719/ 2014 (genbank accession no. lm645058) and pedv/ ger/l00721/2014 (genbank accession no. lm645057). the pedv positive samples selected for genetic characterization showed to be negative for rotavirus group a and tgev. in conclusion, pedv infection was confirmed in a pig herd in the south of portugal in january 2015 and found to be spreading northwards affecting a total of 32 farms. comparative analyses of a 440 bp region of the spike protein gene showed that the isolates were identical to the ones reported in 2015 in germany. the findings of an identical pedv strain in the south of europe, substantially distant from germany where the novel strains have been reported seem to indicate that a single strain (different from the american) is circulating in europe. as with germany, in portugal there is no active surveillance scheme for pedv, hence we cannot state with confidence that this strain has not been circulating in portugal for a longer time. also, the origin of this pedv variant still needs to be elucidated and further studies in the remaining european countries may contribute to the knowledge. the re-emergence of pedv in europe with altered virulence seems to be a relevant issue in swine health and may justify active surveillance by the official entities. comparison of porcine epidemic diarrhea viruses from germany and the united states virus taxonomy: classification and nomenclature of viruses; ninth report of the international committee on taxonomy of viruses porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines 2013: emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences mega6: molecular evolutionary genetics analysis version 6.0 federal order: reporting, herd monitoring and management of novel swine enteric coronavirus diseases new variant of porcine epidemic diarrhea virus an apparently new syndrome of porcine epidemic diarrhea the authors would like to acknowledge all pig farmers that participated in this study. this work was supported by european union funds (qren/feder) under the project ovislab ict-2013-05-004-5314 id-64757. key: cord-281081-rifr5uub authors: deng, junhua; jin, yipeng; liu, yuxiu; sun, jie; hao, liying; bai, jingjing; huang, tian; lin, degui; jin, yaping; tian, kegong title: serological survey of sars‐cov‐2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals date: 2020-05-07 journal: transbound emerg dis doi: 10.1111/tbed.13577 sha: doc_id: 281081 cord_uid: rifr5uub the pandemic sars‐cov‐2 has been reported in 123 countries with more than 5,000 patients died from it. however, the original and intermediate hosts of the virus remain unknown. in this study, 1,914 serum samples from 35 animal species were used for detection of sars‐cov‐2‐specific antibodies using double‐antigen sandwich elisa after validating its specificity and sensitivity. the results showed that no sars‐cov‐2‐specific antibodies were detected in above samples which excluded the possibility of 35 animal species as intermediate host for sars‐cov‐2. more importantly, companion animals including pet dogs (including one dog the sars‐cov‐2 patient kept and two dogs which had close contact with it) and cats, street dogs and cats also showed serological negative to sars‐cov‐2, which relieved the public concerns for the pets as sars‐cov‐2 carriers. deng et al. giant panda, masked civet, porcupine, bear, yellow-throated marten, weasel, red pandas and wild boar). the results showed that no sars-cov-2-specific antibodies were detected in above species of animals including pangolin which has been reported as an intermediate host of sars-cov-2 (kangpeng xiao, 2020) . more importantly, we found companion animals including dogs and cats were serologically negative to sars-cov-2 including one dog kept by the sars-cov-2 patient and two dogs with close contact with it during the quarantine. the sars-cov-2 double-antigen sandwich elisa was purchased from luoyang putai biotechnology co., ltd. the coating was based on s1 protein of sars-cov-2. the same antigen was linked to horseradish peroxidase (hrp) to function as conjugate. the serum samples were tested according to the manufacture manual instructions. briefly, 100 µl serum sample was added into each well of elisa plate and incubated at 37°c for 30 min. after washing the plate with washing buffer for five times, hrp-labelled antigen was added into the wells at 37°c for 30 min before 100 μl of the substrate solution was added to each well and incubated at 37°c for 10 min to stop the reaction. the optical density (od) was measured at 450 nm. to test the specificity of elisa kit, the serum samples of spf chicken (28), duck (25), mouse (23), rat (20) and pig (20) were applied. the final value of od 450 of samples ranged from 0.005 to 0.103 (median 0.007), 0.004 to 0.008 (median 0.006), 0.005 to 0.190 (median 0.007), 0.004 to 0.050 (median 0.007) and 0.005 to 0.134 (median 0.007) for chicken, duck, mouse, rat and pig, respectively. serum samples from other species of experimental animals including guinea pig (30), rabbits (34), beagle dogs (130) and rhesus monkeys (38) were also tested. there were no sars-cov-2 antibodies detected in above animals (data not shown). next, the potential cross-reaction with other coronavirus including ibv (26) we next tested the sensitivity of elisa kit. the sars-cov-2 experimental-infected ferret positive sera were tested. as shown in table 1 , the neutralizing antibody titres of 5 infected ferret (f1-f5) were between 1:128 and 1:256 at 22 days post-infection (dpi). by contrast, the neutralizing antibody titres of 5 placebo ferrets (c1-c5) were all negative at 22 dpi. in the line with the results of neutralizing antibodies, the final od 450 of 5 positive sera detected by elisa was all above 3, which indicated strongly serological positive to sars-cov-2. to further test the dynamic changes of elisa titre of infected ferret, serum samples from one ferret were collected from 0, 7, 12, 17 and 22 dpi, respectively. the positive elisa results were shown at 7 dpi and lasted until 22 dpi when the ferrets were humanely euthanized (table 1) . the above results showed that the elisa has good specificity and sensitivity and suitable for different species of animals. after confirming the specificity, sensitivity and suitability of sars-cov-2 elisa kit for different species of experimental animals, clinical serum samples from domestic livestock (pig, cow, sheep, horse), poultry (chicken, duck, goose), experimental animal (mice, rat and rhesus monkey), companion animal (dog and cat) and wild animals (camel, fox, mink, alpaca, ferret, bamboo rat, peacock, eagle, tiger rhinoceros, pangolin, leopard cat, jackal, giant panda, masked civet, porcupine, bear, yellow-throated marten, weasel, red pandas and wild boar) were used for antibody detection. as shown in table 2 unknown. since sars-cov-2 is genetically close to sars-cov, it has been proposed that bat could be the natural host (phan, 2020) . snake is also presumed as wildlife animal reservoir for sars-cov-2 based on the virus relative synonymous codon usage (rscu) bias (ji, wang, zhao, zai, & li, 2020) . however, there is no report of sars-cov-2 isolation or molecular and serological confirmation of infection from snake samples. pangolins recently was suggested to be direct animal source of sars-cov-2 for humans since the sars-cov-2-related coronaviruses were isolated from malayan pangolins which shared 97.4% similarity with sars-cov-2 in virus receptor-binding domain in s gene (kangpeng xiao, 2020) . in our study, we did not detect sars-cov-2 antibodies in 17 pangolin serum samples. consistent with our results, li et al., (2020) reported the coronavirus carried by pangolins did not have the rrar motif, a unique peptide insertion in the human sars-cov-2 virus. the rrar motif may be involved in the proteolytic cleavage of spike protein and host range and transmissibility which suggests human sars-cov-2 virus did not come directly from pangolins . masked civet and camel were confirmed to be natural hosts for sars-cov and mers-cov, and no specific sars-cov-2 antibodies were detected in 10 masked civets and 31 camels in this study. to the sars-cov-2 has been major concern for the public. one pet dog was reported to be sars-cov-2-positive detected by rt-pcr in hongkong (https://www.news.gov.hk/eng/2020/02/20200 228/20200 228_093205_796.html). later, the serological result of the dog showed negative after quarantine of 14 days. in our study, 87 cats including 66 pet cats and 21 street cats showed serological negative to sars-cov-2 (table 2) . at the same time, 487 dogs including 90 beagle dogs, 147 pet dogs and 250 street dogs during the outbreak of sars-cov-2 were also tested serological negative. among them, 15 pet dog and 99 street dog sera were collected from wuhan city. it should be noted that one pet dog from confirmed sars-cov-2-infected patient showed serologically negative, and other two dogs which had close contact with this dog also tested to be negative. however, we cannot rule out of susceptibility of cats and dogs to sars-cov-2, which need to be tested by experimental infections. molecular techniques such as reverse-transcriptase pcr tests and viral genome sequencing are widely used for the confirmation of human infection. these techniques are also used to explore the potential hosts of sars-cov-2 (pfefferle, reucher, norz, & lutgehetmann, 2020) . compared to these molecular methods, serological test such as elisa has several advantages. first, the host generates sars-cov-2-specific antibodies after infection which could last longer than the viraemia. it provides a wider detection window for elisa than rt-pcr. second, rna extraction from susceptive infected samples has to be performed in a bsl-3 laboratory. by abbreviation: dpi, days post-infection. *the neutralizing antibody titre of positive samples was ≥4. contrast, elisa can be performed in a safety level 2 laboratory and does not require high containment facilities after the serum samples were inactivated at 56°c for 30 min. third, double-antigen sandwich elisa based on recombinant s1 protein could detect both igm and igg antibodies and is not limited to species. to find the host of sars-cov-2, the screening of other wild animals using elisa is undergoing in our laboratory. we want to thank dr. zhigao bu, director of harbin veterinary research institute, chinese academy of agricultural sciences for providing inactivated sars-cov-2-negative and sars-cov-2positive ferret serum samples. this study was supported by luoyang heluo talent plan (kegong tian). we declare that ethical statement is not applicable. there was no conflict of interest with others. the data that support the findings of this study are available from the corresponding author upon reasonable request. kegong tian https://orcid.org/0000-0001-5362-1415 three emerging coronaviruses in two decades cross-species transmission of the newly identified coronavirus 2019-ncov the emergence of a novel coronavirus (sars-cov-2), their biology and therapeutic options evolutionary history, potential intermediate animal host, and cross-species analyses of sars-cov-2 evaluation of a quantitative rt-pcr assay for the detection of the emerging coronavirus sars-cov-2 using a high throughput system genetic diversity and evolution of sars-cov-2. infection a novel coronavirus outbreak of global health concern novel coronavirus 2019, an emerging public health emergency serological survey of sars-cov-2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals key: cord-253850-e3l5xtc2 authors: wang, m.; wang, y.; baloch, a. r.; pan, y.; tian, l.; xu, f.; shivaramu, s.; chen, s.; zeng, q. title: detection and genetic characterization of porcine deltacoronavirus in tibetan pigs surrounding the qinghai–tibet plateau of china date: 2018-01-23 journal: transbound emerg dis doi: 10.1111/tbed.12819 sha: doc_id: 253850 cord_uid: e3l5xtc2 porcine deltacoronavirus (pdcov) is a recently discovered rna virus that belongs to the family coronaviridae and genus deltacoronavirus. this virus causes enteric disease in piglets that is characterized by enteritis and diarrhoea. in our present investigation, 189 diarrhoeic samples were collected between july 2016 and may 2017 from tibetan pigs inhabiting in three different provinces surrounding the qinghai–tibet plateau of china. we then applied the molecular‐based method of reverse transcription polymerase chain reactions (rt‐pcrs) to detect the presence of pdcov in collected samples, and rt‐pcr indicated that the prevalence of pdcov was 3.70% (7/189) in tibetan pigs. four of 7 pdcov‐positive pigs were monoinfections of pdcov, three samples were co‐infections of pdcov with porcine epidemic diarrhoea virus (pedv), and 52 (27.51%) samples were positive for pedv. four strains with different full‐length genomes were identified (chn/gs/2016/1, chn/gs/2016/2, chn/gs‐/2017/1 and chn/qh/2017/1), and their genomes were used to analyse the characteristics of pdcov currently prevalent in tibetan pigs. we found a 3‐nt insertion in the spike gene in four strains in tibetan pigs. phylogenetic analysis of the complete genome and spike and nucleocapsid gene sequences revealed that these strains shared ancestors with the strain chn‐ah‐2004, which was found in pigs from the anhui province of china mainland. however, pdcov strains from tibetan pigs formed different branches within the same cluster, implying continuous evolution in the field. our present findings highlight the importance of epidemiologic surveillance to limit the spread of pdcov in livestock at high altitudes in china. the subfamily comprises four genera that is alphacoronavirus, betacoronavirus, gamacoronavirus and deltacoronavirus. porcine deltacoronavirus (pdcov) was first reported in hong kong in 2012 as an emerging genus prevalent in certain animal species, including swine (woo et al., 2012) . infection by pdcov can be symptomatically compared with other porcine enteric coronavirus diseases caused by transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhoea virus (pedv); however, pdcov exhibits milder symptoms and lower mortality rates in affected neonatal piglets (wang, byrum, & zhang, 2014) . after the first report of pdcov infection (woo et al., 2012) , its prevalence was detected in pigs in several states of the united states, south korea, mainland china, southern china and thailand with majority of piglets having clinical diarrhoeal disease (dong et al., 2015; lee et al., 2016; mai et al., 2017; saeng-chuto et al., 2017; wang et al., 2014) . the prevalence of pdcov in certain regions of the world is intriguing with regard to its epidemiology, evolution and pathogenicity. here, we are the first to report pdcov infection in tibetan pigs from the qinghai-tibet plateau of china. tibetan pigs mainly live in gansu, qinghai, sichuan and tibet provinces, which surround the qinghai-tibet plateau of china (altitude > 3,000 m, average annual temperature <0°c). due to the cold and harsh environment, few viral infections have been reported in animals in these areas until recent years. tibetan pigs had no history of travel to areas where a prevalence of covs had been reported earlier. nevertheless, livestock such as pigs and yaks (bos grunniens), which have been associated with clinical diarrhoeal disease have been reported in these provinces (gong et al., 2014; wang, lan, & yang, 2016 (table 1) . soon after sampling, 10% (wt/vol) faecal suspensions were prepared using sterile phosphate-buffered saline (pbs). supernatants were separated after samples were centrifuged, and samples were then stored at à80°c for rna extraction. total rnas were extracted using trizol reagent (invitrogen, carlsbad, ca, usa) and then used as templates to generate full-length cdna by reverse transcription pcr (rt-pcr; superscript iii synthesis kit, invitrogen) according to the manufacturer's instructions. for higher specificity, two pairs of specific primers were used to detect pdcov as described previously (wang et al., 2014) but with some modifications (table 2) . rt-pcr was performed in a 20-ll volume containing 1 ll of template and 0.1 lmol/l each primer; the reactions were subjected to 95°c for 5 min, followed by 35 cycles of 95°c for 30 s, 58°c for 25 s and 72°c for 30 s, with a final extension step of 10 min at 72°c. rt-pcr-amplified dna fragments of expected sizes were submitted to a commercial company and sequenced in both directions by sanger sequencing (sangon biotech, shanghai, china). before the presence of pdcov was determined, the presence of pedv and tgev was examined with primers specific for the spike gene of pedv and the n gene of tgev as described previously (kim, choi, kim, & chae, 2000; sinha, gauger, zhang, yoon, & harmon, 2015; temeeyasen et al., 2014) . t a b l e 2 primers used for detection and full-length genome amplification of porcine deltacoronavirus (pdcov) in tibetan pigs, as described previously (5), with some modifications pdcov-m-67f 5 0 -atcctccarggaggctatgc-3 0 23104-20597 494 pdcov detection (4,5) pdcov-25420-r 5 0 -tgctccatcccccctataag-3 0 of all examined samples were positive for pedv. in seven pdcovpositive samples, three were positive for pedv; of these, one sample was from gansu province, and two were from qinghai province. the prevalence of pdcov+pedv was only 1.59% in tibetan pigs that were associated with clinical diarrhoeal disease. we also found that all tibetan pigs infected with pdcov were under 1 month of age. nevertheless, both tibetan pigs under 1 month of age and older than 1 month could be infected with pedv. tibetan pigs were also located in the same separate subcluster (figure 2b and c). in our present study, we found that the prevalences of pdcov, et al., 2015) . additionally, all tibetan pigs infected with pdcov were under one month of age, therefore, indicating that the prevalence of pdcov was related to the ages of tibetan pigs (table 1) , consistent with previous studies (mai et al., 2017; song et al., 2015) . nevertheless, pdcov-positive tibetan pigs showed mild clinical diarrhoeal disease, and no mortality was recorded among the infected pigs with clinical diarrhoeal disease, which is inconsistent with previous reports (janetanakit et al., 2016; jang et al., 2017) . these results suggest that subclinical infection of pdcov occurs in tibetan pigs, which are emerging but imperfect hosts for the pdcov. tibetan pigs have evolved for thousands of years as a unique and indigenous breed in china. living in cold and harsh environments on the plateau for a long period of time, the tibetan pigs have undergone a specific selection to enrich disease resistance-related genes in their genome (li et al., 2013; megens et al., 2008) . the lower prevalence of pdcov and pedv in tibetan pigs that is demonstrated in our study is consistent with the prevalence of other pathogens infecting the tibetan pigs (fan et al., 2016; liu et al., 2014 ing the continuous evolution and adaption of pdcov to its hosts in special field conditions. our results also complement the geographical lineage theory of global pdcov distribution. furthermore, investigating variations in other genes will also provide additional data to determine the diversity of the pdcov genome, and we strongly suggest that effective vaccination against pdcov is not ignored in tibetan pigs in the studied areas. we thank the china animal health and epidemiology center for their valuable assistance in sample collection. the authors declare no conflict of interests. pathogenesis of highly pathogenic porcine reproductive and respiratory syndrome virus in chinese tibetan swine molecular investigation of bovine viral diarrhea virus infection in yaks (bos gruniens) from qinghai porcine deltacoronavirus prevalence, complete genome sequencing and phylogenetic analysis of porcine deltacoronavirus in south korea detection and differentiation of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex rt-pcr. the veterinary record mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets detection and phylogenetic analysis of porcine deltacoronavirus in korean swine farms complete genome characterization of korean porcine deltacoronavirus strain kor/knu14-04 genomic analyses identify distinct patterns of selection in domesticated pigs and tibetan wild boars first report of seroprevalence of swine influenza a virus in tibetan pigs in tibet, china. tropical animal health and production the detection and phylogenetic analysis of porcine deltacoronavirus from guangdong province in southern china biodiversity of pig breeds from china and europe estimated from pooled dna samples: differences in microsatellite variation between two areas of domestication retrospective investigation and evolutionary analysis of a novel porcine deltacoronavirus strain detected in thailand from pcrbased retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in us swine newly emerged porcine deltacoronavirus associated with diarrhoea in swine in china: identification, prevalence and full-length genome sequence analysis genetic diversity of orf3 and spike genes of porcine epidemic diarrhea virus in thailand detection and genetic characterization of deltacoronavirus in pigs molecular epidemiological investigation of porcine kobuvirus and its coinfection rate with pedv and sav in northwest china discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus hepatitis e virus genotype 4 in yak, northwestern china first detection of ungulate tetraparvovirus 1 (bovine hokovirus 1) in domestic yaks in northwestern china detection and genetic characterization of porcine deltacoronavirus in tibetan pigs surrounding the qinghai-tibet plateau of china key: cord-260212-m80dkzm4 authors: lee, j. h.; chung, h. c.; nguyen, v. g.; moon, h. j.; kim, h. k.; park, s. j.; lee, c. h.; lee, g. e.; park, b. k. title: detection and phylogenetic analysis of porcine deltacoronavirus in korean swine farms, 2015 date: 2016-03-10 journal: transbound emerg dis doi: 10.1111/tbed.12490 sha: doc_id: 260212 cord_uid: m80dkzm4 this study applied molecular‐based method to investigate the presence of porcine deltacoronavirus (pdcov) in 59 commercial pig farms in south korea. the results of rt‐pcr screening on a relatively large collection of faeces samples (n = 681) from january 2013 to march 2015 did not reveal the presence of pdcov until the end of 2014. however, on march 2015, pdcov‐positive samples (sl2, sl5) were detected from sl swine farm in gyeongbuk province. the phylogenetic trees based on the complete spike‐ and nucleocapsid protein‐coding genes showed that sl2 and sl5 closely related to the us pdcov strains rather than those in china. thought korean strains of pdcov isolated in 2014 (knu14.04) and in 2015 (sl2 and sl5) grouped within us pdcov cluster, the reconstruction of ancestral amino acid changes suggested that they are different. coronaviruses are single-stranded, positive-sense enveloped rna viruses belonging to the coronaviridae family and are divided into 4 genera (alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus) (woo et al., 2012) . until 2014, three members of the alphacoronavirus genus such as porcine epidemic diarrhoea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) are known to cause enteric and respiratory diseases of swine. more recently, a novel emerging porcine deltacoronavirus (pdcov) was demonstrated to be enteropathogenic and causes severe diarrhoea resemble those of pedv and tgev infections jung et al., 2015) , and mild interstitial pneumonia (ma et al., 2015) . since the first report of pdcov in hong kong in 2012 (woo et al., 2012) , the virus is identified in the united states (wang et al., 2014a,b) , south korea (lee and lee, 2014) and china (song et al., 2015) . in this study, we further report the presence and genetic characterization of pdcov from cases showing symptoms of diarrhoea in korean swine farms. in this study, faecal samples of pigs showing signs of diarrhoea (n = 681) collected from january 2013 to march 2015 were screened for the presence of porcine deltacoronavirus (pdcov). the sampling locations were given in the fig. s1 . total rna was extracted using trizol ls (invitrogen, usa) following the manufacturer's instructions. the rna was then converted into cdna with the use of random hexamers and commercial rna to cdna ecodry premix kit (clontech, otsu, japan) following the manufacturer's protocol. to enhance the specificity, two pairs of pdcov primer were utilized. the first method designed primer set of reference (woo et al., 2012) . the other pdcov-specific primers were designed in this study, targeting a region of 587 bp of the nucleocapsid protein-coding gene (pdcov-587f 5 0 -cccagctcaaggtttcagag-3 0 , pdcov-587r 5 0 -ccc aatcctgtttgtctgct-3 0 ). the thermal profile was initial denaturation at 94°c for 5 min, followed by 38 cycles of 94°c for 30 s, 56°c for 30 s, 72°c for 30 s and a final extension at 72°c for 7 min. the screening for other porcine enteric viruses was performed with pathogen-specific primers using accupower â profi taq pcr premix (bioneer ltd., daejeon, korea). the detection of kobuvirus and group a rotavirus was following the previous studies (reuter et al., 2009; lee et al., 2013) . for porcine epidemic diarrhoea virus (pedv) and transmissible gastroenteritis virus (tgev), we used i-tgev/ pedv detection kit (intron ltd., daejeon, korea). for sequencing of genes encoded spike protein (s) and nucleocapsid protein (n), we followed the protocol described in the previous study . pdcov-positive samples were amplified with primer sets (pdcov-sf2, pdcov-sr2 and pdcov-nf1, pdcov-nr1). the specific pcr bands were purified by qiaquick gel extraction kit (qiagen, daejeon, germany), cloned utilizing ta cloning kit (topcloner ta kit; enzynomics, daejeon, korea) and subsequently transformed into competent escherichia coli cells (dh5a). the purified recombinant plasmids were sequenced by macrogen inc (seoul, korea). new sequences of pdcov generated in this study were addressed in genbank accession no. kr060082-kr060085. the genetic relationship of two pdcov strains (sl2, sl5) with other pdcovs was inferred from a codon-based alignment of 31 sequences of complete s gene (3483 bases) and 31 sequences of complete n gene (1029 bases). the details of the data set are summarized in table s1 . the phylogenetic tree was reconstructed by the maximum likelihood model with 1000 bootstrap replicates implemented in iq-tree version 1.3.8 (nguyen et al., 2015) . the best-fitting nucleotide substitution model for each alignment was determined automatically by specifying '-m test' option. amino acid changes on the evolutionary path of pdcov (based on s and n genes) were inferred using the codeml program implemented in paml 4.8 (yang, 2007) . substitutions occurred on a given node of a phylogeny were annotated by treesub program (tamuri, 2013) . fig. 1 . maximum likelihood phylogeny of pdcovs based on the spike protein-coding gene (a) and the nucleocapsid protein-coding gene (b). the numbers at the nodes of the phylogenies denote the bootstrap values to which they belong (for clarity, labels of some terminal nodes were omitted). the phylogenetic trees showed that korean pcdov isolates in 2014 (knu14.04) and in 2015 (sl2, sl5) were grouped within us pdcov cluster, but they located at different branches (highlights). fig. 2 . the maximum likelihood trees based on the s gene (a) and the n gene (b) with reconstructed non-synonymous substitutions were mapped to the nodes of the phylogeny. for clarity, only branches leading to korean pdcov isolates were highlighted (black lines). the nodes where non-synonymous substitutions occurred were indicated by # (for the highlighted branches) and by @ (for the others). the nodes without non-synonymous substitutions were marked by • (for the highlighted branches) and were not marked (for the others). it was observed that the branch which leaded to 2015 isolates (sl2, sl5) accumulated further mutations in comparing to the branch which leaded to 2014 isolate (knu14.04). the screening results by rt-pcr carried out on 681 samples of 59 swine farms (table 1) showed that until the end of 2014 all of tests were negative for nucleic acid of pdcov. it was on march 2015, pdcov-positive samples were detected in a 600-scale sow farm (sl farm) in gyeongbuk province. this farm was reported to be infected by pedv in 2014 and had severe diarrhoea with 100% mortality in piglets. in early 2015, it was observed that up to 20% pigs of all ages had diarrhoea and 10% died. the diagnosis of porcine enteric viruses (table 2) revealed the dual infection of pdcov and pedv, while tgev, group a rotavirus and kobuvirus were not detected. in the literature, it was reported that pdcov co-infected with others enteric viruses, such as: group c rotavirus (marthaler et al., 2014) , tgev (dong et al., 2015) and pedv (song et al., 2015) . combining the detection results of this study with the above-mentioned reports, it could be inferred that pedv was the most frequent co-infected viruses. for the genetic characterization, the maximum likelihood phylogenetic trees reconstructed from the s and n genes (fig. 1a, b) showed a clear separation between chinese and us strains of pdcov and is similar to the previous studies (marthaler et al., 2014; wang et al., 2016) . of which, korean strains of pdcov isolated in 2014 (knu14.04) and in 2015 (sl2 and sl5) were grouped within us pdcov cluster; however, they located at different branches (highlights, fig. 1a, b) . based on the s gene, the inferred ancestral amino acid changes along the nodes of the phylogeny (fig. 2a) showed that the branches leading to korean pdcov isolates in 2014 and in 2015 shared 1 back substitution (node #40: q106l, node #37: l106q) and four unique substitutions (node #39: s697a, node #38: v550a, i669l and node #37: i1014v). however, the branch that leaded to 2015 isolates (sl2 and sl6) had further 2 mutations locating near the tip of the phylogeny (node #59: i110v, t582a). based on the n gene, it was observed only amino acid mutations (six changes) near the tip of the phylogeny, on the node leading to sl2 and sl5 (fig. 2b) . the details of non-synonymous substitutions at every node of the phylogeny can be found in tables s2, s3 . at present, the significance of these substitutions is almost obscured. of the all, the phylogenetic analyses suggested that the pdcovs strains (sl2, sl5) detected in early 2015 are different with the previously emerged virus (knu14.04). in summary, by screening the samples collected from january 2013 to march 2015, this study confirmed the presence pdcov in korean swine farms. the phylogenetic analyses suggested that the korean pdcov isolated in 2014 and in 2015 are closely related to us strains of pdcov, but they are different. additional supporting information may be found in the online version of this article: figure s1 . sampling sites for retrospective detection of pdcov in 9 provinces from 2013 to march 2015. table s1 . list of sequences used in this study. table s2 . list of non-synonymous substitutions at the nodes of the pdcov phylogeny based on the spike protein coding gene (shown in figure 2a) . table s3 . list of non-synonymous substitutions at the nodes of the pdcov phylogeny based on the nucleocapsidprotein coding gene (shown in figure 2b ). pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets porcine deltacoronavirus in mainland china isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the united states pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs complete genome characterization of korean porcine deltacoronavirus strain kor/knu14-04/ 2014 phylogenetic analysis of porcine astrovirus in domestic pigs and wild boars in south korea 2015: origin, evolution, and virulence of porcine deltacoronaviruses in the united states rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus, a member of a new species in the genus kobuvirus, family picornaviridae newly emerged porcine deltacoronavirus associated with diarrhoea in swine in china: identification, prevalence and full-length genome sequence analysis treesub: annotating ancestral substitution on a tree detection and genetic characterization of deltacoronavirus in pigs porcine coronavirus hku15 detected in 9 us states porcine deltacoronavirus: histological lesions and genetic characterization discovery of seven novel mammalian and avian coronaviruses in deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus paml 4: phylogenetic analysis by maximum likelihood this study was supported by a grant (no. pj011184) from biogreen 21 program, rural development administration. the authors declare that there are no conflict of interests. key: cord-309734-m8miwtha authors: vergara‐alert, j.; raj, v. s.; muñoz, m.; abad, f. x.; cordón, i.; haagmans, b. l.; bensaid, a.; segalés, j. title: middle east respiratory syndrome coronavirus experimental transmission using a pig model date: 2017-06-26 journal: transbound emerg dis doi: 10.1111/tbed.12668 sha: doc_id: 309734 cord_uid: m8miwtha dromedary camels are the main reservoir of middle east respiratory syndrome coronavirus (mers‐cov), but other livestock species (i.e., alpacas, llamas, and pigs) are also susceptible to infection with mers‐cov. animal‐to‐animal transmission in alpacas was reported, but evidence for transmission in other species has not been proved. this study explored pig‐to‐pig mers‐cov transmission experimentally. virus was present in nasal swabs of infected animals, and limited amounts of viral rna, but no infectious virus were detected in the direct contact pigs. no virus was detected in the indirect contact group. furthermore, direct and indirect contact pigs did not develop specific antibodies against mers‐cov. therefore, the role of pigs as reservoir is probably negligible, although it deserves further confirmation. middle east respiratory syndrome coronavirus (mers-cov) was first detected in 2012 in saudi arabia, and it causes severe acute respiratory illness with fever, cough and shortness of breath (zaki, van boheemen, bestebroer, osterhaus, & fouchier, 2012) . up to date, it has caused 1952 human infections, including 693 related deaths (world health organization (who), 2017). dromedaries are the natural reservoir of mers-cov (sabir et al., 2016) . however, other animal species such as non-human primates (rhesus macaques and common marmosets), members of the family camelidae (alpacas and llamas), rabbits and pigs have been demonstrated to be susceptible to mers-cov infection (crameri et al., 2016; falzarano et al., 2014; haagmans et al., 2015; vergara-alert, van den brand, et al., 2017; de wit et al., 2013 de wit et al., , 2017 . the finding that pigs can be infected with mers-cov would suggest that other suidae might be susceptible to the virus. indeed, common warthogs (phacochoerus africanus), bushpig (potamochoerus larvatus) and wild boars are commonly found in the greater horn of africa or the middle east, sharing the same habitats and water sources with dromedaries (cumming, 2008; vergara-alert, vidal, bensaid, & segal es, 2017) . a recent study in alpacas demonstrated efficient animal-to-animal transmission (adney, bielefeldt-ohmann, hartwig, & bowen, 2016) but, to our knowledge, evidence for transmission between animals from other species has not been reported. to study whether mers-cov might be transmitted between pigs, an experimental transmission study in this animal model was designed and performed under direct and indirect contact settings. 2.1 | experimental design 3 (bsl-3) animal facilities (irta-cresa, barcelona, spain), and divided into three groups: g1, mers-cov-inoculated pigs (p1-p5); g2, direct contacts (p6-p10); g3, indirect contacts (p11-p15). three extra animals were used as negative controls (g4, p16-p18). animals from g1, g2 and g3 were housed in the same experimental box unit but placed in two different pens. the pens were separated by two fences with a 30 cm distance among them ( figure 1 ). tarpaulin, from the ceiling to the floor, was used to avoid contact between pen 1 and pen 2. tarpaulin was also placed in the front doors of both pens. at the beginning of the experiment, g1 was housed in pen 1, and g2 and g3 in pen 2. g1 was inoculated with 10 7 tcid 50 (50% tissue culture infectious dose) mers-cov (passage 7 human isolate hcov-emc/2012) in 3 ml saline solution via intranasal route (1.5 ml in each nostril). two days later, all tarpaulins were removed and g2 pigs were moved from pen 2 to pen 1 until the end of the study. all animals were monitored daily for clinical signs (sneezing, coughing, nasal discharge and/or dyspnoea), as well as rectal temperatures until day 10 post-inoculation (pi). nasal swabs (ns) were obtained on days 0, 1, 2, 3, 4 pi from all animals and at days 7, and 10 pi from g1. animals from g2 and g3 were also sampled at 5, 6, 9 and 12 days pi, corresponding to days 3, 4, 7 and 10 after direct (g2) and indirect (g3) contact with g1. two independent ns were collected and placed in pbs (for pcr analysis) and dmem containing antimicrobial drugs (for detection of infectious virus); swabbing was performed deep in both sides of the nasal cavity. sera were obtained before challenge and at 7, 15 and 26 days pi, and they were subsequently used to detect the presence of mers-cov-specific antibodies. negative control pigs were sampled (ns and sera) and euthanized before the start of the experiment. daily environmental samples (es) between day 0 and 10 pi were obtained from air sampling and wall surface swabbing (figure 1 ). briefly, swabs pre-moistened with transport medium (copan universal transport medium utm-rt system) were collected from walls in pen 1 and 2 (es1 and es2). air sampling was performed using an air sampler (airport md8 sartorius device) located between pens, which suctioned 50 l/min air volume for 20 min through a gelatin membrane filter (es3). air from the box unit was sampled with 10 9 10 cm dry membrane filters located in the air extraction of the box (es4). es were tested for the presence of viral rna. viral rna from ns and es was extracted with nucleospin â rna virus kit (macherey-nagel, germany) following the manufacturer's f i g u r e 1 schematic representation of the experimental animal box. boxes in the animal facility of the biosafety level 3 at irta-cresa are behind two sets of doors (with a shower in between) following the standards of a negative pressure room. animals were distributed into two pens separated by two fences with a 30 cm distance between them. g1 (p1-p5) was allocated in pen 1 and g2 (p6-p10) and g3 (p11-p15), in pen 2. two days after inoculation of g1 with mers-cov, g2 was cohoused with g1 until the end of the experiment. tarpaulin was used to prevent contact between g1 and the other two groups during the firsts 2 days after inoculation. environmental samples (es) were obtained from different locations, as represented in the scheme [colour figure can be viewed at wileyonlinelibrary.com] vergara-alert et al. instructions. the rna extracts were tested by the upe pcr (raj et al., 2013) , and the techniques were carried on as previously (vergara-alert, van den brand, et al., 2017) . ns were also evaluated for the presence of infectious virus by titration in vero cells, following previous protocol (vergara-alert, van den brand, et al., 2017). serum samples from days 0, 7, 15 and 26 pi were tested to determine the specific s1-antibodies by a mers-cov s1-elisa, and by a specific virus neutralization assay, as previously described (haagmans et al., 2016) . similar to a previous experiment (vergara-alert, van den brand, et al., 2017) , none of the pigs had appreciable rise in rectal temperature upon challenge, nor any clinical signs (data not shown). all mers-cov-experimentally infected animals (p1-p5) shed viral rna at least from 1 to 4 days pi, and three of five pigs had detectable viral rna until 7 days pi (figure 2a ). most importantly, all five animals shed infectious virus during the first 4 days pi (figure 2b ). viral rna was detected in four of five cohoused, direct contact animals (g2) at least one time pi. the mers-cov rna load of g2 pigs, however, was lower than those of g1 (figure 2a) . no viral rna or infectious virus was detected in swabs from g3 (p11-p15) and control pigs (p16-p18).to test whether seroconversion occurred, serum samples were tested with a specific recombinant mers-cov s1-elisa and for neutralizing antibodies against mers-cov. all five mers-cov infected animals (p1-p5) had detectable levels of s1-antibodies 2-and 3-weeks after the infection (figure 2c ). the specificity of the response was confirmed by virus neutralization assay. in p1-p4 (but not in p5), serum neutralizing mers-cov-specific titres (1:40-1:160) were detected at 1-and 2-week pi (figure 2d) . however, at week 3 pi, the virus neutralizing antibodies decreased (1:20-1:40). no mers-cov-specific antibodies were detected in serum of g2, g3 and control pigs. in environmental samples, very low levels of viral rna were detected at different time points, with a peak at day 5 pi (table 1) . other livestock besides dromedaries are susceptible to mers-cov infection (crameri et al., 2016; falzarano et al., 2014; haagmans et al., 2015; munster et al., 2013; vergara-alert, van den brand, et al., 2017; de wit et al., 2013 de wit et al., , 2017 ; thus, they might be potential intermediate we thank all animal caretakers from the irta-cresa biosecurity level 3 laboratories and animal facilities for technical assistance. this work was performed as part of the zoonotic anticipation and preparedness initiative (zapi project) [innovative medicines initiative (imi) grant 115760] with assistance and financial support from imi and the european commission and contributions from efpia partners. the funding from cerca programme/generalitat de catalunya to irta is also acknowledged. infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus phacochoerus africanus. the iucn red list of threatened species infection with mers-cov causes lethal pneumonia in the common marmoset asymptomatic middle east respiratory syndrome coronavirus infection in rabbits an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels pneumonia from human coronavirus in a macaque model dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia livestock susceptibility to infection with middle east respiratory syndrome coronavirus. emerging infectious diseases searching for animal models and potential target species for emerging pathogens: experience gained from middle east respiratory syndrome (mers) coronavirus. one health domestic pig unlikely reservoir for mers-cov middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques middle east respiratory syndrome coronavirus (mers-cov): infection, prevention and control measures are critical isolation of a novel coronavirus from a man with pneumonia in saudi arabia how to cite this article: vergara-alert j, raj t a b l e 1 viral rna from air sampling and wall surface swabbing at different times after mers-cov infection. swabs were collected from the walls in pen 1 and pen 2 (es1 and es2), air sampling was performed with an air device (es3), and circulating air from the box was sampled with filters located in the ceiling air extraction of the room (es4) key: cord-293082-fw7deem8 authors: zhang, guangzhi; li, bin; yoo, dongwan; qin, tong; zhang, xiaodon; jia, yaxiong; cui, shangjin title: animal coronaviruses and sars‐cov‐2 date: 2020-08-16 journal: transbound emerg dis doi: 10.1111/tbed.13791 sha: doc_id: 293082 cord_uid: fw7deem8 covid‐19 is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2). it has rapidly spread to 216 countries and territories since first outbreak in december of 2019, posing a substantial economic losses and extraordinary threats to the public health worldwide. although bats have been suggested as the natural host of sars‐cov‐2, transmission chains of this virus, role of animals during cross‐species transmission, and future concerns remain unclear. diverse animal coronaviruses have extensively been studied since the discovery of avian coronavirus in 1930s. the current article comprehensively reviews and discusses the current understanding about animal coronaviruses and sars‐cov‐2 for their emergence, transmission, zoonotic potential, alteration of tissue/host tropism, evolution, status of vaccines, and surveillance. this study aims at providing guidance for control of covid‐19 and preventative strategies for possible future outbreaks of zoonotic coronavirus via cross‐species transmission. the coronavirus disease 2019 broke out in wuhan, china in december 2019, and 46 spread rapidly across the world. on march 11th of 2020, world health organization (who) 47 announced covid-19 a pandemic. as of april 7, just four months since its first outbreak, more 48 than 3.4 million confirmed cases and 238,000 deaths have been recorded in 215 countries, areas, 49 and territories, and moreover it seems that severe acute respiratory syndrome coronavirus 2 50 (sars-cov-2) that causes covid-19 will probably continue to circulate around the globe 51 (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). the health 52 authorities and governments of affected countries have paid attention to current pandemic and 53 have taken immediate measures to block covid-19 transmission, including utilization of personal 54 protective equipment, quarantine, epidemiological investigation, isolation, clinical data analysis 55 and sharing, public health education, maintaining social distance, the creation of diagnostics, 56 therapeutics, and vaccines, etc (xiao and torok 2020) . the new information is rapidly 57 accumulating and uncovers the nature of sars-cov-2, but many questions remain to be 58 answered. (table 1) . these six covs usually cause infections in pigs, but pdcov seems to have the ability 141 to infect other species such as badgers, calves, and cats (li et al. 2018) . moreover, it was reported our phylogenetic analysis with s protein shows that pedv and tgev share only 42.8%-43.5% of 154 genome similarity with sars-cov-2, and phev and pdcov share 49.2%-49.3% and 155 40.3%-40.4% genome similarity with sars-cov-2, respectively (table 2) . obviously, porcine covs are unlikely the origin of sars-cov-2. however, the rbd of sars-cov-2 is likely to 157 recognize porcine ace2 based on the high similarity of critical virus-binding residues between 158 porcine ace2 and human ace2 (wan, shang, graham, et al. 2020 this article is protected by copyright. all rights reserved the investigation needs to be explored. ibv normally binds to cell receptors via sialic acid for its 185 attachment and entry (shahwan et al. 2013; toro, van santen, and jackwood 2012) (table 1) . live-attenuated vaccines for ibv are usually adopted by the farms, which are developed by serial 187 passages of virulent strains in embryonated chicken eggs (laconi et al. 2020; masoudi, pishraft 188 sabet, and shahsavadi 2020; baron, iqbal, and nair 2018 kong. an oie report shows that the antibody in a pet dog living with a receptor for both fecv and fipv (hohdatsu et al. 1998 ) ( table 1 ). as shown in table 2 showed that the receptor-binding motif of sars-cov-2 rbd can engage with ace2 of humans 260 and cats at similar efficiencies (wan, shang, graham, et al. 2020 for neonates through colostrum (nemoto et al. 2017; kanno et al. 2013) . as for the receptors utilized by sars-cov-2, biophysical and structural data showed that the s 374 protein engages with ace2 with at least 10 times higher affinity compared to that of sars-cov 375 (wrapp et al. 2020 ). in addition to ace2, sars-cov-2 may also invade the cell via another 376 receptor: cd147 ibrahim et al. 2020 accepted article ibrahim et al. 2020; hao et al. 2020) . it was reported that furin is involved in the cleavage of 381 sars-cov-2 s protein and further facilitate viral entry . in principle, tissues 382 with consistently high expression of ace2 or cd147 are likely susceptible to sars-cov-2, 383 including small intestine, kidney (renal tubular cells), testis (leydig cells and cells in seminiferous 384 ducts), gall bladder, heart, stomach, and liver (fagerberg et al. 2014 ). according to a recent report shen et al. 2020 phylogenetic analysis by researchers from institut pasteur in paris showed that sars-cov-2 409 might already circulated in france prior to first local case (gámbaro et al. 2020 ). all of these data 410 undoubtedly showed an intricate situation of this virus. thus, more surveillance needs to be taken 411 to explore the origin and diversity of this virus worldwide. importantly, the careful analyses demonstrate that some of the animal covs have evolved to all data generated or analyzed in this study are included 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'epidemiologic features and clinical course of patients infected with sars-cov-2 in sars-cov-2 spike protein variant d614g increases infectivity and retains sensitivity to antibodies that target the receptor binding domain the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity sars-cov-2 neutralizing serum antibodies in cats: a serological investigation probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child tissue tropisms opt for transmissible reassortants during avian and swine influenza a virus co-infection in swine a genomic perspective on the origin and emergence of sars-cov-2 evolution of infectious bronchitis virus in china over the past two decades fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva', medrxiv: accepted article this article is protected by copyright immunogenicity and safety of a recombinant adenovirus type-5-vectored covid-19 vaccine in healthy adults aged 18 years or older: a randomised contribution of porcine aminopeptidase n to porcine deltacoronavirus infection virulence factors in porcine coronaviruses and vaccine design this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-294021-x8avmtef authors: pérez‐rivera, claudia; ramírez‐mendoza, humberto; mendoza‐elvira, susana; segura‐velázquez, rene; sánchez‐betancourt, josé ivan title: first report and phylogenetic analysis of porcine deltacoronavirus in mexico date: 2019-04-16 journal: transbound emerg dis doi: 10.1111/tbed.13193 sha: doc_id: 294021 cord_uid: x8avmtef porcine deltacoronavirus has caused great economic losses in the swine industry worldwide. in this study, we carried out the first detection, sequencing and characterization of this virus in mexico. we analysed 885 rectal samples by multiplex rt‐pcr to determine coinfections. in addition, the spike gene was amplified, sequenced and analysed phylogenetically. we found 85 positive samples for porcine deltacoronavirus, representing 9.6% of the total samples, and we determined that the most frequent coinfection was with porcine epidemic diarrhoea virus (54.1%). four sequences of mexican isolates were most closely related to those of the united states. the antigenic regions and the glycosylation site of the strains obtained coincide with those previously reported. this relationship is probably related to the commercial exchange of pigs between the us and mexico and the geographical proximity of these two countries. porcine deltacoronavirus (pdcov) is an emerging virus that was recently described. this viral disease together with porcine epidemic diarrhoea (ped) have caused a significant economic impact due to the high mortality rate in piglets (jung, hu, & saif, 2016; zhang, 2016) . pdcov belongs to the recently classified subgenus buldecovirus of the deltacoronavirus genus (ictv, 2018) . the subfamily orthocoronavirinae is divided into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. the first two genera originate in bats, while the third and fourth genera originate in birds (woo et al., 2012) . these viruses can affect several species of mammals and birds and cause various clinical conditions (neurological, digestive and respiratory) (wang, byrum, & zhang, 2014) . coronaviruses are unique within rna viruses because their genome is particularly large (~27.5 to ~31 kb). these enveloped viruses, with a single chain in the 5′-3′ direction and a helical nucleocapsid, contain at least seven open reading frames (orf), which code for four structural proteins (masters, 2006; sawicki, 2009 ). coronaviruses received their name due to one of their surface proteins, which is spicule-shaped, giving the appearance of a crown. this so-called s or "spike" protein is glycosylated and plays a fundamental role in the binding and entry of the virus into the cell (masters, 2006) . the genome organization of pdcov is in the following order: 5′ untranslated region (utr), replicase (orf 1ab), spike gene (s), envelope gene (e), membrane gene (m), nonstructural gene 6 (ns6), nucleocapsid gene (n), ns7 gene and 3´utr. particularly, pdcov lacks orf 3 and non-structural protein 1 (lee & lee, 2014; woo et al., 2012; zhang & yoo, 2016) . in pigs, at least six coronaviruses are known to cause diseases. these belong to three of the four known genera. within the alphacoronaviruses are the transmissible gastroenteritis virus (tgev), the porcine epidemic diarrhoea virus (pedv), the porcine respiratory coronavirus and the hku2-related bat coronavirus, which were described in china in 2016 related to acute diarrhea in pigs . in the betacoronavirus, there is the porcine hemagglutinating encephalomyelitis virus, and finally, in the genus of the deltacoronaviruses, there is the newly described porcine deltacoronavirus (pdcov) ictv, 2018) . kong in samples collected in 2009. this study aimed to determine the variability of bat coronaviruses due to the importance that the coronaviridae family has in hong kong since the presentation of acute respiratory diseases in humans. in this work, pigs did not present an apparent clinical sign; however, pdcov was detected (woo et al., 2012) . subsequently, in early 2014, pdcov was reported from the united states of america (usa) and canada, and it caused heavy economic losses to the swine industry due to the presentation of a clinical enteric disease song et al., 2015; wang et al., 2014) . the described infection was indistinguishable from that caused by pedv or tgev. however, the first reports in the usa mentioned that the mortality in piglets was lower (30%-40%) than that normally observed in outbreaks of ped (marthaler, raymond, et al., 2014; wang et al., 2014) . experimental studies in piglets show that the most frequent signs related to pdcov are watery diarrhoea, vomiting and dehydration (ma et al., 2015) . the authors note that pdcov infections are common in pigs and that coinfections are frequent, especially with the porcine epidemic diarrhoea virus and rotavirus c (hu et al., 2015; marthaler, raymond, et al., 2014; song et al., 2015) . likewise, some authors speculated that coinfections are likely to cause higher mortality rates (song et al., 2015) . despite the impact that the disease has had in several pig-producing countries, until this year, we did not know about its presence or the genetic characteristics of the virus in mexico. the objective of this work was to identify the presence of this virus in mexico and to analyse the genomic sequence of the s gene (spike). to monitor the prevalence and sequence properties of pdcov in mexico, 885 porcine rectal swabs were collected from five different regions of the country from 2014 to 2017. the sampling locations are shown in figure 1 . these samples were preserved at −70°c until use, later they were diluted 1:5 with 1x pbs (ph 7.4) and then centrifuged at 5,000 rpm for 15 min. once the sample was centrifuged, the supernatant was collected and the total rna was extracted using a commercial kit following the manufacturer's recommendations (kit qiaamp viral rna. qiagen cat. 52906). the resulting yield of rna extracted was 60 µl at an average concentration of 7 ng/µl. rt-qpcr was carried out to address the frequency of pdcov monoinfection and coinfection(s) with pedv and tgev. we used the vetmax™ pedv/tgev/sdcov kit (applied biosystems cat. a33402) following the manufacturer´s recommendations. to amplify the s gene, rna from the samples that were positive by rt-qpcr, was reverse-transcribed (rt) using the reverse primer 5′cactatgtctgacgcagaag3′ and superscript ii (invitrogen, san diego, ca). the rt products were then used to perform pcr using primers specifically targeting the s gene of the cdna obtained from the s gene amplification was subjected to sequencing using the ion personal genome machine platform (ion torrent thermo fisher scientific) following the supplier's specifications for dna sequencing. the obtained readings were filtered with the fastqc plug-in v3.4.1.1, selecting only those with a q score ≥20 (6,074,916 reads); the obtained depth was greater than 300×. the alignment of the sequences obtained from the amplification of the s gene was performed using the clustal w program in mega7 software (kumar, stecher, & tamura, 2016) . for the construction of the tree, the neighbour-joining distance-based method was used, and the bootstrap analysis consisted of 1,000 repetitions using the same software. to predict the epitopes, the s protein was analysed with protean, dnastar v.7.1 (madison, wi, ee. uu.). the jameson-wolf method predicted the potential antigenicity sites, which were compared with those reported by mai, k and collaborators based on the sequence hku15-155 (genbank accession no. afd29194.1). likewise, the surface properties, including hydrophobicity, accessibility and flexibility, were analysed by the kyte-doolittle, plot-emini and karplus-schulz methods respectively also in protean software. furthermore, the positions of the epitopes were predicted using the online service http://www.cbs.dtu.dk/services/bepipred/. the modelling of the amino acid sequence of the protein was carried out in swiss-model (https://swissmodel.expasy.org/). the glycosylation sites were analysed with netnglyc 1.0 server (http://www.cbs.dtu. dk/services/netnglyc). pdcov infections in mexico have probably not been adequately addressed due to the high prevalence ped in the country since 2013. pig farms have experienced sporadic outbreaks of diarrhoea in pigs less than 3 weeks old, often without determining the causative agent, while assuming that the causative agent is pedv, possibly masking the presence of deltacoronavirus. a total of 885 samples were analysed from five different regions of mexico, of which 85 (9.6%) were pdcov positive (table 1, figure 1 ). the most common coinfection was pdcov/pedv, found in 54.1% of the total deltacoronavirus-positive cases (46/85), a result that coincides with that reported by other authors (song et al., 2015; zhang, 2016 figure 1 ). in addition, it is interesting that a considerable number of samples (16/85) were positive for the three viral agents studied (pdcov/pedv/tgev). the s protein has the same structure in all coronaviruses. it is the most studied protein due to the role it plays in binding to the receptor and thus determining tropism, in addition to mediating the fusion of membranes for entry of the virus into the cell shang et al., 2018) . within the coronaviruses, the genus deltacoronavirus is the one with the smallest s protein; however, the structure is the same; it is divided into two subunits, s1 (1-573 aa) and s2 (574-1160 aa) (thachil, gerber, xiao, huang, & opriessnig, 2015) . and ohio137/2014 (genbank accession no. aib07807), which have structures known by electron microscopy (shang et al., 2018; xiong et al., 2018) . we observed the mutation of amino acids at six sites (110 e/d, 221 n/k, 510 ni, 534 kn, 550 il, 624 av) . we built a phylogenetic tree using the neighbour-joining method with 48 sequences from all countries that are available in genbank. three main groups were formed. in the first group are sequences from china (in green, figure 2 ) and in the second, sequences from thailand, laos and vietnam are grouped (in orange). finally, in the third group, we found sequences from the us, japan and south korea we observed that at positions 38 to 53, a 1 amino acid insertion (n) increased the antigenic index, surface probability and hydrophilic level (figure 3c and figure s1 ) (mai et al., 2018) . likewise, 17 glycosylations were located throughout the s1 region, similar those predicted by xiaoli, x in 2018 to with the illinois 2014 strain ( figure 3a ). knowing the glycosylation sites is important in the study of the antigenicity of the virus because the sites are part of a viral strategy to evade the host immune system (shang et al., 2018) . we know that the immune response of b cells is against the spike protein (mai et al., 2018) ; however, deeper studies would be necessary to evaluate whether these predictions coincide with the real antigenicity and pathogenicity of the protein. in conclusion, we determined that porcine deltacoronavirus occurs in mexico and that it is frequently associated with other pathogens, mainly pedv and tgev. to date, recombinations of pdcov with other coronaviruses have not been reported, but the recombinant capacity of these viruses is known, as demonstrated by the event reported in italy, where porcine enteric coronavirus (secov) was found. the genome of the s gene of this virus has greater homology with the ped virus, while the rest of its genome has homology with the tge virus (boniotti et al., 2016) . likewise, the mexican strain is phylogenetically closer to those strains reported in the us. this supports the theory of the current global distribution of porcine deltacoronavirus. further analysis of the structure and changes found in the s protein of the pdcov from mexico are necessary to determine its degree of pathogenicity and antigenicity. porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus pathogenicity and pathogenesis of a united states porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the united states virus taxonomy: 2018 release ictv porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis mega 7: molecular evolutionary genetics analysis version 7.0 for bigger datasets complete genome characterization of korean porcine deltacoronavirus strain kor/knu14-04 broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility origin, evolution, and virulence of porcine deltacoronaviruses in the united states the detection and phylogenetic analysis of porcine deltacoronavirus from guangdong province in southern china porcine deltacoronavirus from the united states rapid detection, complete genome, and phylogenetic analysis of porcine deltacoronavirus (technical appendix) the molecular biology of coronaviruses coronavirus genome replication cryo-electron microscopy structure of porcine deltacoronavirus spike protein in the prefusion state newly emerged porcine deltacoronavirus associated with diarrhoea in swine in china: identification, prevalence and full-length genome sequence analysis development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies detection and genetic characterization of deltacoronavirus in pigs discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavi glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin first report and phylogenetic analysis of porcine deltacoronavirus in mexico the authors declare no financial or commercial conflicts of interest. rene segura-velázquez https://orcid.org/0000-0001-8392-4919josé ivan sánchez-betancourt https://orcid. org/0000-0001-8201-9669susana mendoza-elvira https://orcid.org/0000-0003-3672-6471 key: cord-289584-rbp7p8s9 authors: zhou, ling; sun, yuan; lan, tian; wu, ruiting; chen, junwei; wu, zixian; xie, qingmei; zhang, xiangbin; ma, jingyun title: retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern china date: 2019-01-09 journal: transbound emerg dis doi: 10.1111/tbed.13008 sha: doc_id: 289584 cord_uid: rbp7p8s9 swine acute diarrhoea syndrome coronavirus (sads‐cov), a novel coronavirus, was first discovered in southern china in january 2017 and caused a large scale outbreak of fatal diarrheal disease in piglets. here, we conducted a retrospective investigation of 236 samples from 45 swine farms with a clinical history of diarrheal disease to evaluate the emergence and the distribution of sads‐cov in pigs in china. our results suggest that sads‐cov has emerged in china at least since august 2016. meanwhile, we detected a prevalence of sads‐cov (43.53%), porcine deltacoronavirus (8.83%), porcine epidemic diarrhoea virus (pedv) (78.25%), rotavirus (21.77%), and transmissible gastroenteritis virus (0%), and we also found the co‐infection of sads‐cov and pedv occurred most frequently with the rate of 17.65%. we screened and obtained two new complete genomes, five n and five s genes of sads‐cov. phylogenetic analysis based on these sequences revealed that all sads‐cov sequences in this study clustered with previously reported sads‐cov strains to form a well defined branch that grouped with the bat coronavirus hku2 strains. this study is the first retrospective investigation for sads‐cov and provides the epidemiological information of this new virus in china, which highlights the urgency to develop effective measures to control sads‐cov. swine acute diarrhoea syndrome coronavirus (sads-cov) is a newly discovered coronavirus which is an enveloped, positive and singlestranded sense rna virus with a genome size of approximately 27 kb (gong et al., 2017; pan et al., 2017; zhou et al., 2018) . sads-cov belongs to the family coronaviridae which contains four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (woo, huang, lau, & yuen, 2010; woo et al., 2012) . so far, six coronaviruses have been identified from pigs, which include porcine epidemic diarrhoea virus (pedv), porcine respiratory coronavirus (prcv), sads-cov and transmissible gastroenteritis virus (tgev) that all belong to the alphacoronavirus genus, as well as one betacoronavirus, porcine hemagglutinating encephalomyelitis virus (phev) and one deltacoronavirus, porcine deltacoronavirus (pdcov) (lin, saif, marthaler, & wang, 2016; wesley, woods, & cheung, 1991; woo et al., 2010) . among these viruses, sads-cov is the most newly discovered coronavirus, which has been first reported in 2017 in china and is considered to be an hku2-related coronavirus with a bat-origin (gong et al., 2017; zhou et al., 2018) . in january 2017, sads-cov was detected in a swine farm and subsequently spread rapidly to three other farms in *these authors contributed equally to this work. guangdong province and caused the fatal swine acute diarrhoea syndrome (sads) characterized by the clinical signs with severe, acute diarrhoea and rapid weight loss of piglets. the symptoms of sads-cov are similar to those that caused by other swine enteric coronaviruses such as pdcov and pedv, but sads-cov is more harmful than these viruses because it has led to the death of almost 25,000 piglets in a short time and resulted in more significant economic losses (dong et al., 2015; sun, wang, wei, chen, & feng, 2016; zhou et al., 2018) . so, it is urgent to investigate the molecular epidemiology and transmission patterns of sads-cov for establishing effective controls for this new coronavirus. in the present study, we performed the retrospective pcr testing on diarrheal samples from 45 swine farms in guangdong province to evaluate the emergence and the distribution of sads-cov in pigs in china. the prevalence and co-infection information of sads-cov from eleven sads-cov-positive farms was provided. the sequences of sads-cov, including two complete genomes, five nucleocapsid protein (n) genes and five spike protein (s) genes, were also identified and characterized to investigate the phylogenetic relationships of sads-cov. samples were homogenized in phosphate-buffered saline (pbs) (20% w/v), frozen and thawed three times, then centrifuged for 10 min at 10,000 g. viral nucleic acid was extracted following the manufacturer's recommendations of axyprep tm body fluid viral dna/rna miniprep kit (axygen scientific, inc). the virus nucleic acid was stored at −80°c until pcr was performed. a pair of primers (forward primer 5′-ggtccctgtgaccgaagttttag-3′, reverse primer 5′-gcgttctgcgataaagcttaaaactatta-3′) was designed to detected sads-cov based on the conserved n gene of this virus. one step rt-pcr using primescript ™ one step rt-pcr kit ver.2 with dye plus (takara, biotechnology, dalian, china) was carried out to amplify the target fragments by the following thermal profile of 50°c for 30 min, 94°c for 3 min, 35 cycles of denaturation at 94°c for 30 s, annealing at 55°c for 30 s, an extension at 72°c for 30 s, and a final step of 72°c for 5 min. four other diarrheal pathogens including pedv, pdcov, rotavirus (rv), and tgev from sads-covpositive farms were also tested by rt-pcr according to the previously described methods (liu, zhu, liao, xu, & zhou, 2015; mai et al., 2017; stevenson et al., 2013) . specific primer pairs based on reported sads-cov strains (genbank accession numbers: mf094681-mf094684) were designed for s genes, n genes and complete genome amplifications, respectively (table s1 ). pcr assays were performed with the following thermal profile: 95°c for 5 min, 35 cycles of 95°c for 30 s, 50°c for 30 s, and 72°c for 1 min 15 s, followed by a final 10 min extension at 72°c. the products were purified following the manufacturer's the nucleotide sequences were assembled and aligned using the dnastar program (dnastar v7.1, madison, wi, usa). phylogenetic trees were constructed using the neighbour-joining method in mega 7.0 software with bootstrap analysis of 1,000 replicates. percentages of replicate trees in which the associated taxa clustered are shown as nearby branches (chenna et al., 2003; kumar, stecher, & tamura, 2016; tamura, nei, & kumar, 2004 ). the two complete genomes (accession number mg605090 and f i g u r e 4 phylogenetic analysis of the s genes of sads-cov and reference coronavirus species. the tree was constructed as per figure 2 above. five new sequences of s genes studied in this work were indicated with "black solid circles" diarrhoea samples. our results showed that the first sads-cov positive sample was collected in august 2016 from the farm ls with a history of diarrhoea, as well as from other two farms tp and zw, which indicates that sads-cov has emerged in pigs in china at least since august 2016. and this time point is 5 months earlier than the first discovered time reported by our previous study (zhou et al., 2018) . as the same time, clinical signs of sads-cov during the retrospective investigation included sever and acute vomiting and diarrhoea, leading to death in piglets that were less than 5 days of age with a mortality rate of around 50%. these clinical presentations were similar to those signs in the large scale outbreak of sads-cov reported by zhou et al. (2018) , except the mortality rate in piglets later increased to 90%. based on the rates of infection documented in our work, it revealed that pedv (78.25%) was still the primary cause of the porcine diarrhoea, which is consistent with previous studies that pedv has been considered to be the major pathogen responsible for the porcine diarrhoea epidemic in china since 2010 (ge et al., 2013; sun et al., 2012; zhao et al., 2016) . the phylogenetic relationships of sads-cov sequences were also identified in this study. the results showed that all sads-cov sequences clustered together to form an independent branch and separated from other viral sequences in the genus alphacoronavirus. our results also indicated that both the complete genomes, n genes and s genes of all sads-cov strains shared the highest nucleotides identifies with those corresponding sequences of four bat coronavirus hku2 strains. in this work, the phylogenetic trees of full length genomes and s genes of sads-cov sequences showed that the sads-cov branch clustered with these four hku2 strains, which is same to previous results (gong et al., 2017; pan et al., 2017; zhou et al., 2018) . besides the genomes and s genes, the tree of n genes in our study revealed the identical result too. so far, a total of eight full-length genomes of sads-cov have been reported in guangdong province of china (gong et al., 2017; pan et al., 2017; zhou et al., 2018 ; this study). the two new genomes of sads-cov sequences in this work shared 100% nucleotides identities with the sequence mf167434 published by gong et al. (2017) and our four previously reported sequences (zhou et al., 2018) , and shared 99.8% nucleotides identities with the sequence mf370205 studied by pan et al. (2017) . the results suggest that these eight sads-cov sequences may come from the same origin. only the phylogenetic tree of s genes in our work showed that sequences of the alphacov were divided into two sublineages, alphacov1 which contained all sads-cov sequences and alphacov2, clustering together with sequences of the beltacov and the delatcov, respectively. and this result was consistent with the study of pan et al. (2017) . as a newly discovered coronavirus, the availability of sads-cov sequences data is limited which prevents better understandings of the molecular epidemiology of this virus. meanwhile, being a rna virus, sads-cov may mutate rapidly and exhibit high genetic differences (drummond, pybus, rambaut, forsberg, & rodrigo, 2003; kühnert, wu, & drummond, 2011 the authors declare no conflict of interests with any organization. ma https://orcid.org/0000-0001-6285-312x infectious diseases, 23, 1607-1609. https://doi.org/10.3201/eid2309. porcine enteric alphacoronavirus gds04 | mf167434 rhinolophus bat coronavirus hku2 ch/gd-01/2017/p2 | mf370205 bat coronavirus hku2 | nc009988 bat coronavirus hku2 strain hku2/hk/46 bat coronavirus hku2 strain hku2/hk/33 human coronavirus 229e | nc002645 camel alphacoronavirus isolate riyadh/ry141/2015 | nc028752 229e-related bat coronavirus strain btky229e-1 | ky073747 porcine enteric alphacoronavirus gds04 | mf167434 rhinolophus bat coronavirus hku2 ch/gd-01/2017/p2 | mf370205 bat coronavirus hku2 strain hku2/hk/33 bat coronavirus hku2 strain hku2/hk/46 bat coronavirus 1a | nc010437 bat coronavirus hku8 strain afcd77 | eu420139 rousettus bat coronavirus hku10 | nc01887 porcine epidemic diarrhoea virus strain gds01 | km089829 porcine epidemic diarrhoea virus strain cv777 | kt323979 porcine epidemic diarrhoea virus| nc003436 human coronavirus nl63 | nc005831.2 nl63-related bat coronavirus strain btkynl63-9a | nc032107 229e-related bat coronavirus strain btky229e-1 | ky073747 camel alphacoronavirus isolate riyadh/ry141/2015 | nc028752 human coronavirus 229e | nc002645 porcine hemagglutinating encephalomyelitis virus | nc007732 human coronavirus oc43 strain sc2481 | ky983583 mouse hepatitis virus strain mhv-a59 c12 mutant | nc001846 murine hepatitis virus strain jhm complete genome | ac000192 middle east respiratory syndrome coronavirus | nc019843 bat sars coronavirus hku3-1 | dq022305 bat sars-like coronavirus rsshc014 | kc881005 bat sars-like coronavirus wiv1 | kf367457 european turkey coronavirus 080385d | kr822424 bulbul coronavirus hku11-796 | fj376620 porcine deltacoronavirus isolate pdcov/chjxni2/2015 | kr131621.1 porcine coronavirus hku15 strain hku15-44 | nc016990 rhinolophus bat coronavirus hku2 ch/gd-01/2017/p2 | mf370205 bat coronavirus hku2 strain hku2/hk/46 bat coronavirus hku2 strain hku2/hk/33 human coronavirus oc43 strain sc2481 | ky983583 porcine hemagglutinating encephalomyelitis virus | nc007732 mouse hepatitis virus strain mhv-a59 c12 mutant | nc001846 murine hepatitis virus strain jhm complete genome | ac000192 middle east respiratory syndrome coronavirus | nc019843 bat sars coronavirus hku3-1 | dq022305.2 sars coronavirus | nc004718 bat sars-like coronavirus rsshc014 | kc881005 bat sars-like coronavirus wiv1 | kf367457 european turkey coronavirus 080385d | kr822424 porcine deltacoronavirus isolate pdcov/chjxni2/2015 | kr131621.1 porcine coronavirus hku15 strain hku15-44 | nc016990 bulbul coronavirus hku11-796 | fj376620 bat coronavirus hku8 strain afcd77 | eu420139 bat coronavirus 1a | nc010437 camel alphacoronavirus isolate riyadh/ry141/2015 | nc028752 multiple sequence alignmentwith the clustal series of programs porcine deltacoronavirus in mainland china measurably evolving populations epidemiological survey of porcine epidemic diarrhea virus in swine farms in a new bat-hku2-like coronavirus in swine phylogenetic and epidemic modeling of rapidly evolving infectious diseases mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains the porcine microrna transcriptome response to transmissible gastroenteritis virus infection the detection and phylogenetic analysis of porcine deltacoronavirus from guangdong province in southern china discovery of a novel swine enteric alphacoronavirus (sea-cov) in southern china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review prospects for inferring very large phylogenies by using the neighbor-joining method genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus coronavirus genomics and bioinformatics analysis discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus the rate of co-infection for piglet diarrhea viruses in china and the genetic characterization of porcine epidemic diarrhea virus and porcine kobuvirus fatal swine acute diarrhea syndrome caused by an hku2-related coronavirus of bat origin key: cord-318237-22s13v2y authors: mira, francesco; purpari, giuseppa; di bella, santina; colaianni, maria loredana; schirò, giorgia; chiaramonte, gabriele; gucciardi, francesca; pisano, patrizia; lastra, antonio; decaro, nicola; guercio, annalisa title: spreading of canine parvovirus type 2c mutants of asian origin in southern italy date: 2019-07-14 journal: transbound emerg dis doi: 10.1111/tbed.13283 sha: doc_id: 318237 cord_uid: 22s13v2y canine parvovirus type 2 (cpv‐2) emerged as dog pathogen in the late 1970s, causing severe and often fatal epizootics of gastroenteritis in the canine population worldwide. although to date cpv‐2 is circulating in all continents, most of the current studies have analysed the amino acid changes accounted in the vp2 gene sequence, with limited information on virus introductions from other countries. the aim of this study was to analyse the genetic features of cpv‐2c strains currently spreading in italy. swabs and tissue samples were collected from dogs suspected of cpv infection. the nearly complete genome sequence from the cpv‐positive samples was obtained. the co‐circulation of two different but related cpv‐2c strains, with amino acid changes characteristic of cpv strains of asian origin (ns1: 60v, 544f, 545f, 630p – ns2: 60v, 151n, 152v ‐ vp2: 5a/g, 267y, 297a, 324i, 370r), were observed. the phylogenetic analyses inferred from the ns1 and vp2 gene sequences confirmed the relationship with asian cpv‐2c strains. this study reports the spread of novel cpv‐2c mutants in italy and supports further studies to evaluate the coexistence of genetically divergent cpv strains in the same geographical environment. . after its emergence, the original type cpv-2 was replaced by three antigenic variants termed cpv-2a, and cpv-2c (buonavoglia et al., 2001; parrish et al., 1991; parrish, o'connell, evermann, & carmichael, 1985) . during the years, several amino acid (aa) changes were accounted in the vp2 gene sequence (battilani et al., 2001; geng et al., 2015; jeoung, ahn, & kim, 2008; nakamura et al., 2004; truyen, 1999) and, only recently, the analysis of the ns1 gene sequence was included in the cpv phylogenies (canuti, rodrigues, whitney, & lang, 2017; grecco et al., 2018; han et al., 2015; li et al., 2018; mira et al., 2019; pérez et al., 2014; zhuang et al., 2019) . previous studies provided information on the cpv strains spreading in italy (decaro, desario, et al., 2007; decaro et al., 2013 decaro et al., , 2006 dei giudici et al., 2017; mira, dowgier, et al., 2018; purpari et al., 2018; tucciarone et al., 2018) , suggesting the need of a continuous epidemiological survey to evaluate the cpv circulation and evolution, whereas limited data are available on the spread of novel strains imported from other continents (mira, purpari, lorusso, et al., 2018) . the aim of this study was the detection and molecular analysis of cpv strains displaying genetic features of asian viruses spreading in southern italy. during an epidemiological survey, rectal swabs (n = 3) and tissue samples (n = 19) from seven dogs suspected of cpv infection (table 1) , collected in southern italy (sicily) from august 2018 to march 2019, were analysed at the istituto zooprofilattico sperimentale della sicilia "a. mirri" (palermo, italy) for diagnostic purposes. dna and rna were extracted from swab/organ homogenates, obtained as previously described presence of cpv dna was evaluated by a diagnostic pcr using a primer pair targeting the vp2 gene (touihri et al., 2009) , as previously described (mira, purpari, lorusso, et al., 2018) , and each amplicon was analysed by electrophoresis on a 3% agarose gel supplemented with ethidium bromide. sequencing encompassing both orfs (ns and vp genes) was carried out using primer pairs developed by pérez et al. (2014) , as previously described . sequences were assembled according to an overlapping strategy and analysed using bioedit ver 7.0.5.3 software (hall, 1999) . assembled nucleotide sequences were submitted to nblast program (zhang, schwartz, wagner, & miller, 2000) to search related sequences in public domain databases. these sequence data have been submitted to the ddbj/embl/ genbank databases under accession numbers mk802679-85. the obtained sequences were aligned with reference sequences retrieved from the genbank database, which included the sequence (accession number mf510157) of a cpv-2c strain collected from the same region and previously analysed (mira, purpari, lorusso, et al., 2018 to elucidate the genetic relationships of the analysed cpv strains, two phylogenetic trees, based on the full-length vp2 and ns1 gene sequences, were constructed with the mega x software (kumar, stecher, li, knyaz, & tamura, 2018) , using the maximum-likelihood f i g u r e 1 maximum-likelihood tree based on 50 full-length vp2 gene sequences of canine parvovirus type 2 strains (bootstrap 1,000 replicates; bootstrap values greater than 65 are shown). black dots markings (•) indicate cpv strains analysed in this study. each sequence is indicated with virus type (fplv: feline panleukopenia virus-cpv: canine parvovirus) or variant (cpv-2, cpv-2a, cpv-2b, cpv-2c), country and year of collection, and accession number method according to the tamura 3-parameter (t92) and hasegawa-kishino-yano (hky) models, with discrete gamma distribution (five rate categories) (g) and invariant sites (i) (bootstrap analyses with 1,000 replicates). the models selection was performed using the best-fit model of nucleotide substitution with mega x software (vp2 gene: t92+g+i; ns1 gene: hky+g). extracted dna/rna were also amplified using a set of pcr assays for the detection of canine distemper virus (cdv) , canine adenovirus (cadv) type 1 and type 2 (dowgier et al., 2016) , canine herpesvirus (cahv-1) (decaro et al., 2010) , canine coronavirus (ccov) (decaro et al., 2004) and canine rotavirus (crov) (freeman, kerin, hull, mccaustland, & gentsch, 2008) . sequence analysis revealed amino acid changes previously described in asian cpv-2c strains (ns1: 60v, 544f, 545f, 630p-ns2: 60v, 151n, 152v-vp2: 5a/g, 267y, 297a, 324i, 370r) (table s1 ). cpv strain izssi_pa5632/19 evidenced an additional change at residue 492 of ns1 protein (table s1 ). only one mutation (a/g) was observed among the analysed strains at residue 5 of the vp2 protein, which suggests the circulation of two different but related cpv-2c strains in southern italy. amino acid change i60v in ns1 also lies at the same residue in the ns2-encoding sequence, while change at codon 630 of ns1 sequences did not result in any changes in the encoded ns2 protein. additional two amino acid changes in the ns2-encoding sequences were observed among the analysed strains: d151n and m152v (table s1 ). these changes resulted in silent mutations in the corresponding encoded ns1 protein. phylogenetic analysis inferred from vp2 sequences indicated that analysed strains are more related to asian than to european cpv strains, clustering in a separate clade (figure 1 ). phylogenetic tree inferred from ns1 gene sequences shows that strains clustered within the phylogeny according to the geographical origin and the year of collection rather than to the cpv variant ( figure 2 ). the present molecular analysis of cpv strains detected in southern italy provides new data about the viral spread and dynamics of cpv mutants circulating in italy. in the last decades, several studies analysed the spread of cpv strains in italy, and in 2001, the emergence of the cpv-2c variant was firstly reported (buonavoglia et al., 2001) . in the following years, all three cpv variants were described in italy, with a slightly higher prevalence of the cpv-2a and cpv-2c variants (decaro, desario, et al., 2007; decaro et al., 2013 decaro et al., , 2006 tucciarone et al., 2018) . more recently, a cpv-2c strain displaying genetic signatures typical of asian viruses was detected in southern italy (mira, purpari, lorusso, et al., 2018) , thus suggesting the introduction of the virus from other countries, as reported for other canine viruses (decaro, campolo, et al., 2007; martella et al., 2006; . therefore, a continuous molecular survey was assessed to point out eventual introduction and spread of cpv strains originated from other geographic areas in the italian canine population. since (hoelzer & parrish, 2010) . according to this study, the spread of asian cpv strains in a separate geographical area different from asian countries could be suggested, as previously described in south america (grecco et al., 2018; maya et al., 2013) . whereas cpv-2a and cpv-2b are the prevalent variants circulating in asia (yi, tong, cheng, song, & cheng, 2016) , and more recently, cpv-2c has been described in the same continent (chiang, wu, chiou, chang, & lin, 2016; geng et al., 2015; nakamura et al., 2004; wang et al., 2016; zhao et al., 2017; zhou, zeng, zhang, & li, 2017; zhuang et al., 2019) , showing molecular signatures different from those of other continents. indeed, the asian cpv-2c variant shows specific amino acids in ns1 (60v, 544f, 545v, 630p) and vp2 (5a/g, 267y, 297a, 324i, 370r) gene sequences. most of these amino acids have been described in the vp2 of cpv-2a/2b/2c strains collected in china, vietnam, india, taiwan, south korea, thailand and japan (chiang et al., 2016; geng et al., 2015; han et al., 2015; jeoung et al., 2008; lin et al., 2014; mukhopadhyay et al., 2014; nakamura et al., 2004; phromnoi, sirinarumitr, & sirinarumitr, 2010; soma, taharaguchi, ohinata, ishii, & hara, 2013; xu et al., 2015; yi et al., 2016; zhang et al., 2010) . in particular, cpv-2c strains displaying the amino acid glycine (g) instead of the highly conserved alanine (a) at residue 5 of the vp2 have been previously detected in china (wang et al., 2016) and italy (mira, purpari, lorusso, et al., 2018) . more recently, molecular analyses including the ns1 gene sequence showed the presence of molecular signatures of the asian cpv strains even in this region (han et al., 2015; mira, purpari, lorusso, et al., 2018; zhuang et al., 2019) . indeed, it results critical to extend the analysis to other genomic regions to properly infer the spread of the genetic cpv variants (grecco et al., 2018) . (ns1: 60v, 544f, 545f, 630p -ns2: 60v, 151n, 152v) could contribute to further elucidate its evolution ). the classification system based on single amino acids (426 and 297) of the vp2 protein does not clearly reflect the phylogenetic relationships of the strains, better supported to proposed "clade" or "lineage/sub-lineage" new classification criteria (grecco et al., 2018; zhuang et al., 2019; mira et al., 2019) . as observed also in this study, phylogeny lacks any clustering based on the single vp2 aa residue 426 (cpv-2a/2b/2c), as well as on the geographic origin and period of sample collection. therefore, a wider evolutionary analysis further supports the thesis to consider the cpv antigenic variants as variants of cpv-2a rather than distinct subtypes (organtini, allison, lukk, parrish, & hafenstein, 2015) and could be considered as a more reliable tool in outbreak tracing. this study reported the early evidence and spread of cpv-2c strains of asian origin in the italian canine population. as observed in south america (grecco et al., 2018) , studies based on the complete coding genome are useful to monitor the spread of cpv strains with asian origin in a different continent and highlight the need of further studies to evaluate the cpv evolution due to the coexistence of genetically divergent strains in the same geographical environment. the authors would like to thank the centro veterinario darwin analysis of canine parvovirus sequences from wolves and dogs isolated in italy evidence for evolution of canine parvovirus type 2 in italy introduction of canine parvovirus 2 into wildlife on the island of newfoundland identification of a novel canine parvovirus type 2c in taiwan ictv virus taxonomy profile: parvoviridae development and validation of a real-time pcr assay for specific and sensitive detection of canid herpesvirus 1 canine parvovirus-a review of epidemiological and diagnostic aspects, with emphasis on type 2c infectious canine hepatitis: an "old" 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china in 2011 phylogenetic analysis of canine parvovirus vp2 gene in china phylogenetic analysis of the vp2 gene of canine parvoviruses circulating in china a greedy algorithm for aligning dna sequences typing of canine parvovirus strains circulating in north-east china the genetic evolution of canine parvovirus -a new perspective genome sequence characterization of canine parvoviruses prevalent in the sichuan province of china additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article key: cord-284377-hsju2shr authors: li, meng; zhao, lin; ma, jiajun; zhao, na; luo, jing; wang, chengmin; chen, lin; ma, guoyao; wang, yutian; he, hongxuan title: vibrio vulnificus in aquariums is a novel threat to marine mammals and public health date: 2018-07-26 journal: transbound emerg dis doi: 10.1111/tbed.12967 sha: doc_id: 284377 cord_uid: hsju2shr vibrio vulnificus is a gram‐negative, curved, obligate halophilic marine bacterium that exclusively exists in coastal seawaters. previous studies revealed that v. vulnificus is one of the most dangerous foodborne zoonotic pathogens for human beings. however, it remains unknown whether marine mammals can be infected by v. vulnificus. in may 2016, a captive spotted seal (phoca largha) died due to septicemia induced by v. vulnificus. upon post‐mortem examination, v. vulnificus was isolated, identified, and named as bj‐ph01. further analysis showed that bj‐ph01 belongs to biotype 1 and the clinical genotype. furthermore, we performed an epidemiological investigation of v. vulnificus in six aquariums in northern china. as a result, v. vulnificus was successfully isolated from all investigated aquariums. the positive rates ranged from 20% to 100% in each investigated aquarium. during the investigation, 12 strains of v. vulnificus were isolated, and all 12 isolates were classified into biotype 1. eleven of the 12 isolates belonged to the clinical genotype, and one isolate belonged to the environmental genotype. all 12 isolated v. vulnificus strains showed limited antibiotic resistance. overall, our work demonstrated that v. vulnificus is frequently distributed in aquariums, thus constituting a threat to captive marine mammals and to public health. salinity, (b) season, (c) gender and age, (d) preexisting chronic diseases, and (e) bacterium virulence. as reported, the majority of human infections occur in the subtropical regions from april to november. people over the age of 40 are predominantly infected. moreover, males are more susceptible than females (ito et al., 2012) , perhaps due to the role of estrogen in protecting against the bacterium's endotoxins (miyamoto et al., 1999; soucy, boivin, labrie, & rivest, 2005) . as mentioned previously, patients with underlying chronic diseases, including alcoholism, diabetes, cancer, or renal diseases, are more susceptible to v. vulnificus (bross, soch, morales, & mitchell, 2007a) . several other risk factors contribute to the high pathogenicity of v. vulnificus in humans, such as capsule, iron, and the vcg gene (jones & oliver, 2009 ). marine mammals are important inhabitants of tropical and temperate regions, especially offshore areas of the sea (schipper et al., 2008) . there is an overlap in the distributions of v. vulnificus and marine mammals. no evidence has shown that marine mammals can be infected by v. vulnificus; however, the concerns cannot be excluded. here, we provide evidence that marine mammals can also be infected by v. vulnificus. further investigation showed that v. vulnificus is ubiquitous in aquariums, thus revealing a novel threat to captive marine animals and human beings in aquariums. animal studies were performed in strict accordance with the guidelines for the care and use of animals in research, which is issued by the institute of zoology, chinese academy of sciences. this study was evaluated and approved by the animal ethics committee of institute of zoology, chinese academy of sciences. all experiments were conducted in a biosafety level 2 (bsl-2) facility. an autopsy was performed after the spotted seal died. pathological lesions were observed and recorded. tissue samples (i.e., lung, liver, stomach, spleen, intestine, kidney, and heart) were sterilely collected. a festered sample was collected from the trauma injury. all samples were transported on ice and analyzed immediately. tissue samples were cultured on blood agar (oxoid, uk), macconkey agar (oxoid, uk), chocolate agar (oxoid, uk), and thiosulphatecitrate-bile-salt-sucrose (tcbs) agar (oxoid, uk). all plates were placed in aerobic or anaerobic conditions at 37°c for 18-24 hr. from each plate, at least 24 single colonies were selected for identification, and all the selected colonies were preliminarily identified by 16s rdna sequencing. the results were further confirmed by the bd phoenix automated microbiology system (bd diagnostic systems, sparks, md.) (stefaniuk, baraniak, gniadkowski, & hryniewicz, 2003) and species-specific pcr amplification. several known viral pathogens that have been reported to infect marine mammals were also detected by virus-specific pcr, including type a influenza virus (iav), phocine distemper virus (pdv), coronavirus, and rotavirus. the primers used in this study are listed in table 1 (chatzidaki-livanis, jones, & wright, 2006; gómara, wong, blome, desselberger, & gray, 2002; hill et al., 1991; lau et al., 2005; miller et al., 2011; reynaud, pitchford, de decker, wikfors, & brown, 2013; sea, 2015; warner & oliver, 2008 ccatcatcagatagaatcatcata animals (i.e., spotted seal, turtle, dolphin, shark, whale, and saltwater fish) were kept (table 2) . animal (i.e., turtle, spotted seal) body surface swabs were collected using medical degreasing cotton. freshwater samples and freshwater fish body surface swabs were also collected as negative controls. in total, 54 samples were collected. all the samples were stored on ice, transported to the institute of zoology, chinese academy of sciences, and analyzed immediately. all samples were examined using procedures in the bacteriological analytical manual of the food and drug administration (fda) (kaysner & depaola, 2004) . in detail, water samples were serially diluted and cultured by 1% nacl alkaline peptone water (apw) at 37°c for 18-24 hr. body surface swabs were diluted in a 5-ml volume of sterile phosphate-buffered saline (pbs) followed by vortexing for 45 s. the supernatant was cultured in 1% nacl alkaline peptone water (apw) at 37°c for 18-24 hr. the resulting products were diluted and cultured on blood agar and then detected by v. vulnificus vvha gene-specific pcr (warner & oliver, 2008) , 16s rdna sequencing, and the bd phoenix automated microbiology system (bd). biotyping of isolated v. vulnificus was performed as previously reported (bisharat, agmon, finkelstein, raz, ben-dror, lerner, soboh, colodner, cameron, & wykstra, 1999) . briefly, onpg testing, indole production, 1% nacl ornithine decarboxylase testing, d-sorbitol fermentation, d-mannitol fermentation, and lactose fermentation analysis were performed to identify the biotypes of the isolated v. vulnificus. genotyping was conducted to identify the virulence genes of the isolated v. vulnificus. in our study, vcg (virulence-correlated gene), sere (serovar e) gene, cps (capsular polysaccharide) gene, bt2 (biotype 2) gene, ary (arylsulfatase) gene, mtlabc (mannitol/fructose-specific phosphotransferase system iia protein) gene, and nana (n-acetylneuraminate lyase) gene were detected using virulence gene-specific pcr as listed in table 1 . the v. vulnificus isolated from the seal (bj-ph01) was cultured in 1% apw and quantified by a colony forming unit (cfu) assay on blood agar. the inoculum was serially diluted to 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , and 10 3 cfu/ml 6-week-old female balb/c mice were intraperitoneally (for each group, n = 4) (strom & paranjpye, 2000) or intramuscularly (for each group, n = 4) ( several virulence factors of v. vulnificus have been reported previously, such as toxin, lps, and capsule (jones & oliver, 2009 ). possession of an antiphagocytic capsule is one of the absolute requirements for virulence. encapsulated cells produce opaque colonies, and only opaque cells are able to utilize transferrin-bound iron. as shown in figure 2b , the v. vulnificus isolate bj-ph01 grew opaque colonies on blood agar. this result prompted us to consider that bj-ph01 is virulent in animal models (simpson, white, zane, & oliver, 1987) . several animal models have been constructed to explore the increased susceptibility to v. vulnificus infections after the injection of iron-containing compounds. here, we conducted an having demonstrated that v. vulnificus can be detected in aquariums, we next asked whether this lethal bacterium is widespread in aquariums. to explore the prevalence of v. vulnificus in aquariums, we performed an epidemiological investigation of v. vulnificus. in this analysis, we successfully collected 54 samples from six aquariums in northern china ( genotypes based on its virulence-correlated gene (vcg) (warner & oliver, 2008) ; our analysis showed that 11 of the 12 isolated v. vulnificus strains were classified into the c genotype, and only one of the 12 belonged to the e genotype (table 4) . a previous study demonstrated that genotypic markers cannot unequivocally predict virulence, although several genes were putatively reported to be linked to virulence. here, we investigated the genes ary (arylsulfatase), mtlabc (mannitol/fructose-specific phosphotransferase system iia protein), and nana (n-acetylneuraminate lyase) by pcr in our study (table 4 ). the ary gene has been demonstrated to be associated with virulence of clinical strains by providing a pathogen with sulfur within the host, thus providing an immune evasion approach (morrison et al., 2012) . positive ary pcrs were obtained in all of the isolated strains. although mtlabc appears to be linked to pathogen virulence, the precise role of mtlabc is not bj-sar01 | 1867 yet understood (reynaud et al., 2013) . in our experiments, we obtained positive mtlabc results for all of the isolated strains. the nana gene has been demonstrated to be involved in sialic acid metabolism and is essential for v. vulnificus virulence (kim, hwang, kim, & choi, 2011) . positive nana gene pcr results were obtained for all of the isolated strains. these results indicate that the 12 isolated v. vulnificus strains display a potential threat to mammals, including marine animals and humans. the disease progression of v. vulnificus infection is often acute, and it is therefore important to provide timely treatment with proper antibiotics. we conducted a further analysis to understand its sensitivity to major antibiotics. the result showed that all isolated v. vulnificus strains were sensitive to antibiotics. in detail, v. vulnificus was sensitive to levofloxacin, tetracycline, piperacillin, and gentamicin (table 5) . vibrio vulnificus is a foodborne pathogen of humans. here, we provide evidence that marine mammals can also be infected by v. vulnificus. epidemiological investigations of v. vulnificus showed that this lethal bacterium can frequently be detected in aquariums, thus constituting a novel threat to marine animals, workers, and tourists in relevant aquariums. antibiotic drug resistance is an increasing concern due to the overuse of antibiotics. fortunately, all of the isolated v. vulnificus isolates in our study were sensitive to levofloxacin, tetracycline, piperacillin, and gentamicin. no drug-resistant v. vulnificus isolates were isolated. the minimum dose capable of causing human infection is currently unknown (strom & paranjpye, 2000) . previous studies have suggested that the dose may be fewer than 1,000 organisms. animal experiments have been helpful in researching disease syndromes produced by v. vulnificus. however, they have no instructive value for determining the infectious dose 50 (id 50 ) for human infections (strom & paranjpye, 2000) . in our research, we found that the med(gao et al., 2017) . vibrio fluvialis has been considered an emerging pathogen that induces foodborne diarrhea (ramamurthy, chowdhury, pazhani, & shinoda, 2014) . these results suggest that v. vulnificus is not the only threat to marine animals and public health in aquariums. the major limitation of our research is that we failed to perform koch's postulate test because it was impossible for us to confirm the infection using a healthy seal. due to limited background information, the typical clinical manifestations of v. vulnificus infected seals are poorly understood. we provided a diagnosis mainly based on the detection and isolation of v. vulnificus from the visceral tissues and blood, which indicated that v. vulnificus induced septicemia (bross, soch, morales, & mitchell, 2007b we demonstrated that v. vulnificus can infect and kill marine mammals. however, it was difficult to evaluate the risk of infection of marine mammals by v. vulnificus: (a) v. vulnificus is an opportunistic pathogen, and one or more predisposing factors are required to initiate disease; (b) the minimum dose for v. vulnificus to cause an infection is poorly understood, even in humans; (c) the interface between v. vulnificus and marine mammals in the wild has never been studied. nevertheless, the prevalence of v. vulnificus is a persistent threat to marine mammals. this work was funded by grants from the state forestry administration of china, chinese academy of sciences (czbzx-1). no conflict of interest exists in the submission, and all authors approved the publication. li http://orcid.org/0000-0002-2453-1821 rapidly developing and fatal vibrio vulnificus wound infection. idcases, 6, 13 clinical, epidemiological, and microbiological features of vibrio vulnificus biogroup 3 causing outbreaks of wound 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of vibrio vulnificus biotype 1 vibrio vulnificus in aquariums is a novel threat to marine mammals and public health key: cord-294698-mtfrbn87 authors: kim, h. k.; yoon, s.‐w.; kim, d.‐j.; koo, b.‐s.; noh, j. y.; kim, j. h.; choi, y. g.; na, w.; chang, k.‐t.; song, d.; jeong, d. g. title: detection of severe acute respiratory syndrome‐like, middle east respiratory syndrome‐like bat coronaviruses and group h rotavirus in faeces of korean bats date: 2016-05-23 journal: transbound emerg dis doi: 10.1111/tbed.12515 sha: doc_id: 294698 cord_uid: mtfrbn87 bat species around the world have recently been recognized as major reservoirs of several zoonotic viruses, such as severe acute respiratory syndrome coronavirus (sars‐cov), middle east respiratory syndrome coronavirus (mers‐cov), nipah virus and hendra virus. in this study, consensus primer‐based reverse transcriptase polymerase chain reactions (rt‐pcrs) and high‐throughput sequencing were performed to investigate viruses in bat faecal samples collected at 11 natural bat habitat sites from july to december 2015 in korea. diverse coronaviruses were first detected in korean bat faeces, including alphacoronaviruses, sars‐cov‐like and mers‐cov‐like betacoronaviruses. in addition, we identified a novel bat rotavirus belonging to group h rotavirus which has only been described in human and pigs until now. therefore, our results suggest the need for continuing surveillance and additional virological studies in domestic bat. bats are considered reservoirs of several emerging infectious disease. recently emerging human viruses, such as severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), nipah virus, hendra virus and ebola virus, are thought be bat-borne viruses (han et al., 2015) . in addition, new species of influenza a viruses, lyssaviruses, paramyxoviruses and coronaviruses continue to be discovered in bats around the world (tong et al., 2013; banyard et al., 2014; yang et al., 2014; mortlock et al., 2015) . reassortant group a rotavirus was also detected in the faeces of a straw-coloured fruit bat (esona et al., 2010) . therefore, viruses that originate in bats may be a source of additional spill over from wildlife into domestic animals and humans (plowright et al., 2015) . since the outbreak of mers-cov in korea in 2015, the prediction and control of newly emerging viruses has gained urgency. however, in korea, there have been a few studies on viruses in domestic bat species. serological evidence of japanese encephalitis virus (jev) was reported in the early 1990s (lee and lee, 1992) . in 1995, there was a report on the isolation and genetic characterization on a hantavirus from bats (jung and kim, 1995) . more information of viruses in bats is needed to clarify the role of these animals in infectious diseases epidemiology. therefore, in this study, we investigated viruses in bat species in korea, using 49 faecal samples collected from july to december 2015 in 11 sites in natural bat habitats. from july to december in 2015, 49 faecal samples were collected at 11 sites in natural bat habitats, such as caves, an abandoned mine and under a bridge (table 1) . fresh bat faeces were placed into transport medium in a 10% suspension and were transported to the laboratory for further laboratory analysis. the major bat species at the collection sites were determined based on morphology and on previous data from bats' roosting sites (han et al., 2012) . in this study, the major bat species in the collecting sites were rhinolophus ferrumequinum, myotis macrodactylus, myotis aurascens kuzyakin, myotis petax and miniopterus schreibersii. consensus primer-based reverse transcriptase polymerase chain reactions (rt-pcrs) were performed to detect influenza a viruses, coronaviruses, lyssaviruses and paramyxoviruses (fouchier et al., 2000; poon et al., 2005; v azquez-mor on et al., 2006; tong et al., 2008) . the detailed information on the rt-pcrs is in table s1 . a total of 49 bat faecal samples were tested. the amplicons from positive rt-pcrs were sequenced using target-specific forward and reverse primers synthesized by cosmogenetech co. ltd (daejeon, korea). the nucleotide data obtained from sanger sequencing of the pcr fragments were further analysed with related sequences in genbank using bioedit (hall, 1999) and mega version 6 (tamura et al., 2013) . in total, 16 bat faecal samples that were collected in four sites were used for high-throughput sequencing ( table 1) . the supernatants from suspensions of one, four, five and six faecal samples collected in danyang, wonju, taebaek and uljin, respectively, were pooled by sampling site. each of the four pooled samples was filtered through a 0.2-lm filter and ultracentrifuged at 252 000 g for 1 h. each pellet was resuspended in 500 ll of 19 digestion buffer (turbo dna free kit; ambion, darmstadt, germany), and a total of 10 ll of turbo dnase was added. after incubating for 30 min at 37°c, suspensions were transferred to a 1.5-ml reaction tube and centrifuged at 1500 g for 3 min. the supernatants were used for rna extraction. rna was extracted using trizol ls (invitrogen corp., carlsbad, ca, usa), following the manufacturer's manual. the extracted rna was submitted to macrogen (seoul, korea) for high-throughput sequencing in a hiseq 2000 sequencing system based on the transcriptome de novo sequencing platform. the obtained viral contigs were analysed and annotated by the mg-rast server (meyer et al., 2008) . the cut-off for the annotation was 10 à5 , 60% and 15 for maximum e-value, minimum percentage identity and minimum alignment length, respectively. the contigs annotated as mammalian viruses were validated through blastn (http:// www.ncbi.nlm.nih.gov). the genbank accession numbers for the nucleotide sequences in this study are ku528584-ku528594. there were no samples positive for influenza a viruses, lyssaviruses or paramyxoviruses. among 14 cases of the submitted samples, three cases were positive for coronavirus by rt-pcr, showing the expected 440 nt target. these included the following: (case no. 1) one of four samples from ho cave in wonju where rhinolophus ferrumequinum and myotis macrodactylus were the major species present, (case no. 6) one of six samples bm abandoned mine in inje where rhinolophus ferrumequinum was the major species found and (case no. 12) two of four samples bgs (case no. 12) cave in munkyung where miniopterus schreibersii was found to be the major species (table 1) . in the ho cave, one of four faecal samples (b15-8) was positive by coronavirus-specific rt-pcr. when the pcrpositive fragment was sequenced, it was found to be a bat coronavirus (bat cov) high-throughput sequencing from the pooled bat faecal samples as shown in table 1 , cases 1, 2, 3 and 4 were further analysed by high-throughput sequencing. by hiseq 2000 sequencing, 4341, 162 559, 7690 and 16 657 contigs were obtained from cases 1, 2, 3 and 4, respectively. based on the annotation by the mg-rast system, several viral sequences that are related to mammalian viruses, insect viruses, plant viruses, fungal viruses and bacteriophages were identified. the mammalian viral sequences annotated by the mg-rast system were further validated by blastn (http:// www.ncbi.nlm.nih.gov). the blastn result is presented in table 2 . fifteen viral sequences from case no. 1 were similar to porcine rotavirus ska-1 (69-73% nucleotide identity), bat cov sc2013 (70-91% nucleotide identity), bat cov hku5-5 (82% nucleotide identity), alphacoronavirus eptesicus fuscus-related strains (95-96% nucleotide identity) and rhinolophus pearsoni bunyavirus shaanxi 2011 (72% nucleotide identity). three viral sequences from case no. 2 were similar to banna virus 02vn078b (90% nucleotide identity), bat cov cdphe15 (80% nucleotide identity) and bat cov neixiang-31 (82% nucleotide identity). there was no mammalian viral sequences in cases 3 and 4. partial rna-dependent rna polymerase (rdrp) sequences obtained from b15-8 (sample in case no. 2), b15-21 (sample in case no. 6), b15-40 and b15-41 (sample in case no. 12) were phylogenetically compared with other reference sequences (fig. 1a) . three of these sequences clustered with the alphacoronaviruses (b15-8, 40 and 41 because an rdrp sequence from betacoronavirusrelated bat cov b15-1-6 (detected from the pooled sample in case no. 1) was not obtained, a partial spike gene sequence (c10151) was compared with the spike genes of reference betacoronaviruses (fig. 1b) . in addition, the full spike gene sequence obtained from bat cov b15-21 was also analysed. bat cov b15-1-6 grouped with mers-covlike bat covs (bat cov strains hku5, a434, sc2013 and hku4), showing 80.9-82.2% nucleotide identities, and was found to be closely related to mers-cov emc, showing 76.6% of nucleotide identity. the amino acids sequence encoded by the full spike gene of b15-21 was aligned and compared with human sars-cov and sars-cov-like bat cov sequences ( figure s1 ). the s2 domain is more conserved than the s1 domain in the spike protein. the receptor binding domain (rbd) of the sars-cov-like bat covs, except for rashc014, had two major deletion sites, tgnyn (446-450) and pfspdgkpctppa (472-484), compared to human sars coronaviruses sequences. the receptor binding motif (rbm) and human ace2 (hace2)-interacting residue were highly variable between human sars-covs and sars-cov-like bat covs but rashc014 strain. porcine rotavirus-related sequences from pooled bat faeces in case no. 1 were analysed with reference sequences of rotaviruses in human and animals (fig. 2) . based on the partial sequences of the vp1, vp3 and vp4 regions, the rotavirus-related sequences were found to belong to rotavirus group h. the group h bat rotavirus in this study according to a recent report, there are 23 species of bats in korea (han et al., 2012) . most korean bats are thought to be insectivores, as no fruit bats have been found in korea to date, except for some fruit bats as pets or indoor exhibitions (han et al., 2010 (chu et al., 2008) . as bat covs b15-40 and 41 were also found in faeces in a cave where miniopterus schreibersii was dominant, bat cov strains in the same lineage with bat cov 1a and 1b may be adapted to the miniopterus spp. it was interesting that bat cov b15-21 found in this study was closely related to sars-cov-like bat covs, such as bat cov rf1, based on the partial rdrp and s genes, respectively. the newly identified bat cov b15-21 clustered with the sars-cov-like bat cov group in the phylogenetic analysis. the major bat species in the site where bat cov b15-21 was collected was rhinolophus ferrumequinum, which is consistent with a previous report that sars-covlike bat covs were mostly found in rhinolophus spp. (yuan et al., 2010) . the amino acids of its full spike gene showed that the b15-21 strain is closely related to sars-cov-like bat covs rp3, rf1, hku3-2 and 273 rather than human sars-covs and bat cov rashc014, which was reported to have high potential for human emergence (menachery et al., 2015) and showed two specific deletion sites in the rbd of the spike protein. one additional betacoronavirus (b15-1-6), which was not detected by the consensus primer-based rdrp-specific rt-pcr (poon et al., 2005) , was detected by high-throughput sequencing as shown in table 2 . three contigs were similar to bat cov sc2013, which is a mers-related betacoronavirus that was found in bats in china (yang et al., 2014) . one contig was also similar to bat cov hku5-5, which clustered with mers-cov (woo et al., 2007; annan et al., 2013) . phylogenetic analysis based on partial spike gene sequences of bat cov b15-1-6 showed that it belonged to a mers-cov-like clade with previously known mers-cov and mers-cov-like bat covs. although information of the associated bat species was not clearly available, this study first report that sars-cov and mers-cov-like bat covs exist in korea. this may imply that other strains or types of those coronaviruses may circulate in bat species in korea. therefore, we cannot ignore appearance of novel or variant coronavirus through local emergence in korea, like sars and mers in china and middle east, respectively. in addition to coronaviruses, this study is the first to determine the existence of group h rotaviruses in bat faeces collected in korea. group h rotavirus is a recently proposed group of rotaviruses that include strains adrv-n and b219, which infect human adults (alam et al., 2007; matthijnssens et al., 2012) . so far, group h rotaviruses have only been reported in human and pigs (molinari et al., 2015) , but this study provides evidence that bat species may be a host of group h rvs. to confirm that, there should be follow-up studies including virus isolation and characterization, genomic analysis, continuous surveillance and vp6-based classification (matthijnssens et al., 2012) to find its prevalence, epidemiology and zoonotic potential. localized emergences of zoonotic diseases are usually due to expansion of the wildlife-human interface (morse et al., 2012) . the human-bat interface in korea has not been fully described. people usually recognize that bats are flying and roosting in the deep forest or caves. however, the sites where the bat faecal samples were collected showed some evidence of human-bat contact ( figure s2 ). some caves or abandoned mine where bats are roosting are located near human habitats. people living there frequently visit into the caves or the abandoned mine to take a rest or to ferment foods. some caves were also used for some religious ceremony by korean traditional shamans. in this study, sars-cov-like and mers-cov-like bat covs and group h rotavirus were detected for this first time in korea, which may be of interest because of their zoonosis potential. several kinds of evidence for humanbat contact were also found. therefore, the potential for novel or variant human viruses from bats in korea should not be ignored. continuous surveillance and additional virological research should be a priority to prevent future emergences of zoonotic viral diseases. intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans. j. gen. virol. 91, 1058-1062. additional supporting information may be found in the online version of this article: table s1 . information on targets and primers for viral detection. figure s1 . amino acid-based multiple alignment of spike proteins from b15-21 and other reference strains. figure s2 . possible evidence of human-bat interaction in the bat habitats in this study. genetic analysis of an adrv-n-like novel rotavirus strain b219 detected in a sporadic case of adult diarrhea in bangladesh ska-1(porcine)-ab576629 j19(human)-dq113897 b219(human)-ef453355 03v0567(chicken)-nc_021590 cal-1(human)-eu490414 bang373(human)-nc_021541 03v0568(chicken)-nc_021625 05v0049(chicken)-nc_014511 osu(porcine)-gu199514 j19(human)-dq113900 b219(human)-ef453357 ska-1(porcine)-ab576631 03v0567(chicken)-nc_021589 cal-1(human)-eu490417 bang-373(human)-nc_021551 osu(porcine)-ay277921 human betacoronavirus 2c emc/2012-related viruses in bats lyssaviruses and bats: emergence and zoonotic threat genomic characterizations of bat coronaviruses (1a, 1b and hku8) and evidence for co-infections in miniopterus bats reassortant group a rotavirus from straw-colored fruit bat detection of influenza a viruses from different species by pcr amplification of conserved sequences in the matrix gene bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/ nt nucl isolation of a zoonotic pathogen kluyvera ascorbata from egyptian fruit-bat rousettus aegyptiacus sounds of the bats in korea. national institute of biological resources press bats as reservoirs of severe emerging infectious diseases genomic 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twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features mers-related betacoronavirus in vespertilio superans bats key: cord-303289-qoukiqr7 authors: hemida, m. g.; chu, d. k. w.; perera, r. a. p. m.; ko, r. l. w.; so, r. t. y.; ng, b. c. y.; chan, s. m. s.; chu, s.; alnaeem, a. a.; alhammadi, m. a.; webby, r. j.; poon, l. l. m.; balasuriya, u. b. r.; peiris, m. title: coronavirus infections in horses in saudi arabia and oman date: 2017-03-13 journal: transbound emerg dis doi: 10.1111/tbed.12630 sha: doc_id: 303289 cord_uid: qoukiqr7 equine coronaviruses (ecov) are the only coronavirus known to infect horses. so far, data on ecov infection in horses remain limited to the usa, france and japan and its geographic distribution is not well understood. we carried out rt‐pcr on 306 nasal and 315 rectal swabs and tested 243 sera for antibodies to detect coronavirus infections in apparently healthy horses in saudi arabia and oman. we document evidence of infection with ecov and hku23 coronavirus by rt‐pcr. there was no conclusive evidence of middle east respiratory syndrome coronavirus infection in horses. serological data suggest that lineage a betacoronavirus infections are commonly infecting horses in saudi arabia and oman but antibody cross‐reactivities between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections. infect or cause disease in horses (balasuriya, 2013) . it belongs to the genus betacoronavirus lineage a, as does human coronavirus oc43, bovine coronavirus and porcine hemagglutinating encephalomyelitis virus. equine coronavirus was first isolated from faeces of a diarrhoeic foal in 1999 (ecov-nc99) in north carolina, usa (guy, breslin, breuhaus, vivrette, & smith, 2000) , and was initially believed to only affect foals. since 2010, there have been several reports of ecov-associated respiratory and enteric infections in adult horses in japan, europe and the united states, but its global distribution is still poorly defined (kooijman, mapes, & pusterla, 2016; miszczak et al., 2014; oue, morita, kondo, & nemoto, 2013; pusterla, holzenkaempfer, mapes, & kass, 2015) . middle east respiratory syndrome coronavirus (mers-cov) is a betacoronavirus within lineage c first identified in humans in 2012 and continues to pose a threat to global health (who, 2016 may 27) . the evidence points to dromedary camels as a natural host and a major source for human mers-cov infections (alexandersen, kobinger, soule, & wernery, 2014; chu et al., 2015; gutierrez, tejedor-junco, gonzalez, lattwein, & renneker, 2015; memish et al., 2014; perera et al., 2013; reusken et al., 2014) . there is no convincing evidence of mers-cov infections in other domestic livestock species so far (adney et al., 2016; alexandersen et al., 2014; hemida et al., 2013; meyer et al., 2015; perera et al., 2013) . sequence similarity comparisons of dipeptidyl peptidase-4 (dpp4 [cd26]), the functional receptor for mers-cov, have revealed that equine dpp4 is phylogentically closely related to human dpp4 and the binding affinity of mers-cov spike s1 domain for equine dpp4 is similar to that of human and camel dpp4, raising the possibility that mers-cov infections may occur among horses (barlan et al., 2014) . serological studies of horses originating from spain and the united arab emirates gave negative results for mers-cov (alexandersen et al., 2014; meyer et al., 2015) , but it is still relevant to investigate for evidence of mers-cov in horses in areas endemic for mers-cov. we have tested for coronavirus infections in apparently healthy horses in saudi arabia and oman by testing nasal and rectal swabs by rt-pcr assays for mers-coronavirus and using a pan-coronavirus rt-pcr with potential to detect all coronaviruses, hitherto known and unknown. following the detection of ecov and hku23 coronavirus (hku23) by the pan-coronavirus rt-pcr, we tested the swab specimens with specific rt-pcr assays for ecov and hku23 and tested the sera with serological assays to detect ecov, bovine cov (bcov) (closely related to hku23), as well as mers-cov. (guy et al., 2000; zhang et al., 2007) and the bovine coronavirus (atcc brcv-ok-0514-2). viral rna was extracted from nasal and rectal swabs using the qiaamp viral rna minikit (qiagen, hilden, germany) following the manufacturer's instructions. rna extracts were tested for evidence of conserved coronavirus nucleic acid genetic sequences using previously reported rt-pcr assays (chu et al., 2014) , rtqpcr assay for mers-cov upe gene (corman et al., 2012) , rtqpcr assay for ecov (miszczak et al., 2014) , and a rtqpcr assay for hku23 reported below. rna was reverse transcribed in a 20 ll reaction mixture containing 19 first-strand buffer, 5 mm dtt, 0.5 mm deoxynucleotide triphosphates (dntp), 2.5 ng/ll random hexamers and 200 units of superscript iii (life technologies). the pan-coronavirus nested pcr targeted the rna-dependent rna polymerase (rdrp) gene of coronaviruses developed by us as previously described (chu et al., 2014) . using the cdna synthesized as described above, first round pcr was carried out using forward primer 5 0 -ggktgggaytaycckaartg-3 0 (position 15,287 of ecov strain nc99; genbank accession number ef446615) and reverse priof ecov strain nc99) and 5 ll of cdna as template for the reaction. pcr products with expected size of 658 bp were purified for dna sequencing using the forward and reverse primers. we developed a two-step real-time quantitative rt-pcr (rt-qpcr) assay for the detecting hku23 targeting n gene of the virus. virus rna was reverse transcribed as detailed above. real-time pcr the details of all rt-pcr and real-time pcr assays used in this study are summarized in table s1 . the primers used for sequencing were those used to generate the pcr products. dna amplicons were purified and sequenced in both forward and reverse directions with the pcr primers using bigdye all sera were tested using previously validated mers-cov spike pseudoparticle neutralization test (ppnt) (hemida et al., 2014; perera et al., 2013) , and the positive sera were confirmed using microneutralization (mn) test and plaque reduction neutralization tests (prnt) using live mers-cov strain emc in a biosafety level 3 containment laboratory (park et al., 2015) . selected sera were tested for antibodies to ecov and bcovs . the north american ecov-t a b l e 1 geographic location of specimens collected and animal management practices bcov is ubiquitous in cattle worldwide and is genetically and antigenically related with hku23, a coronavirus endemic in dromedaries (woo et al., 2014 (woo et al., , 2016 . antibody to bcov was tested using mn assay as described previously (park et al., 2015; perera et al., 2013) . serology for bcov and ecov was carried out with culture medium without foetal calf serum. known positive and negative control sera were included in all serology assays. (woo et al., 2014) . (table 4 ). in the confirmatory prnt which is considered the "gold standard" serological test for mers-cov, horse serum 9-2 reduced plaque numbers by 90% (prnt 90 ) up to a titre of 40. this serum was negative in ecov and bcov mn assays (table 4 ). this serum was from a racing horse stable in al-qassem and may represent a rare example of transmission of mers-cov to horses or cross-reaction with another yet undocumented coronavirus infecting horses. this horse was not known to frequently come into contact with camels. three of the other sera (serum id nos 5.35; 5.41; 2.12) positive in the mers-cov ppnt assay had detectable ecov mn antibody and one of these also had detectable bcov mn antibody (table 4 ). thus, these may well represent evidence of cross-reactive responses. t a b l e 5 cross-neutralization titres (denoted as reciprocal titres) for middle east respiratory coronavirus (mers-cov), bovine coronavirus (bcov) and equine coronavirus (ecov) in hyperimmune or naturally infected sera known to be positive for different coronaviruses nr460pig antiserum to porcine respiratory coronavirus 1,200 a <20 <20 <20 <20 nr2518 -guina pig antisera feline infectious peritonitis virus 2,000 a <20 <20 <20 <20 nr458pig antisera for porcine transmissible gastroenteritis virus 1,400 a <20 <20 <20 <20 ecov-negative sera 2 (horse, hong kong) not relevant <20 <20 <20 <20 bcov-positive camel serum #740293 e not relevant <20 nd 640 80 bcov-positive camel serum #467468 e not relevant <20 nd 80 <20 c99-10 -bcov antisera from guinea pig 20,480 b <20 <20 320 <20 bcov antisera from germ free bovine calf 580 bneutralisation titre <20 <20 1,280 80 nr456 -bcov antisera from gnotobiotic calf 10,000 a <20 <20 80 <20 nrc772 -rabbit antisera sars s protein high titre 640 <20 <20 <20 <20 mouse hepatitis virus(jhm strain) hyperimmunized mouse dam 1 1,778 cneutralisation titre <20 <20 <20 <20 mouse hepatitis virus(jhm strain) hyperimmunized mouse dam 2 363 cneutralisation titre <20 <20 <20 <20 mouse hepatitis virus(a59 strain)-infected mice 1,000 cneutralization titre <20 <20 <20 <20 nrc774antisera for sars coronavirus sero titre <10 <20 <20 <20 <20 nrc769 -rabbit antisera to sars s protein sero titre <10 <20 <20 <20 <20 gamma coronavirus nr2515-guina pig antiserum to infectious bronchitis virus 50,000 a <20 <20 <20 <20 7-3 <20 <20 7-4 <20 20 7-1 <20 <20 7-2 <20 <20 al-qassem 9 (racing) 9-a3 20 <20 9-a4 <20 40 9-a5 <20 <20 9-b1 <20 <20 9-c1 20 20 9-c2 80 640 9-e1 <20 <20 9-e2 <20 <20 qateef 10 (farming) 10-13 <20 80 10-14 <20 20 10-15 <20 80 10-16 20 160 11 (farming) 11-8 80 40 11-9 <20 320 11-11 <20 <20 11-12 <20 <20 11-13 <20 80 (continues) the extent of the serological cross-reactivity of mers-cov, ecov and bcov was systematically investigated. we did not have an isolate of hku23 virus available for serological testing and bcov was used as a virus that is genetically closely related to hku23 with pairwise amino acid similarity of 94.1% in the spike protein gene, the determinant of antigenic cross-reaction in neutralization tests (woo et al., 2014) . horse sera obtained from the usa reported to be strongly reactive by elisa (optical density ranging from 1.458 to 1.939) for ecov showed high titre (>1,280) for ecov in mn assay as expected, but also had high antibody titres (ranging from 160 to 1,280) in bcov mn tests (table 5) . as these horse sera were from horses in the "field," they may well have been exposed to multiple coronaviruses. thus, these results may reflect cross-reactivity or coinfection of us horses with ecov-and bcov-like viruses. two of these sera showed low titres (20) in the mers-cov ppnt assay but were negative in the more stringent mers-cov mn assay. as mers-cov is not circulating in the usa, these results suggest crossreactivity between mers-cov and ecov or related covs may occur, albeit at low titre. similarly, a bcov immune serum from an experimentally infected gnotobiotic calf showed detectable, but 16-fold reduced antibody titre with ecov but no cross-reaction with mers-cov. spike proteins of equine coronavirus and bovine coronavirus have an overall amino acid similarity of 80.8% and 73.1% similarity of the s1 region which contains the receptor-binding domain and neutralization epitopes. this probably explains the large difference in neutralization titres observed in table 5 . a dromedary serum from kazakhstan, a region known to be free of mers-cov activity (miguel et al., 2016) , had detectable antibody to bcov (probably reflecting hku23 or bcov infection) and also to ecov. we cannot conclude whether this reflects cross-reactivity or infection with bcov, hku23 and/or ecov. none of the beta coronavirus immune sera to sars-cov or mouse hepatitis virus, nor immune sera to alpha or gamma coronaviruses gave cross-reactions in bcov or ecov mn assays (table 5) . as had been previously reported, none of these dromedary sera cross-reacted with mers-cov (hemida et al., 2014) . fifty-four horse sera from saudi arabia and oman were selected to represent different collection locations, excluding those five sera previously reported to be mers-cov ppnt positive (table 6 ). these sera were seronegative for mers-cov (as expected); 40 (74%) of them had detectable mn antibody to ecov (titres ranging from <20 to 640; median 40), and 18 (33.3%) had mn antibodies to bcov (titres ranging from <20 to 160; median <20). the reciprocal geometric mean antibody titres (assigning sera with titres <20 the value of 10 for computational purposes) for ecov and bcov were 44 and 25.5, respectively. there were twenty-four sera with undetectable antibody to bcov with detectable ecov antibody titres ranging from 20 to 640. the titres to ecov were higher than or equal to titres to bcov, with two exceptions where the titre to bcov (20) was marginally higher than that with ecov (table 6 ). these results are compatible with the rt-pcr detection of ecov in rectal swabs in horses and probably suggest that ecov or a closely related virus is commonly infecting horses in many regions of the arabian peninsula. two of the five horses in whom ecov rna was detected by rtqpcr had low ecov mn antibody titres (20) while the other three had titres ranging from 160 to 640 (table 6 ). this may reflect virus shedding persists into convalescence or that re-infection of previously seropositive horses can occur. the rtqpcr detection of two hku23-like viruses indicates that hku23 which is common in dromedaries in the region is also infecting horses. in the absence of an hku23 isolate for use in neutralization tests and given the potential serological cross-reactivity between these three viruses, it is difficult to conclude how commonly equines are infected with hku23. the antibody titres to bcov, which is genetically closely related to hku23 in the spike protein gene, may suggest that bcov-or hku23-like virus infection of horses does occur in saudi arabia. mers-cov did not result in virus replication or seroconversion (adney et al., 2016) . in conclusion, the serological data suggest that lineage a betacoronaviruses are commonly infecting horses in saudi arabia and oman but antibody cross-reactions between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections. rt-pcr detection of ecov and hku23 in equine swabs confirms the circulation of these two viruses in horses in saudi arabia. cocirculation of related viruses may provide potential for recombination, a potential means of generating genetic diversity and facilitating host jumps in coronaviruses. this is the first report of hku23 being detected in horses and the first detection of ecov in asia outside of japan. inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding middle east respiratory syndrome coronavirus antibody reactors among camels in dubai coronaviridae receptor variation and susceptibility to middle east respiratory syndrome 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supporting information tab for this article. how to cite this article key: cord-330364-ye02hwhy authors: semenza, jan c.; sewe, maquines odhiambo; lindgren, elisabet; brusin, sergio; aaslav, kaja kaasik; mollet, thomas; rocklöv, joacim title: systemic resilience to cross‐border infectious disease threat events in europe date: 2019-05-17 journal: transbound emerg dis doi: 10.1111/tbed.13211 sha: doc_id: 330364 cord_uid: ye02hwhy recurrent health emergencies threaten global health security. international health regulations (ihr) aim to prevent, detect and respond to such threats, through increase in national public health core capacities, but whether ihr core capacity implementation is necessary and sufficient has been contested. with a longitudinal study we relate changes in national ihr core capacities to changes in cross‐border infectious disease threat events (idte) between 2010 and 2016, collected through epidemic intelligence at the european centre for disease prevention and control (ecdc). by combining all ihr core capacities into one composite measure we found that a 10% increase in the mean of this composite ihr core capacity to be associated with a 19% decrease (p = 0.017) in the incidence of cross‐border idte in the eu. with respect to specific ihr core capacities, an individual increase in national legislation, policy & financing; coordination and communication with relevant sectors; surveillance; response; preparedness; risk communication; human resource capacity; or laboratory capacity was associated with a significant decrease in cross‐border idte incidence. in contrast, our analysis showed that ihr core capacities relating to point‐of‐entry, zoonotic events or food safety were not associated with idte in the eu. due to high internal correlations between core capacities, we conducted a principal component analysis which confirmed a 20% decrease in risk of idte for every 10% increase in the core capacity score (95% ci: 0.73, 0.88). globally (eu excluded), a 10% increase in the mean of all ihr core capacities combined was associated with a 14% decrease (p = 0.077) in cross‐border idte incidence. we provide quantitative evidence that improvements in ihr core capacities at country‐level are associated with fewer cross‐border idte in the eu, which may also hold true for other parts of the world. global health security has been undermined by infectious disease threat events (idte) such as severe acute respiratory syndrome (sars) during 2002 (sars) during -2003 (dzau, fuster, frazer, & snair, 2017; morens, folkers, & fauci, 2008; paules & fauci, 2017) . these idte have caused substantial human suffering, placed considerable pressure on government resources, and inflicted significant economic damage. in financial terms, the cost of potential pandemics can amount to us$60 billion per year (sands, mundaca-shah, & dzau, 2016; sands, turabi, saynisch, & dzau, 2016) . however, if mortality costs are also taken into account, the annual cost can be as high as us$490 billion (fan, summers, & jamison, 2016) . to prevent, protect against, control and provide a public health response to the international spread of disease, the world health organization (who) led efforts to update the international health regulations (ihr), and the updated regulations were adopted in 2005 and came into force in 2007 (gostin, debartolo, & friedman, 2015; world health organisation, 2005) . the aim was to prepare 'states parties' to be able to detect and respond to these threats more quickly and effectively. to prevent public health emergencies of international concern (pheic) that can be a threat to global health security, the ihr oblige all 'states parties' to establish ihr core capacities (table 1) to detect, assess, notify and report events, and to respond to public health risks and emergencies. however, the persistent occurrence of idte post ihr implementation has raised questions about the implementation, compliance, and enforcement of these measures (commission on a global health risk framework for the future, 2016; gostin et al., 2015; gostin, debartolo, & katz, 2017; hoffman, 2014; suthar, allen, cifuentes, dye, & nagata, 2018; world health organisation, 2015) help to identify deficiencies in ihr core capacities and ihr non-compliance, additional factors might be responsible for the emergence of idte (gostin et al., , 2017 . it is possible that ihr core capacities are necessary but not sufficient to prevent the spread, control or response to such threats. they might not comprehensively identify and mitigate all the underlying drivers and determinants of idte in an increasingly interconnected and interdependent world (glaesser, kester, paulose, alizadeh, & valentin, 2017; jones et al., 2008; morens et al., 2008; paules & fauci, 2017; semenza, rocklov, penttinen, & lindgren, 2016; weiss & mcmichael, 2004 in europe as a result of global resurgence, increasing mobility and low vaccine uptake, in part related to vaccine hesitancy (leong, 2018; massad, 2018) . in 2017, the chikungunya virus was introduced into france and italy by viraemic passengers and spread by aedes albopictus mosquitoes, in part due to favourable climatic conditions (lillepold, rocklov, liu-helmersson, sewe, & semenza, 2019; rezza, 2018; rocklöv et al., 2019; semenza & suk, 2018) . international donors invested us$0.88 billion in outbreak preparedness, response and management of cross-border externalities in 2013 (schaferhoff et al., 2015) and national governments have allocated substantial resources to ihr core capacity implementation. the ecdc is an eu agency with a mission to monitor, identify (early warning and assessment) and respond to serious cross-border health union, 2013) this is analogous to the world health organization (who) ihr, where countries are also committed to further build their capacities to detect, assess and notify, and report on public health emergencies of international concern. thus, the cross-border idte we analyse here lend themselves to an analysis of ihr core capacities. european centre for disease prevention and control conducts epidemic intelligence, a process of systematic collection and collation of information on threats from health from a variety of sources. cross-border idte are assessed and verified to ensure they correspond to real public health events (for examples of idte see semenza, rocklov, et al., 2016) ). the assessment is based on an analysis, using ihr and early warning and response system (ewrs) criteria and expert opinion (table s1 ). ecdc initiated data collection for epidemic intelligence in june 2005. we analysed cross-border idte that originated in one of the 28 eu member states (eu28) from 2010 to 2016. this time period included the migrant wave of 2015 (semenza, carrillo-santisteve, et al., 2016) . we included idte with a risk of introduction to or propagation between member states within the eu/eea and idte that may require timely and coordinated eu action to contain (table s1 ). we also analysed cross-border idte for other parts of the world that were recorded by ecdc epidemic intelligence, despite the low numbers of idte identified in those countries. we excluded travelassociated legionnaires' disease outbreaks not originating in the eu28 from the analysis due to changes in reporting during the study period. who has developed an analytical framework for monitoring the achievement of ihr core capacities (world health organisation, 2013) . it allows country data for each core capacity, poe and potential hazards to be analysed in detail (table 1) organisation, 2011). the main purpose of the framework is to enable countries to measure their current status and assess progress over time. although individual ihr core capacities do not necessarily carry the same weight in an assessment of capabilities, all attributes are given the same weight in the framework. the scores range from 0% to 100% and were available from 2010 to 2016 (world health organisation). the analysis also included potential hazard 1 (zoonotic events) and potential hazard 2 (food safety); however, potential hazard 3 (chemical events) and potential hazard 4 (radiation emergencies) were not included in this analysis as they do not relate directly to idte. we determined the incidence of cross-border idte per capita in different countries based on the annual number of idte in a country divided by the annual population of the country. we modelled the relative change in the incidence of cross-border idte that originated in one country of the eu28, with a panel study, using a longitudinal general estimation equation framework (gee) (hanley, negassa, edwardes, & forrester, 2003) with a poisson log-link using random effects by country of origin to adjust for unmeasured confounders. we used an exchangeable correlation structure of the observations within countries, not to make prior assumptions of the temporal covariance structure. initially, we performed univariate analysis of the association of each of the ihr core capacities to cross-border a total of 135 cross-border idte in the eu28 met the study inclusion criteria between 2010 and 2016 (tables 2; s1). over the study period, the composite measure of the ihr core capacities, which averages 11 capacities ( analysis of idte in other parts of the world (besides eu28) was constrained by few cross-border idte detected in these countries and reported to ecdc. nevertheless, a 10% increase in the mean of all ihr core capacities combined was associated with a 14% decrease (p = 0.077) in the incidence of cross-border idte in countries other than the eu28. due to sample size constraints, a regional analysis was not possible. the results for specific ihr core capacities for all non-eu countries combined is provided in the supporting information (table s3) . with respect to the association of a 10% increase in individual core capacities with the incidence of cross-border idte in the eu28, core capacity 1 (national legislation, policy and financing) was associated with a 10% decrease (95% ci: 0.84, 0.98) in the incidence of cross-border idte ( figure 2 ); core capacity 2 (coordination and figure 2) . a bivariate analysis adjusted for gdp per capita yielded essentially the same point estimates (table s4 ). the principal component analysis revealed that three components explained the majority of variability of the ihr core capacities (table s5) . however, only the first pca score was significantly related to idte with an estimated 20% decrease in risk for every 10% increase in the core capacity score (95% ci: 0.73, 0.88). the individual core capacity weights related to this component was in line with the univariate analysis by relating mainly to ihr core capacities and less so to hazards. the irr estimate from the first pca score was also very similar to the estimate from the average composite measure of the ihr core capacities (decrease 20% vs. 19%), and further indicated that the irr from the univariate analysis of ihr core capacities cannot be combined, due to the strong inter-core capacity correlations. & semenza, 2012; wolicki et al., 2016) . to this end, surveillance needs to be flexible and sensitive and to encompass syndromic, laboratory-based, population-based and sentinel systems (wolicki et al., 2016) . in our analysis, national surveillance was associated with fewer idte, presumably because they were intercepted prior to international spread. response (core capacity 4) was highly significant in our analysis. systemic resilience to idte entails management and coordination of operations to rapidly respond to epidemic events that could develop into public health emergencies of national or international concern. it also includes active case management, infection control and decontamination, the importance of which were demonstrated during the mers-cov and ebola outbreaks in 2012 and 2014, respectively (siedner, gostin, cranmer, & kraemer, 2015; zaki, boheemen, bestebroer, osterhaus, & fouchier, 2012) . in our analysis, preparedness (core capacity 5) was also prosharing of infectious agents must occur through national or collaborating centres (gostin et al., 2017 to the contrary, vaccination coverage for example, has declined in certain countries (e.g. italy). it is important to bear in mind that this longitudinal study has a much stronger plausibility of an inference of a causal association than a simple cross-sectional study. we relate a change in ihr core capacities to a change in idte over time. thus, due to this temporal association, the causal inference is high, but nevertheless potentially subject to biases. another potential bias is reporting bias due to the self-assessment of ihr core capacities (gostin et al., 2017) which could have shifted our results to the null. selective reporting could also have contributed to the high inter-core capacity correlations, which decreases the granularity of our results. to overcome this lack of objective metrics, who has introduced a joint external evaluation (jee) as part of the ihr monitoring and evaluation framework (bell et al., 2017; world health organisation, 2019a ). this is a voluntary, multi-sectoral, peer-to-peer process with external experts to assess country capacity to prevent, detect and rapidly respond to public health risks. such an assessment is likely to be more objective than a self-assessment of ihr core capacities. as of april 2019, 82 countries had conducted a jee, but only five of these countries were eu member states (belgium, finland, latvia, lithuania and slovenia) (world health organisation, 2019b). once all eu28 countries have completed a jee, analysis of the association with idte will need to be revisited. we would like to thank dr. piotr kramarz (ecdc) and two anonymous reviewers for critical feedback on our manuscript. the views and opinions expressed herein are the authors' own and do not necessarily state or reflect those of ecdc. ecdc is not responsible for the data and information collation and analysis and cannot be held liable for conclusions or opinions drawn. no conflict of interest. jcs conceived the study, developed the study design, led the data analysis and data interpretation, and wrote the manuscript. mos conducted the analysis and contributed to the writing. el contributed to the writing. sb, kka and tm collected epidemic intelligence data. jr led the data analysis and contributed to the writing of the manuscript. all authors reviewed and approved the final manuscript. jan c. semenza https://orcid.org/0000-0002-4625-874x the neglected dimension of global security -a framework to counter infectious disease crises available from: https ://www.nap.edu/catal og/21891/ the-negle cted-dimen sion-of-global-secur ity-a-frame work-to-counter. investing in global health for our future the iclusive cost of pandemic influenza risk ebola in west africa-cdc's role in epidemic detection, 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evaluation framework ihr monitoring & evaluation isolation of a novel coronavirus from a man with pneumonia in saudi arabia additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article key: cord-279794-hn5vmic0 authors: guo, jiahui; fang, liurong; ye, xu; chen, jiyao; xu, shangen; zhu, xinyu; miao, yimin; wang, dang; xiao, shaobo title: evolutionary and genotypic analyses of global porcine epidemic diarrhea virus strains date: 2018-08-27 journal: transbound emerg dis doi: 10.1111/tbed.12991 sha: doc_id: 279794 cord_uid: hn5vmic0 porcine epidemic diarrhea virus (pedv), which re‐emerged in china in october 2010, has spread rapidly worldwide. detailed analyses of the complete genomes of different pedv strains are essential to understand the relationships among re‐emerging and historic strains worldwide. here, we analysed the complete genomes of 409 strains from different countries, which were classified into five subgroup strains (i.e., gi‐a, gi‐b, gii‐a, gii‐b, and gii‐c). phylogenetic study of different genes in the pedv strains revealed that the newly discovered subgroup gii‐c exhibited inconsistent topologies between the spike gene and other genes. furthermore, recombination analysis indicated that gii‐c viruses evolved from a recombinant virus that acquired the 5′ part of the spike gene from the gi‐a subgroup and the remaining genomic regions from the gii‐a subgroup. molecular clock analysis showed that divergence of the gii‐c subgroup spike gene occurred in april 2010, suggesting that the subgroup originated from recombination events before the pedv re‐emergence outbreaks. interestingly, ascaris suum, a large roundworm occurring in pigs, was found to be an unusual pedv host, providing potential support for cross‐host transmission. this study has significant implications for understanding ongoing global pedv outbreaks and will guide future efforts to develop effective preventative measures against pedv. porcine epidemic diarrhoea (ped) is a devastating enteric disease in pigs that results in severe diarrhoea, vomiting, and dehydration, with very high mortality observed in suckling pigs (pensaert & de bouck, 1978) . porcine epidemic diarrhea virus (pedv), which is the causative agent of ped, belongs to the genus alphacoronavirus within the family coronaviridae and is an enveloped single-stranded positivesense rna virus (woo et al., 2012) . the pedv genome consists of seven open reading frames (orfs) organized in the order orf1a, orf1b, spike (s) glycoprotein gene, orf3 hypothetical protein gene, envelope (e) gene, membrane (m) gene, and nucleocapsid (n) gene . among the proteins encoded by the orfs, the s glycoprotein is located on the envelope of the virus in the large surface projections of the virion and plays an important role in the attachment of viral particles to host cell receptors (lee, park, kim, & lee, 2010) . thus, the s gene is considered important for understanding the genetic relatedness and epidemiological status of pedv field isolates, as well as for advancing vaccine development (chen, liu, lang, et al., 2013) . the ped disease was first discovered in pig farms in belgium and the united kingdom in 1976 (pensaert & de bouck, 1978) , with reports of its occurrence in china as early as the 1980s (li et al., 2012) . with the emergence of new pedv strains, however, serious disease epidemics have been observed in china since october 2010 (sun et al., 2012) . beyond china, the disease has rapidly spread to more than 38 states in the usa following its first outbreak in may 2013, affecting more than 4,000 farms accounting for more than 7 million piglets (cima, 2014) . japan, canada, mexico, and colombia have also experienced successive outbreaks, with considerable economic losses to the global pig industry (lara-romero et al., 2018; ojkic et al., 2015; takahashi, okada, & ohshima, 1983; valko et al., 2017) . major global outbreaks since 2012 have renewed concerns about the potential changes in the mode of pedv transfer (chen, liu, lang, et al., 2013; li et al., 2012; sun et al., 2012) . although increasing evidence suggests that pedv routinely undergoes significant changes, especially in spike proteins (lara-romero et al., 2018; stott et al., 2017) , the prevalence and evolution of pedv strains is not well-defined and limited knowledge is known regarding the ways in which pedv subgroups circulate among themselves and how they might influence the evolution of pedv. to better understand the molecular epidemiology and genetic diversity of pedv field isolates, we investigated the genetic characterization, origin, and evolution of emergent pedv strains worldwide, which will provide much needed information for the effective prevention and control of this disease. the complete pedv genome was selected for genetic analysis. to clarify the evolution of pedvs, we obtained four complete genome sequences of pedv from our own lab (i.e., zl29, aj1102, hub1-2017, and hub7-2017) (bi, zeng, xiao, chen, & fang, 2012) firstly, we performed multiple sequence alignment of the 409 complete pedv genomes, as well as the orf1ab, s, orf3-e-m-n genes, and applied the bat coronavirus btcov/512/2005 (genbank accession no. dq648858) sequence as an outgroup (tang et al., 2006) . a maximum-likelihood (ml) phylogenetic tree was constructed using iq-tree v.1.6.5 (nguyen, schmidt, von haeseler, & minh, 2015) , with the best fitting evolutionary model suggested by the program following 1,000 bootstrap replicates. the phylogenetic tree was rooted against the pedv-related bat coronavirus, with removal of the long-branch leading to greater resolution of the viruses of interest. nucleotide and deduced amino acid sequences were aligned using mafft v.7.402 (katoh & standley, 2013) . the resulting tree was visualized using itol v.4 (interactive tree of life, http://itol.e mbl.de/). we preliminarily screened the pedv sequence data set for recombination using rdp, geneconv, chimaera, maxchi, and 3seq, followed by secondary scanning and recombination using bootscan and siscan in recombination detection program version.4.95 (rdp v.4 .95) (martin, murrell, golden, khoosal, & muhire, 2015) . sequences with significant signals for recombination determined by more than two methods were analysed in greater detail. nucleotide sequence similarity was assessed by simplot v.3.5.1 (lole et al., 1999) , with a sliding window size of 500 bp, step size of 100 nucleotides, and 1,000 bootstrap replicates, using gap-stripped alignments and the f84 (ml) distance model. all data were analysed using graphpad prism software (v.5.03, san diego, ca, usa). all s protein sequences from the pedv sample strains were analysed using the meta data-driven comparative analysis tool (meta-cats) (pickett et al., 2013) , with a p-value threshold of 0.05 (this threshold f i g u r e 1 genotyping and origin of the 409 pedv strains based on full-length genomic sequence analyses. (a) phylogram was tested by 1,000 bootstrap replicates, branch lengths were measured by the number of substitutions per site (see scale bars). names of strains, years, places of isolation, genbank accession numbers, genogroups, and subgroups are shown. (b) line chart shows the number of pedv sequences obtained by gene subgroup and year of sampling. yearly percentages of samples positive for pedv are indicated by different coloured lines respectively. data are indicated below sampling years. (c) subgroup distribution of all available complete or partial pedv genome sequences from countries reporting pedv infections (n.a., sequence not available). in the bar charts, counts are shown by country or region. data are indicated below bar charts [colour figure can be viewed at wileyonlinelibrary.com] was the maximum probability level for the likelihood that the position differed among groups simply by chance), to identify significantly different sites between the five subgroups. to characterize the genetic diversity of pedvs circulating globally, we constructed a phylogenetic tree using iq-tree based on the 409 complete pedv genomes (see materials and methods 2.2). consistent with our previous research (wang, fang, & xiao, 2016a) , the phylogenetic tree indicated that the complete pedv genomes evolved into two separate genogroups, gi (classical) and gii (variant), as presented in figure 1a . furthermore, genogroup gi evolved into two subgroups (gi-a and gi-b) and genogroup gii evolved into three subgroups (gii-a, gii-b, and gii-c). the gi-a subgroup mainly included the earlier pedv strains found in europe and belgium (virulent we identified the geographical and temporal distributions of the pedv strains to clarify the evolution of the virus. as shown in figure 1b , only sporadic outbreaks of pedv were reported before 2010, with the pathogens involved in these outbreaks found within the gi genogroup. the pedvs were primarily located in the gi-a (virulent cv777) and gi-b (attenuated dr13) subgroups and were predominantly from asia (figure 1b ). however, considerable pedv outbreaks were reported in asia and the united states after 2010, even for vaccinated piglets (lin, saif, marthaler, & wang, 2016) . based on our examination and assembly of public data, we identified that the pedv strains were primarily from the gii genogroup. interestingly, gii-b subgroup strains were reported predominantly in 2011, whereas gii-a strains were reported more prevalently after 2011 and occupied a larger proportion of strains. moreover, reports of the newly discovered gii-c strains showed a significant increase after 2012 due to further sequencing from europe. the above results indicate that the epidemic strains from different periods were from the five different subgroups. based on the geographical distribution of pedvs (figure 1c) , the gi genogroup (classical and cell culture-adapted vaccine strains) largely originated from the earlier pedv-threatened areas, such as china, south korea, and europe. the different subgroups from the gii genogroup also showed characteristic geographical distribution. while most gii-a subgroup strains were from the americas, a small number were from china and japan or from sporadic outbreaks in a few other isolated areas. the gii-b subgroup strains were mostly endemic to asia, especially china, south korea, and japan. the gii-c subgroup strains were primarily from europe, with some from the usa and china. our study showed that pedv strains from different subgroups were prevalent within the same areas, implying that the coincident "hot spots" in pedv-endemic areas (e.g., china and south korea, figure 1c critical for determining the sources of some pedv variations. these "hot spot" areas have the potential to be important reservoirs for the genetic variation of pedvs, resulting in recombination between different pedv subgroups. we also examined the potential hosts of the 409 pedv strains. results showed an unusual pedv strain (genbank accession no. kx883635) hosted by ascaris suum (supporting information table s1 ) (shi et al., 2016) , a large roundworm in pigs, thus providing novel insight into the possible epidemiology of pedv infection. indeed, parasites have long been regarded as a harmful factor to the pig industry as sources for a variety of infectious agents (jesudoss chelladurai et al., 2017) . for example, metastrongylus larvae are considered a reservoir for various porcine viral pathogens, primarily swine fever virus and swine flu virus (sen, kelley, underdahl, & young, 1961) . however, whether ascaris suum plays a critical role as a pedv reservoir requires further investigation. to further explore the evolution of pedvs, we constructed three phylogenetic trees based on the orf1ab, s, and orf3-e-m-n gene sequences of the 409 pedv strains. the orf1ab and orf3-e-m-n gene alignments confirmed that the gii-c subgroup was deeply nested within the gii-a subgroup (figure 3a and b) . strikingly, the phylogeny of the s gene suggested an entirely different evolutionary history for the gii-c subgroup compared to the other subgroups (figure 3c ). all gii-c subgroup strains showed inconsistent topology in the s gene phylogenetic tree, differing from the orf1ab gene phylogenetic tree. this inconsistent topology, in which outlier sequences were found between two well-defined subgroups in a phylogenetic tree, was attributed to (95%-99% genetic identity to the orf1ab and orf3-e-m-n genes). in contrast, the genetic identity between the s gene sequences of these viruses was only 85% and showed strong similarity with virulent cv777 (95% gene identity). in the s gene phylogeny, virulent dr13 did not cluster with kupe21 but instead with virulent cv777, suggesting that the pedv s gene was subjected to relatively frequent recombination, even between divergent subgroups (figure 3c and supporting information figure s2a ). moreover, the italy/7239/2009 pedv strain intragenogroup recombination provides a mechanism for amalgamation among these distinct subgroups and increases the genetic repertoire of co-circulating pedv strains. the s protein attaches to the cellular receptors of a host, resulting in virus entry by membrane fusion, and contains the domain that stimulates the production of neutralizing antibodies. variations in the s protein are important for understanding the genetic relatedness of pedv field strains (chang et al., 2002; lara-romero et al., 2018; wang et al., 2016b) . to determine the significant s protein sequences among the five different pedv subgroups, meta-cats analysis was performed for spike protein sequences of all 409 pedv strains. we identified 257 amino acid positions with significant variation among the isolates from the five subgroups (supporting information table s2 ). as shown in figure 4 , the gii genogroup contained 11 distinct patterns of aa mutations (i116t, i356t, e365q, t549s, g594s, n724s, a959v, s1044a, g1173d, s1232r, and r1298q), distinguishing it from isolates in the gi genogroup. although the gii-c subgroup s genes shared some aa substitutions with the gi-a and gii-a subgroups (two parents of recombination), they also exhibited three unique patterns (l76, a/s92, and h/t113), which clearly distinguished these isolates from those in the other subgroups, suggesting that the gii-c subgroup s gene evolved gradually through antigenic drift. it is well established that the s protein of coronaviruses induces high levels of neutralizing antibodies. four neutralizing epitopes (499-638, 748-755, 764-771 , and 1,368-1,374 amino acids) have also been identified in the pedv s protein (chang et al., 2002) . to explore whether the gii genogroup acquired substitutions in neutralizing epitopes characteristic of the gi genogroup, we mapped the significantly different positions to the equivalent positions in representative sequences (figure 4) . results identified seven substitutions in the neutralizing epitopes of the s protein in the gii genogroup (l521h, s523g, v527i, t549s, g594s, a605e, and l612f), which may explain why traditional inactivated vaccines and attenuated vaccines against the gi genogroup cannot effectively protect piglets threatened by a pandemic strain from the gii genogroup. in summary, this study revealed the genetic diversity and evolutionary dynamics of pedv strains. our genetic analyses showed that the pedv strains could be categorized into two groups, namely, gi (classical) and gii (variant) . we also discovered a new subgroup (gii-c) with novel genetic, molecular, and phylogenic characteristics. the gii-c subgroup evolved from a recombination event between the gi-a and gii-a subgroups, and we further found recombination in two relatively early strains: virulent dr13 and italy/7239/2009. these recombination events occurred prior to the re-emergence of pedv in 2010. additionally, to explore the potential link between s protein amino acid sequence variations and recombination, we performed a series of comparative analyses of the pedv s protein sequences. we found 10 positions that were localized in a well-known neutralizing epitope and revealed several unique amino acids that could easily distinguish the different subgroups. this study provides critical information to help trace the sources of pedv variants and identify the evolutionary mechanisms involved. furthermore, this research will hopefully facilitate the development of diagnostic kits, vaccines, and new therapeutic 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virosphere evolutionary and epidemiological analyses based on spike genes of porcine epidemic diarrhea virus circulating in thailand outbreak of porcine epidemic diarrhea in suckling piglets an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan. nihon juigaku zasshi prevalence and genetic diversity of coronaviruses in bats from china porcine epidemic diarrhoea virus with a recombinant s gene detected in hungary immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes g1 and g2 porcine epidemic diarrhea in china discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus key: cord-316006-t080mykk authors: kong, dechuan; wang, yuanping; lu, lu; wu, huanyu; ye, chuchu; wagner, abram l.; yang, jixing; zheng, yaxu; gong, xiaohuan; zhu, yiyi; jin, bihong; xiao, wenjia; mao, shenghua; jiang, chenyan; lin, sheng; han, ruobing; yu, xiao; cui, peng; fang, qiwen; lu, yihan; pan, hao title: clusters of 2019 coronavirus disease (covid‐19) cases in chinese tour groups date: 2020-07-27 journal: transbound emerg dis doi: 10.1111/tbed.13729 sha: doc_id: 316006 cord_uid: t080mykk international travel may facilitate the spread of the novel coronavirus disease (covid‐19). the study describes clusters of covid‐19 cases within chinese tour groups travelling in europe january 16–28. we compared characteristics of cases and non‐cases to determine transmission dynamics. the index case travelled from wuhan, china, to europe on 16 january 2020, and to shanghai, china, on 27 january 2020, within a tour group (group a). tour groups with the same outbound flight (group b) or the same tourism venue (group d) and all chinese passengers on the inbound flight (group c) were investigated. the outbreak involved 11 confirmed cases, 10 suspected cases and six tourists who remained healthy. group a, involving seven confirmed cases and six suspected cases, consisted of familial transmission followed by propagative transmission. there was less pathogenicity with propagative transmission than with familial transmission. disease was transmitted in shared outbound flights, shopping venues within europe and inbound flight back to china. the novel coronavirus caused clustered cases of covid‐19 in tour groups. when tourism and travel opens up, governments will need to improve screening at airports and consider increased surveillance of tour groups—particularly those with older tour members. a novel coronavirus (sars-cov-2) was discovered in mainland china in december 2019 (harapan et al., 2020; wu et al., 2020) . the world health organization (who) declared coronavirus disease a pandemic on 11 march 2020 (world health organization, 2020d . in many countries outside of china, the first cases were identified as tourists and students from the city of wuhan travelling abroad. similarly, the spread of sars-cov-2 throughout southwestern and central asia has been facilitated by iranian citizens and residents who were travelling outside of iran (tuite et al., 2020) . shown that there is person-to-person transmission among close contacts since the middle of december 2019 . in recent studies, clusters of covid-19 cases have been principally identified in family settings (bai et al., 2020; chan et al., 2020) . these patterns suggest that familial transmission-through living and eating together-is a key contributor to the spread of covid-19. how disease is spread outside of close contacts is less clear. other infectious diseases with airborne transmission have reported clusters of cases outside of family settings. in 2009, a cluster of influenza a/h1n1 cases occurred in a tour group in tour bus in sichuan province, china (han et al., 2009 ). in the current covid-19 pandemic, two cases were reported at a conference in germany (rothe et al., 2020) . another notable example has been the diamond princess cruise ship, which initially carried approximately 3,700 passengers and crew from more than 50 countries and regions. there have been more than 700 confirmed cases of covid-19 in the ship. the spread of disease thought to be attributable to inappropriate containment measures in the ship (world health organization, 2020a). year, or the spring festival, the most important holiday in china. during this time, chinese people often travel domestically and abroad. before wuhan was closed off, it is believed that a couple of million local people left the city for travel, which could have facilitated the spread of covid-19 to foreign countries. it is not as clear how well the infection is transmitted in tour groups outside of the familial setting. our study describes clusters of covid-19 cases within tour groups travelling in european countries from january 16 through 28. this study conducted a retrospective look at four cohorts of individuals. the index case was a 69-year-old chinese man who had travelled from the city of wuhan, china, to paris, france, on 16 january 2020 and back to shanghai, china, on 27 january 2020, within a tour group of totally 34 members (group a). during the travel, a total of seven confirmed cases and six suspected cases were documented within the tour group. group a were on three flights, af139 (wuhan-paris), af1004 (paris-rome) and af116 (paris-shanghai). we examined two other groups have epidemiological evidence, that is travelling or living in hubei province or communities with a confirmed case of covid-19, or having contact with a confirmed case of covid-19 or having contact with patients with fever and/or respiratory symptoms from hubei province or communities with confirmed case of covid-19 within 14 days. cases' clinical evidence could include fever and/or respiratory symptoms, pneumonia-related radiological finding, normal or reduced white blood cell count or reduced lymphocyte count. a suspected case was defined by at least one of epidemiological criterion combined with at least two clinical criteria or clinical evidence combined with zero epidemiological evidence. a confirmed case was defined by a positive rt-pcr test. based on their exposure history, we categorized cases as having familial or propagative (i.e. within tour group) transmission. we calculated the proportion of pathogenicity by group by counting the number of individuals with a sign or symptom of disease. the data that support the findings of this study are available on request from the corresponding author. the data are not publicly available due to privacy or ethical restrictions. the index case and his wife and parents-in-law lived in two separate apartments in a city 310 km away from wuhan, in hubei province. on january 10, they went to wuhan for a schengen visa application and then had a dinner taking wild eel (monopterus albus) at the aquatic food corner of huanan seafood wholesale market where initial cases were reported in the beginning of the covid-19 epidemic . it is likely that some of the family members were exposed to sars-cov-2. on january 16, the family joined the tour group a and boarded the flight af139. the index case's father-in-law (zero case), the index case, the index case's wife (case 2) and mother-in-law (case 1) successively had symptoms on january 22, 26, 27 and 28. the zero case developed severe symptoms and was hospitalized in paris on january 25, and was taken care of by his daughter (case 2). these two were labelled as the 4th and 5th cases in france. the index case and case 1 went back to china on january 27 and were immediately diagnosed as covid-19 confirmed cases. the timeline was listed in figure 2 . group a was placed under quarantine once they arrived back in shanghai, china. among the rest (n = 30) of the group, a total of nine members successively developed symptoms from january 28 through february 9 in quarantine, including three confirmed cases and six suspected cases. the cases and their family members/ accompanying friends are described in figure 2 . clinical examinations and virological testing results are presented in table 1 . pathogenicity in the group transmission (9/30) was lower compared with the familial transmission (3/3). similarly, pathogenicity seemed to be weakened as all of the family were confirmed cases (3/3), whereas the following cases in the group transmission included both confirmed (3/9) and suspected (6/9) cases. tour group transmission may have occurred in the following sceparis-shanghai, all of the confirmed and suspected cases had seats within two rows ( figure 3 ). furthermore, suspected cases had nearer seat proximity to the family cases, compared with the other three confirmed cases (case 3, 4, 5) which may be attributable to the suspected cases being less contagious. considering time sequence of onset and exposure/ contact history, we inferred that there was initially a familial transmission, followed by a propagative transmission within the group, which possibly occurred during the tour and on the inbound flight. in addition, we identified that five members had close contact with the cases, who remained healthy during the 12-day tour in europe and in 14-day quarantine in shanghai ( figure 2) . similarly, some members who had seats close to the confirmed cases remained healthy (figure 3 ). on af139, there was a cluster consisting of two covid-19 confirmed cases and one suspected case, which all belonged to tour group b (table 1) . they shared the same outbound flight from wuhan with group a and then had different itineraries in europe (figure 2 ). on board, the cases in group b had a seat proximity of >3 rows to the family in group a. at this point, the zero case did not have any symptoms. the onset of disease in the first case (case 12) in group b occurred on january 29, which was 7 days later than the zero case in group a. thus, we could not completely exclude the connection to group a. on af116, there were approximately 200 chinese passengers (group c; they shared the same flight but not belonged to a same tour group), in which two were diagnosed as suspected case in quarantine after arrival in shanghai (figure 2 ; table 1 ). case 15 was an independent traveller, and case 16 was a guide of a tour group of 19 members (the rest of the tour group have not developed symptoms). their seats were <3 rows away from the cases in group a. neither of them reported travel or living history in hubei province/ communities with confirmed case of covid-19; contact with confirmed case of covid-19; or contact with patients with fever and/ or respiratory symptoms from hubei province/ communities with confirmed case of covid-19, within 14 days before the onset. they might have a connection to group a. we identified another group of 41 tourists (group d), which had been visiting interlaken, switzerland, in the afternoon of january 22, sharing the same shopping venue with the group a. the group d was not placed under quarantine after they arrived in shanghai. later, two confirmed cases and one suspected case were successively diagnosed, which composed a cluster (figure 2 ; table 1 a large concern is if sars-cov-2 is able to efficiently spread in settings beyond the family or other sustained close contact. we reported three clusters of covid-19 confirmed cases in three tour groups travelling in european countries and one cluster of suspected cases on one flight in late january 2020. the outbreak in total involved 11 confirmed cases, 10 suspected cases and more than two hundred persons placed in quarantine. in group a, the cluster was initiated by a familial transmission, followed by spread of disease to 13 out of 34 members in the 12-day tour. group a was characterized by lower pathogenicity in the following group transmission, compared with the familial transmission which might be attributable to exposure to the huanan seafood wholesale market. in our study, the first case in group d might have been infected through a casual meeting with the zero case in group a at a shopping venue. a similar finding had been reported in a previous study that reports that chinese tourists wearing facial masks may have infected a thai taxi driver (pongpirul, pongpirul, ratnarathon, & prasithsirikul, 2020) . thus, there may be two conditions facilitating efficient transmission, (1) confined space and (2) consistent close contact. however, we found five tour group members who remained healthy even after sustained contact with the cases, such as sharing same hotel rooms, taking dinner together and having neighbouring seats. one explanation is that children are less susceptible to the virus (del rio & malani, 2020) . further studies on how the virus transmits across different age groups are urgently warranted. this is especially important considering the possible role of pre-symptomatic transmission . furthermore, we supposed that group c had a connection to group a and could not exclude a connection between groups b and a. these connections suggest potential transmission on the plane. recent studies have documented conflicting findings, including possible transmission on planes (liu, liao, chang, chou, & lin, 2020) or in cars (pongpirul et al., 2020) , but also no observed transmission in vehicles (phan et al., 2020) . previous studies have shown that planes have high-efficiency particulate air filters that may avoid airborne transmission of pathogens, whereas buses have about 70% recirculated inside air (han et al., 2009 ). we did not precisely examine the seats in tour bus. the confirmed and suspected cases had an obvious cluster of seats on the inbound flight, suggesting the infection was very likely to occur on plane, which may be associated with high affinity of sars-cov-2 spike glycoprotein binding ace2 (wrapp et al., 2020) . however, we also identified healthy members after taking seats close to the family cases on board. thus, we hypothesized that the zero case and case 2 were hospitalized in paris; thus, we did not obtain detailed clinical examination and virological testing results. b case 18 was kept in quarantine for 10 days after her arrival in china and then diagnosed another 9 days later, due to a stay-home notice sent by shanghai municipal center for disease control and prevention; thus, we did not obtain detailed clinical examination and virological testing results. disabling the efficient transmission of sars-cov-2 may depend on social distance and personal protection, in addition to air conditioning system and air filters. the tour groups included in our study had visited france, italy, switzerland, spain, portugal, germany and morocco, which has raised a public health concern of spreading the virus internationally. currently, china is the number 3 sender of tourists globally and is estimated to be 1 by 2030 (arcibal, 2018) . however, the entry-exit quarantine in airports has limited capability in detecting cases whose course of infection is in the incubation period. this problem of detection has been documented in the influenza a/h1n1 (priest, duncan, jennings, & baker, 2011) and current covid-19 pandemic (gostic, gomez, mummah, kucharski, & lloyd-smith, 2020) . the spread of sars-cov-2 into south korea, italy and iran has increased attention towards those tourists (gostic et al., 2020; pongpirul et al., 2020; tuite et al., 2020) . however, it is possible that the typical activities undertaken by tourists make them less likely to infect compared with other members of the tour group. the zero case in group a was diagnosed in france. however, we did not identify any local cases epidemiologically linked to the tour group members in the above-mentioned countries. this lack of transmission may be attributed to the absence of sustained contact in a confined space between local people and chinese tourists. there of course could be existing cases with mild or no obvious symptoms. since february 22, covid-19 cases have increased dramatically in italy, iran and south korea, which have been retrospectively traced to some locals who reported no known exposure history (world health organization, 2020b, 2020c). they might have been exposed to asymptomatic carriers of sars-cov-2. thus, improvement in the continuous surveillance on international tourists remains crucial. the study has several limitations. the sensitivity of the pcr assays could be low and result in a large number of false-negative results in the diagnosis of covid-19 (sheridan, 2020; wang et al., 2020) . all of the suspected cases reported in this study were finally excluded from the diagnosis of covid-19 due to a negative rt-pcr test, though some of them had pulmonary inflammation according to chest ct examination. because our diagnostic methods may have underestimated the incidence of disease in the study population, we combined suspected cases with confirmed cases to explore potential transmission dynamics. another limitation was recall bias and concealment in the investigation. we checked the activities with each case, cross-checked activities between cases, illustrated the transmission chains and then determined the potential connections between cases and groups. we also may have missed asymptomatic carriers on the passenger flights. in conclusion, we reported a cluster of 13 covid-19 cases, which was initiated by within-family transmission followed by propagative transmission into tour groups travelling in european countries. the study findings show that clustered cases in tour groups may be more propagative than simple familial transmission. currently, the covid-19 pandemic is spreading to increased numbers of countries and regions worldwide. if we consider each country as a group, such as china, south korea, iran and italy, we could understand the pandemic of the covid-19 as a propagative transmission within a 'group' and then between 'groups'. thus, the government should improve screening at airports and consider increased surveillance of tour groups-particularly those with older tour members. we thank all the investigators in the pudong new area, huangpu tion, analysis and interpretation of data; the writing of the manuscript; the decision to submit the manuscript for publication. all authors declare no competing interests. this study involved the use of existing, routinely collected data from a public health outbreak investigation, under the national health commission of the people's republic of china. all data included in the study were kept confidential without person identifiers. no additional interviews were conducted, and no data were collected independently for this study. thus, this study is exempt from ethical review and there was no need of obtaining informed consent. the data that support the findings of this study are available on request from the corresponding author. the data are not publicly available due to privacy or ethical restrictions. yihan lu https://orcid.org/0000-0003-4651-9433 hao pan https://orcid.org/0000-0002-7566-8158 mainland chinese will overtake americans to become largest group of international tourists by 2030, says euromonitor a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster 2019 novel coronavirus-important information for clinicians diagnostic and treatment protocol for novel coronavirus pneumonia (trial version 6 estimated effectiveness of symptom and risk screening to prevent the spread of covid-19 lack of airborne transmission during outbreak of pandemic (h1n1) 2009 among tour group members, china coronavirus disease 2019 (covid-19): a literature review pre-symptomatic transmission of novel coronavirus in community settings. influenza and other respiratory viruses early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a locally transmitted case of sars-cov-2 infection in taiwan importation and human-to-human transmission of a novel coronavirus in vietnam journey of a thai taxi driver and novel coronavirus thermal image scanning for influenza border screening: results of an airport screening study transmission of 2019-ncov infection from an asymptomatic contact in germany coronavirus and the race to distribute reliable diagnostics estimation of covid-2019 burden and potential for international dissemination of infection from iran clinical diagnosis of 8274 samples with 2019-novel coronavirus in coronavirus disease 2019 (covid-19): situation report -37 missi on-in-italy -to-suppo rt-covid -19-contr ol-and-preve ntion -efforts. world health organization (2020c). who director-general's opening remarks at the mission briefing on covid-19 who director-general's opening remarks at the mission briefing on covid-19-11 who director-general's remarks at the media briefing on 2019-ncov on 11 cryo-em structure of the 2019-ncov spike in the prefusion conformation a new coronavirus associated with human respiratory disease in china clusters of 2019 coronavirus disease (covid-19) cases in chinese tour groups key: cord-339871-jso21mbx authors: lee, sunhee; lee, changhee title: genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from south korea, 2017 date: 2018-05-16 journal: transbound emerg dis doi: 10.1111/tbed.12904 sha: doc_id: 339871 cord_uid: jso21mbx porcine epidemic diarrhoea virus (pedv) is a globally emerging and re‐emerging enteric coronavirus in pigs causing serious economic threats to the world swine industry. since the re‐emergence of massive pedv outbreaks in south korea in 2013−2014, domestic pig farms have continued to experience ped epidemics or endemics. this study represents the molecular characterization of pedv isolates identified in diarrhoeic animals collected across the country in 2017. initial sequencing analysis of the full‐length s genes revealed that 70% of the 2017 isolates (7/10) belong to the g2b subgroup, while the remaining isolates were classified as g1b. the data indicated that both variant g1b and global epidemic g2b strains were responsible for current ped outbreaks in south korea. the 2017 g1b and g2b isolates shared 98.7%–99.4% and 98.1%–99.2% amino acid sequence identity at the s gene level and 99.3% and 99.0%–99.6% nucleotide sequence homology at the genome level compared to the corresponding korean prototype g1b and g2b strains, respectively. in an interesting manner, one g2b‐like knu‐1705 strain was found to possess a large 39‐nucleotide deletion in the orf1a region theoretically encoding nonstructural protein 3. phylogenetic analysis based on the entire genome and spike protein sequences indicated that the 2017 isolates were most closely related to other global g1b or g2b strains but formed different branches within the same genogroup. these results indicate that pedvs undergo continuous evolution in the field. in addition, one 2017 pedv strain, kor/knu‐1705/2017, was successfully isolated and propagated in vero cells. the antisera raised against the korean prototype 2014 g2b strain efficiently neutralized knu‐1705 virus infection, suggesting antigenic homology between the 2014 and 2017 pedv strains. our data advance the understanding of the molecular epidemiology and antigenicity of pedv circulating in south korea. polyadenylated tail and consists of seven canonical coronaviral genes, including open reading frame (orf) 3, in the following conserved order: 5 0 untranslated region (utr)-orf1a-orf1b-s-orf3-e-m-n-3 0 utr (kocherhans, bridgen, ackermann, & tobler, 2001) . the first two large orfs, orf1a and 1b, encompass the 5 0 -proximal two-thirds of the genome and code for nonstructural proteins (nsps). orf1a translation yields a replicase polyprotein (pp) 1a, whereas orf1b is expressed by a à1 ribosomal frame shift that c-terminally extends pp1a into pp1ab. these pp1a and pp1ab are proteolytically matured by internal viral proteases to generate 16 processing end-products, named as nsp1-16. the remaining orfs in the 3 0 -proximal region of the genome encode four canonical structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n), as well as one accessory gene, orf3 (duarte et al., 1994; kocherhans et al., 2001; lai, perlman, & anderson, 2007; lee, 2015; saif et al., 2012) . porcine epidemic diarrhoea was first observed in feeder and fattening pigs in england in 1971 (oldham, 1972) and caused widespread epidemics in multiple swine-producing countries in europe during the 1970s (opriessnig, 2016) . a marked decrease in acute ped epizootics occurred in europe in the 1980s and 1990s, and only sporadic outbreaks have occurred in recent years (opriessnig, 2016) . in asia, ped was first reported in 1982, and unlike in europe, it has since posed a huge economic threat to the asian pork industry (chen et al., 2008; kweon et al., 1993; li et al., 2012; puranaveja et al., 2009; takahashi, okada, & ohshima, 1983) . in may 2013, ped outbreaks suddenly appeared in the united states and swiftly spread across the nation, as well as to adjacent countries. this outbreak caused the death of more than 8 million newborn piglets in the united states alone during a 1-year-epidemic period, leading to annual losses in the range of $900 million to $1.8 billion (langel, paim, lager, vlasova, & saif, 2016; mole, 2013; ojkic et al., 2015; stevenson et al., 2013; vlasova et al., 2014) . the us emergent strain-like viruses further reached east asian countries, resulting in nationwide ped disasters (lee, 2015; lin et al., 2014; maff, 2018) . during the 2013-2014 pandemics, ped rapidly swept across mainland south korea and jeju island, killing hundreds of thousands of piglets in domestic herds , 2017 . since then, ped epizootics or enzootics have regionally occurred through provinces in south korea with intensive swine industries. to investigate the diversity of pedvs responsible for the ongoing outbreaks in south korea, in this study, we determined the full-length sequences of the s proteins of field isolates and complete genome sequences of representative strains identified throughout 2017. in addition, we isolated and serially propagated a kor/knu-1705/2017 strain and assessed the antigenic cross-reactivity between 2014 and 2017 pedv field isolates. the small intestine or stool specimens were collected from piglets showing acute watery diarrhoea at various swine farms located in eight different provinces from march through december 2017. intestinal homogenates were prepared as 10% (wt/vol) suspensions in phosphate-buffered saline (pbs) using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of 15 s at a speed of 8,000 g. faecal samples were also diluted with pbs to 10% (wt/vol) suspensions. the suspensions were then vortexed and centrifuged for 10 min at 4,5009 g (hanil centrifuge fleta5, incheon, south korea). the clarified supernatants were initially subjected to rt-pcr using a tge/ped detection kit (intron biotechnology, seongnam, south korea) according to the manufacturer's instructions. pedv-positive samples were filtered through a 0.22-lm-poresize syringe filter (millipore, billerica, ma) and stored at à80°c until subsequent sequencing analysis and virus isolation. the s glycoprotein gene sequences of the virus isolates were determined by the traditional sanger method. two overlapping cdna fragments spanning the entire s gene of each isolate were amplified by rt-pcr as described previously (lee, park, kim, & lee, 2010) . the individual cdna amplicons were gel-purified, cloned into a pgem-t easy vector system (promega, madison, wi) and sequenced in both directions using two commercial vector-specific t7 and sp6 primers and gene-specific primers. the full-length s sequences of 10 pedv, designated knu-1701 to -1710, were deposited in the gen-bank database under the accession numbers shown in figure 1a . in addition, the complete genomes of representative pedv field strains were sequenced by the traditional sanger method. ten overlapping cdna fragments spanning the entire genome of each virus strain were rt-pcr-amplified as described previously lee et al., 2015 , and each pcr product was sequenced as described above. the 5 0 and 3 0 ends of the genomes of individual isolates were determined by rapid amplification of cdna ends (race) as described previously . general procedures for dna manipulation and cloning were performed according to standard procedures (sambrook & russell, 2001) . the complete genomic sequences of the 2017 viruses were deposited in the gen-bank database under the accession numbers shown in figure 1b. the sequences of the 48 fully sequenced s genes and 31 complete genomes of global pedv isolates were independently used in sequence alignments and phylogenetic analyses. multiple sequence alignments were generated with the clustalx 2.0 program (thompson, gibson, plewniak, jeanmougin, & higgins, 1997) , and the percentages of nucleotide sequence divergences were further assessed using the same software program. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbour-joining method and subsequently subjected to bootstrap analysis with 1,000 replicates to determine the percentage reliability values of each internal node of the tree (saitou & nei, 1987 pedv isolation was conducted from faecal suspensions on vero cells in the presence of trypsin (usb, cleveland, oh) as described previously . virus isolation was confirmed by cytopathic effect (cpe) observation, immunofluorescence assay (ifa) and nucleotide sequencing as described previously . the isolated pedv strain was propagated for serial passages in vero cells, and virus titres were determined as described previously . the cross-reactivity of antisera collected from sows inoculated with a korean pandemic g2b strain knu-141112 isolated in 2014 (baek et al., 2016) was evaluated by a serum neutralization (sn) test in 96-well microtiter plates against the past 2014 and present 2017 isolates as previously described oh, lee, choi, & lee, 2014) . the neutralization titre was calculated as the reciprocal of the highest dilution of serum that inhibited virus-specific cpe in all duplicate wells. the pedv s glycoprotein is a suitable viral gene for investigating genetic relatedness among isolates and the molecular epidemiology of pedv (chen et al., 2014; gerber et al., 2014; lee, 2015; lee et al., 2010; oh et al., 2014) . based on the s gene sequences, therefore, pedv can be genetically separated into two genogroup clusters, genogroup 1 (g1, classical and recombinant: low-pathogenic) and genogroup 2 (g2, field epizootic or panzootic: high-pathogenic), which are further divided into subgroups 1a and f i g u r e 1 phylogenetic analysis based on nucleotide sequences of the spike genes (a) and full-length genomes (b) of porcine epidemic diarrhoea virus strains. a region of the spike protein and complete sequence of tgev were included as an outgroup in each tree. multiple sequence alignments were performed using the clustalx program, and the phylogenetic tree was constructed from the aligned nucleotide sequences using the neighbour-joining method. numbers at each branch represent bootstrap values greater than 50% of 1,000 replicates. names of the strains, countries, years of isolation, genbank accession numbers, and genogroups and subgroups proposed in this study are shown. the pedv isolates identified in this study are indicated by solid circles. scale bars indicate nucleotide substitutions per site lee and lee | 951 1b as well as 2a and 2b (lee, 2015; figure s1 ). in an interesting manner, the g1b virus s genes were well-conserved, sharing 99.0%-99.7% aa identity with each other, whereas the 2017 g2b s genes were relatively variable, exhibiting 97.1%-99.8% aa homology with each other (table 1) (table s1 ). the number of nt/aa differences and percent identity shared between the 2017 isolates and genogroup representative strains is summarized in table s2 . to establish the genetic relationships involved, phylogenetic analyses were carried out using the nucleotide sequences of the s gene and full-length genome of the 2017 isolates, which were determined in this study and are available from genbank (figure 1) . consistent with previous studies (lee, 2015; lee et al., 2015) , phylogenetic analysis based on the pedv s genes revealed clear separation among the g1a, g1b, g2a and g2b subgroups. all g2b strains identified in 2017 were grouped within the g2b clade; however, they were in different branches from the emergent us strains and past re-emergent korean field isolates (figure 1a) . the 2017 g1b isolates were most closely clustered together, forming an independent branch within the g1b subgroup. furthermore, a phylogenetic tree subsequently reconstructed from the complete genome showed the same grouping structure as the s gene-based tree (figure 1b) . as shown previously (lee, 2015; lee et al., 2015) , the entire genome-based phylogenetic tree revealed that the g1b strains including knu-1702 were grouped within the g2 clade because of the similarity between the g1b and g2b genomes, except for the nthe percent nucleotide identity was shown in the upper right and the percent amino acid identity was presented in the lower left. lee and lee | 953 which were less than 1-log 2 lower but not significantly different compared to those against knu-141112. taken together, our data indicate that the antisera cross-reacted well between the homologous g2b field isolates, suggesting antigenic similarity between the 2014 and 2017 pedv strains. pedv has emerged or re-emerged as one of the deadliest and most contagious viral pathogens in swine, leading to large financial losses in the global swine industry. along with strict biosecurity, vaccination is a fundamental tool for managing and eradicating pedv during epidemic or endemic outbreaks. although g1a-based vaccines against pedv were developed and used to combat this disease in south korea over the past decade, their efficacy in the field, as well as the advantages and disadvantages of their use, is continuously debated. furthermore, a growing body of evidence suggests that their incomplete effectiveness may result from antigenic, genetic (>10% aa variation between respective s proteins) and phylogenetic (g1 versus g2) differences between vaccine and field epidemic strains (lee et al., 2010; oh et al., 2014; kim et al., 2015; lee et al., 2015; lee, 2015) . the advent of the 2013-2014 pedv pandemic led to a breakthrough in the development of g2bbased vaccines phenotypically and genotypically homologous to field strains responsible for global ped epidemics, and these g2b vaccines are currently applied to prevent pedv in south korea. another important policy for controlling pedv is to operate a monitoring and surveillance system (moss) to monitor genetic diversity among field isolates and surveil the emergence of novel variants in the field, which will contribute to preventing future outbreaks. to provide insight into the understanding of the current epidemiological status of pedv in south korea, the present study aimed to investigate the genetic, phylogenetic and antigenic characteristics of pedvs responsible for regional outbreaks in south korea in 2017. nucleotide sequencing analysis revealed that two different pedv genotypes, low-pathogenic g1b and high-pathogenic g2b, caused regional outbreaks in south korea, with the latter genotype more 1% nucleotide sequence variations at the genome level with the 2013-2014 pandemic strains. however, field g2b isolates with nearly 2% amino acid sequence divergence compared to previous g2b strains at the s gene level were identified in the present study. furthermore, mutations within the s protein were randomly and extensively distributed in the s1 and s2 regions among the 2017 isolates ( figure s1 ). replication. however, this unique del is in the glu-rich acidic region, which does not affect the authentic roles of nsp3 and thus is nonessential for coronavirus replication (lei, kusov, & hilgenfeld, 2018) . although the virus can tolerate the large nsp3-del which is dispensable for pedv replication as shown in figure conditions. therefore, the timeline of this situation is unclear and it is unknown whether antigenic differences among pedv epidemic strains will contribute to the failure of current g2b vaccines. to counteract the prospective scenario, further studies are critical for securing culturable pedv epidemic strains that are genetically, phenotypically and antigenically characterized in the laboratory. in summary, genetic and phylogenetic analyses indicated that the 2017 epidemic-related isolates are closely related with corresponding global g1b or g2b strains identified in previous outbreaks and that the virus continues to evolve in its host environment. despite their genetic diversity, antigenicity currently seems to remain unchanged among g2b strains, indirectly confirming the efficacy of g2b-based vaccines against homologous g2b pedvs responsible for current epidemics. because the virus is assumed to undergo an evolutionary process to accumulate mutations to ensure viral fitness in the field, new genotypes or variants of pedv, against which the current g2b vaccine may provide partial protection, will eventually emerge. furthermore, this circumstance may advent earlier than expected if pedv outbreaks fade from our attention following sporadic or endemic outbreaks without serious economic problems. therefore, it is important to execute mandatory notification of ped-like outbreaks essentially followed by activating an moss, including early diagnosis, to survey forthcoming pedv strains that may emerge locally or globally through genetic drift (e.g., nonsilent point mutations) or genetic shift (e.g., recombination events) and obtain and characterize epidemic field isolates to predict and prepare for future epizootics or panzootics. we would like to acknowledge the swine veterinarians and choon the authors declare that they have no conflict of interest. lee http://orcid.org/0000-0002-5930-5461 efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets nidovirales: a new order comprising coronaviridae and arteriviridae isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf detection of antibodies against porcine 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swine the neighbor-joining method: a new method for reconstructing phylogenetic trees molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan mega4: molecular evolutionary genetics analysis (mega) software version 4.0 the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools distinct characteristics and complex evolution of pedv strains additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article: lee s, lee c. genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from south korea key: cord-253450-k7p510p4 authors: keha, abi; xue, luo; yan, shen; yue, hua; tang, cheng title: prevalence of a novel bovine coronavirus strain with a recombinant hemagglutinin/esterase gene in dairy calves in china date: 2019-05-31 journal: transbound emerg dis doi: 10.1111/tbed.13228 sha: doc_id: 253450 cord_uid: k7p510p4 bovine coronavirus (bcov) is the causative agent of diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract illnesses in cattle across the world. in this study, a total of 190 faecal samples from dairy calves with diarrhoea were collected from 14 farms in six chinese provinces, and bcov was detected in 18.95% (36/190) of the samples by reverse transcriptase polymerase chain reaction. full‐length spike, hemagglutinin/esterase (he), nucleocapsid and transmembrane genes were simultaneously cloned from 13 clinical samples (eight farms in four provinces), and most of the bcov strains showed a unique evolutionary pattern based on the phylogenetic analysis of these genes. interesting, 10 of the 13 strains were identified as he recombinant strains, and these strains had experienced the same recombination event and carried the same recombination sites located between the esterase and lectin domain. they also shared an identical aa variant (f181v) in the r2‐loop. moreover, 9/10 strains displayed another identical aa variant (p, s158a) in the adjacent r1‐loop of the he gene, which differs from the other available bcov he sequences in the genbank database. our results showed that bcov is widely circulating in dairy cattle in china, contributing to the diagnosis and control of dairy calves diarrhoea. furthermore, a bcov strain that carries a recombinant he gene has spread in dairy calves in china. to the best of our knowledge, this is the first description of an he recombination event occurring in bcov; this is also the first description of the molecular prevalence of bcov in china. our findings will enhance current understanding about the genetic evolution of bcov. bovine coronavirus possesses five major structural proteins: the spike (s), hemagglutinin/esterase (he), nucleocapsid (n), transmembrane (m) and the small membrane (e) (lai & cavanagh, 1997) . the s protein is involved in receptor recognition and carries distinct functional domains near its amino (s1) and carboxy (s2) termini, while the n-terminal s1 domains recognize sugar receptors, and the s2 subunit is a transmembrane protein that mediates viral and cellular membrane fusion during cell invasion (fang li, 2016) . s1 and s2 contain several antigenic domains, but s1 appears to be the most efficient at inducing antibodies with high neutralizing activities in its host (yoo & deregt, 2001) . the he protein contains two important functional domains: the lectin domain and the esterase domain. the lectin domain recognizes sugar receptors in the cell, whereas the esterase domain possesses a receptor-destroying enzyme activity capable of removing cellular receptors from the surfaces of the targeted cells. the receptor-binding (lectin) and receptor-destroying (esterase) domains may be important for virus entry (kienzle, abraham, hogue, & brian, 1990; schultze, wahn, klenk, & herrler, 1991) . therefore, in addition to the s protein, the he protein serves as a second viral attachment protein for infection initiation (groot, 2006) . the primary role of bcov n protein is to package the viral genome into long, flexible, helical ribonucleoprotein (rnp) complexes, protect the genome and ensure its timely replication and reliable transmission, as well as playing a role in viral transcription and translation (hurst, ye, goebel, jayaraman, & masters, 2010) . in contrast, the m protein plays a crucial role in bcov assembly (oostra, haan, groot, & rottier, 2006) . the high genetic diversity in coronaviruses is attributable to the high mutation rates associated with rna replication, the high recombination frequencies within the coronavirus family and the large coronavirus genomes (woo, lau, huang, & yuen, 2009 ). recombination in coronaviruses plays an important role in virus evolution and can result in the emergence of new pathotypes (menachery, graham, & baric, 2017; wang et al., 2015) as well as changing the host ranges and ecological niches (bakkers et al., 2017) . thus far, recombination regions in coronaviruses have been extensively reported for the s gene (kin et al., 2015; lau et al., 2011; minami et al., 2016) , a finding also applicable to bcov (martínez et al., 2012) . recombination events in m (herrewegh, smeenk, horzinek, rottier, & groot, 1998 ), n (kin et al., 2015 , rp3 (lau et al., 2010) and the orf1 gene (chen et al., 2017; kin et al., 2015) have also been reported. however, to date, recombination events in he have only been reported in mhv, a betacoronavirus, and this situation may act as a strong force for generating strains with new genotypes, host spectra and tissue tropisms (groot, 2006; luytjes, bredenbeek, noten, horzinek, & spaan, 1988; smits et al., 2005) . the presence of bcov has been confirmed in chinese dairy cows (genbank accession number fj556872), but the prevalence and molecular characteristics of bcov are still largely unknown. therefore, we sought to investigate the prevalence of bcov in dairy calves with diarrhoea in china. unexpectedly, our results reveal that a bcov containing a recombinant he gene has emerged and spread in dairy calves in china. a total of 190 faecal samples were collected from dairy calves (≤3 months of age) with obvious diarrhoea at 14 farms from six provinces in china during september 2017 and may 2018 (table 1) . the samples were shipped on ice and stored at −80°c. the faecal samples were fully resuspended in phosphate-buffered saline (1:5 w/v) and centrifuged at 10,000 × g for 10 min. viral rna was extracted from 300 μl of the faecal suspension using rnaios plus (takara bio inc) according to the manufacturer's instructions. the cdna was synthesized using the primescript™ rt reagent kit according to the manufacturer's instructions (takara bio inc.) and then stored at −20°c until required. bovine coronavirus nucleic acids in the faecal samples were identified using a pcr assay established in our laboratory that targets the bcov polymerase gene. after validating the specificity and stability of the assay, the detection limit for the viral nucleic acid in the assays was determined to be 1 × 10 −2 pg per μl-1. in detail, a primer pair (f: 5′-cgagttgaacaccc agat-3′, the complete s, he, n and m genes were pcr-amplified from samples already known to be bcov-positive based on rt-pcr assays previously reported (gélinas, boutin, sasseville, & dea, 2001; lau et al., 2011; martínez et al., 2012; park et al., 2006) . all pcr products were purified using the omega gel kit (omega) following the manufacturer's instructions, after which they were ligated to the pmd19-t vector (takara bio inc.) and transformed into dh5α competent escherichia coli cells (yeasen) for sequencing. the s and n gene sequences were assembled using seqmansoftware (version 7.0; dnastar inc). the homologies of the nt and deduced amino acid (aa) sequences were determined using the megalign program in dnastar 7.0 software (dnastar inc). mega 7.0 was used for multiple sequence alignment and to subsequently build the maximum-likelihood phylogenetic tree with bootstrap testing (1,000 replicates). recombination events were assessed using simplot software (version 3.5.1) and the recombination detection program rdp 4.0 (version 4.9.5) with the rdp, geneconv, chimaera, maxchi, bootscan, siscan and 3seq methods (martin, murrell, golden, khoosal, & muhire, 2015) . of the 190 faecal samples from the calves with diarrhoea, 36(18.95%) were found to be bcov-positive, which revealed that the virus was distributed in 13/14 farms across the six provinces (table 1) . full-length s, he, n and m genes were successfully cloned out of 13 positive samples from eight farms in four different chinese provinces (shanxi, two strains; henan, three strains; liaoning, five strains; and sichuan, three strains). the 13 s genes, at 4,092 -bp each, encode a protein of 1,363 aa, the cleavage site of which is located at aa 768 in all 13. sequence comparisons revealed that all 13 s genes share 98.6%-100% nt identity and 98.5%-100% aa identity with each other. they also share 96.8%-100% nt identity and 95.3%-100% aa identity with all 163 full-length bcov s genes available in the genbank database. a phylogenetic tree based on the complete s gene sequences using the maximum-likelihood method showed that 12 of the 13 s genes from this study together with 13 other bcov s genes from china (one strain from cattle, genbank accession number ku886291; 12 strains from yaks, bos grunniens, submitted by our team, genbank accession number mh810151-mh810162) clustered on an independent large branch. the remaining s genes clustered with three north american bcov strains (genbank accession number mh043952, mh043954 and mh043955) on a small independent branch of the tree ( figure 1 ). compared with the other bcov s genes, 9/13 sequences from this study and the 13 other chinese bcov sequences motioned above, which were located in the independent large branch, each had an identical aa variant (n1192y) in the s2 subunit. additionally, 4/13 sequences from this study and the above-mentioned 12 sequences from chinese yaks, which are located in the large independent branch, have an identical aa variant (e121v) in the s1 subunit. compared with the bcov mebus prototype strain, these bcov s genes have a total of 13 aa changes in the s1 subunit and 3 aa changes in the s2 subunit ( figure 2 ). no frame shifts, deletions, insertions or recombination events were observed in the s gene sequences from all the strains in this study. all 13 he genes were 1,275 -bp long, and the protein they encode is 424 aa residues in length. fgds, the putative esterase active site in all he proteins, was located at aa positions 37-40, and nine n all of the 13 n genes were 1,347 -bp in length, each encoding a protein of 230 aa residues. sequence comparison of these genes revealed that they share 99.8%-100% nt sequence identity and 99.3% bovine coronavirus, an important pathogen of calves, is globally responsible for severe economic losses in farming (azizzadeh et al., 2012; bok et al., 2015; johnson & pendell, 2017) . in china, the prevalence of bcov is still largely unknown. therefore, in this study, we screened 190 diarrhoea faecal samples from calves, 36 of which were found to be bcov-positive, and the positive samples were distributed across 13 of the 14 farms we screened across six provinces in the major dairy cattle production areas of china. the results showed that bcov is circulating widely in these dairy cattle, a finding that should help with the diagnosis and control of diarrhoea in these animals. most of the strains from this study are unique in their evolutionary histories based on our analysis of the full-length s, he, n and m genes, a finding similar to that for bcov in korean (ko et al., 2006; park et al., 2010) . this may be the result of geographical, environmental and natural selection patterns (bidokhti et al., 2013; hasoksuz, sreevatsan, cho, hoet, & saif, 2002; martínez et al., 2012) . the bcov s protein is involved in receptor recognition, host specificity, antigenic diversity and immunogenicity (fang li, 2016) . its gene sequences are variable, and mutations in this protein are associated with alterations in viral antigenicity, viral pathogenicity, host range and tissue tropism (gallagher & buchmeier, 2001; peng et al., 2012) . in this study, compared with other bcov s genes, we found that nine out of 13 of our sequences and 13 chinese bcov sequences (one strain from cattle and 12 strains from yaks), which clustered on a large independent branch of the phylogenetic tree, each had an identical aa variant (n1192y) in the s2 subunit. as a transmembrane protein, the s2 subunit mediates the fusion of viral and cellular membranes (luo & weiss, 1998) ; hence, the biological significance of this variant warrants further investigation. in addition, four out of 13 sequences and 12 sequences from chinese yaks were found to have an identical aa variant (e121v) in the s1 receptor-binding region, compared with the other s genes. f i g u r e 2 amino acid variants of the 13 complete s genes in this study. the figures in the box indicate the identical amino acid change sites in all 13 strains in this study compared with the bcov prototype strain mebus s sequences; the figure marked with triangle was an unique aa variant in the four sequences in this study and 12 sequences from chinese yaks; the figure marked with circular was an unique aa variant in the nine sequences in this study and 13 sequences (12 from chinese yaks and one from chinese cattle); the figure marked with line was an unique aa variant in shanxi strains in this study; which compared with the other available bcov s sequences in the genbank database. hp, the first hydrophobic domain of the s2 subunit; hr-n and hr-c, the heptad repeats; s1a and s1b, the immune reactive domain; s1-ntd, receptor-binding domain; sp, signal peptide the s1 subunit in the n-terminal of bcov (aa 1-330) recognizes a sugar receptor (peng et al., 2012) , and aa substitutions in this region can change the receptor-binding capacity (li et al., 2005) and host receptor specificity (sheahan et al., 2008) . two bcov strains (genbank accession number mk095177 and mk095178) from shanxi province were found to have unique aa substitutions (e1051v, s1076p) in the heptad repeat region. this region is crucial for viral entry and for viral and host cell membrane interactions to occur, and it promotes lipid bilayer fusion and nucleocapsid release into the cytoplasm (forni et al., 2015) . thus, aa substitutions in this region may affect the interaction between the coiled-coil structure and the host cell receptor (martínez et al., 2012) . the he protein has a receptor-binding function, which also plays a critical were recovered from eight farms in four provinces across a wide geographical distance, with the two furthest provinces being more than 1,000 km apart. thus, these novel he recombinant strains have been circulating widely in dairy cattle in china. to the best of our knowledge, this is the first report of a recombination event in the he gene from bcov in cattle, a finding that augments current understanding about the evolution of bcov. in fact, mhv, which is another lineage a member of the betacoronavirus genus, has also reportedly undergone a non-homologous recombination event in the he gene (luytjes et al., 1988) . recombination in the he gene from influenza c virus and in toroviruses has also been observed (groot, 2006; smits et al., 2005) . recombination in the he gene may be a strong driving force for generating strains with new genotypes, host spectra and tissue tropisms (groot, 2006; luytjes et al., 1988; smits et al., 2005) . notably, in our study, the he recombinant and non-recombinant strains simultaneously existed on the same cattle farm in liaoning province. interestingly, the reported decrease in the he receptor-binding capacity of hcov-oc43 betacoronaviruses was thought to be caused furthermore, nine of the 10 strains have another identical aa variant (p, s158a) in the adjacent r1-loop of their he genes, which is an identical situation to that seen with the hcov-oc43 (bakkers et al., 2017) . thus, further investigation of the significance of the receptor-binding capacity caused by aa substitutions in the receptor-binding region of the he recombinant strains is warranted. notable, monoclonal antibodies against the bcov he protein efficiently neutralized bcov infectivity in vitro (deregt & babiuk, 1987) and protected the intestinal epithelium of cattle from virus infection in vivo (deregt et al., 1989) , indicating that the he protein of bcov may also play a significant role in the induction of protective effect on in conclusion, the results of our study have shown that bcovs are circulating widely in dairy calves in china and that most of these strains have unique evolutionary pattern based on our phylogenetic analysis of the complete s, he, n and m genes. recombination events between the esterase and lectin domain of he were identified as occurring at remarkably high frequencies, and these recombinant strains are widely prevalent in dairy cattle in china. as far as we f i g u r e 6 phylogenetic tree based on the deduced 448 aa sequences of the complete n gene. sequence alignments and clustering were performed by clustalw in mega 7.0 software. the tree was constructed by the maximum-likelihood method with bootstrap values calculated for 1,000 replicates. the strains in this study were marked with a circle, and the other chinese bcov strains were marked with a triangle are aware, this is the first description of a recombination event in the he gene of bcov, and our findings will enhance current understanding 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phylogenetics and coalescence analysis coronavirus diversity, phylogeny and interspecies jumping a single amino acid change within antigenic domain ii of the spike protein of bovine coronavirus confers resistance to virus neutralization prevalence of a novel bovine coronavirus strain with a recombinant hemagglutinin/esterase gene in dairy calves in china the authors declare that there is no conflict of interest. this study did not involve animal experiments besides the faecal sampling of diarrhoea dairy calves that visited farm for clinical treatment. https://orcid.org/0000-0002-8413-7260cheng tang https://orcid.org/0000-0003-2680-0519 key: cord-317455-6qx0v28w authors: brown, paul a.; courtillon, céline; weerts, erik a. w. s.; andraud, mathieu; allée, chantal; vendembeuche, anthony; amelot, michel; rose, nicolas; verheije, monique h.; eterradossi, nicolas title: transmission kinetics and histopathology induced by european turkey coronavirus during experimental infection of specific pathogen free turkeys date: 2018-09-10 journal: transbound emerg dis doi: 10.1111/tbed.13006 sha: doc_id: 317455 cord_uid: 6qx0v28w numerous viruses, mostly in mixed infections, have been associated worldwide with poult enteritis complex (pec). in 2008 a coronavirus (fr‐tcov 080385d) was isolated in france from turkey poults exhibiting clinical signs compatible with this syndrome. in the present study, the median infectious dose (id (50))(,) transmission kinetics and pathogenicity of fr‐tcov were investigated in 10‐day‐old spf turkeys. results revealed a titre of 10(4.88) id (50)/ml with 1 id (50)/ml being beyond the limit of genome detection using a well‐characterized qrt‐pcr for avian coronaviruses. horizontal transmission of the virus via the airborne route was not observed however, via the oro‐faecal route this proved to be extremely rapid (one infectious individual infecting another every 2.5 hr) and infectious virus was excreted for at least 6 weeks in several birds. histological examination of different zones of the intestinal tract of the fr‐tcov‐infected turkeys showed that the virus had a preference for the lower part of the intestinal tract with an abundance of viral antigen being present in epithelial cells of the ileum, caecum and bursa of fabricius. viral antigen was also detected in dendritic cells, monocytes and macrophages in these areas, which may indicate a potential for fr‐tcov to replicate in antigen‐presenting cells. together these results highlight the importance of good sanitary practices in turkey farms to avoid introducing minute amounts of virus that could suffice to initiate an outbreak, and the need to consider that infected individuals may still be infectious long after a clinical episode, to avoid virus dissemination through the movements of apparently recovered birds. infectious bronchitis virus is a highly contagious virus transmitted very quickly among naive birds in the field. it is responsible worldwide for respiratory diseases, egg drop with poor eggshell quality, reduced hatchability, nephritis and sometimes, in early infection of future breeders, genital atrophy responsible for the syndrome of "false laying" in chicken breeders or layers (jackwood, & wit, 2013) . turkey coronavirus, originally identified in the usa in the 1970s as one of the agents responsible for an acute enteritis named bluecomb (panigrahy, naqi, & hall, 1973; ritchie, deshmukh, larsen, & pomeroy, 1973) and since with a multifactorial disease known as poult enteritis complex of turkeys (pec) , has now been detected in most areas where turkeys are farmed cavanagh et al., 2001; dea & tijssen, 1988; domańska-blicharz, seroka, lisowska, tomczyk, & minta, 2010; martin, vinco, cordioli, & lavazza, 2002; maurel et al., 2009; teixeira et al., 2007) , although tcovs isolated in europe have been shown to have a different genetic lineage to those isolated in the usa (brown et al., 2016; maurel et al., 2011) . pec includes several intestinal disorders that occur in turkeys mostly within the first three weeks of life (guy, 2013) and its clinical signs often include diarrhea, stunting, anorexia, dehydration, weight loss, and immune dysfunction (atrophy of the thymus and the bursa of fabricius) that promotes secondary infections. the wide distribution of both ibv and tcov and their highly contagious nature have considerable economic repercussions. the contagious nature of a disease can be measured by the "reproduction number" (r0) defined as "the expected number of secondary cases produced by a single (typical) infection in a totally susceptible population" (masters & perlman, 2013) . the parameters necessary to calculate r0 are (a) the speed of transmission and (b) the shedding duration of the infectious viruses. generally, a virus with an r0 less than 1 will disappear quickly because an infected individual will have a low ability to infect another. a virus with an r0 greater than one will spread in the susceptible population. for ibv, an r0 of 19.95 has been estimated (de wit, de jong, pijpers, & verheijden, 1998) , which is a figure comparable to the r0 of highly contagious human viruses such as measles virus (r0 12-18) (masters & perlman, 2013) . for tcov, r0 has not yet been fully calculated; however, a study with an american tcov isolate demonstrated that infectious virus particles can be shed up to six weeks post-infection in experimentally infected turkeys . the current study focused on strain fr-tcov 080385d that was detected in france in 2008 in turkeys with clinical signs compatible with pec. fr-tcov is the only european tcov strain isolated to date, although coronaviruses have been detected in turkeys in poland, great britain and italy (cavanagh, 2001; domańska-blicharz et al., 2010; martin et al., 2002) . the aim of this study was to determine the transmission properties of the virus by evaluating its id 50 and reproduction number (r0) under experimental conditions in 10day-old spf turkeys, in order to better understand the diffusion of the disease. histopathological examination and in-situ detection of tcov antigen at the sites of replication in the intestinal tract were also performed. three animal experiments (exp 1, 2 and 3) were performed in agreement with the national regulations of the french ministry for higher education and research on animal welfare and after approval from the french agency for food, environmental and occupational health & safety's (anses) ethical committee. virus fr-tcov 080385d isolated from duodenal contents of 42-dayold turkeys affected by pec in november 2008 was propagated by inoculating embryonated spf turkey eggs (anses, ploufragan, france) via the intra-amniotic route, as previously described (guionie et al., 2013) . because fr-tcov 080385d does not induce clinical lesions in the embryo, the intestines of inoculated embryos were screened 4 days post-inoculation by qrt-pcr (maurel et al., 2011) , and the intestines of positive embryos were collected and pooled to prepare a virus stock (22). five-fold serial dilutions of this stock were inoculated into seven eggs per dilution, and a titre of 10 4.01 eid 50 /ml was calculated according to reed & muench (20) . one hundred microliters of intestinal or cloacal swab material was lysed with 300 μl of buffer rlt (qiagen, france) by mixing and incubating at room temperature for 15 min. rna was extracted using magattract rna tissue mini m48 kit or magattract virus mini m48 kit for biorobot m48 (qiagen, france) and eluted in 100 μl of buffer ave following the manufacturer's instructions. the presence of tcov genome was detected using a qrt-pcr specific for avian coronaviruses (maurel et al., 2011) . the limit of detection (lod) and the linear phase of this qrt-pcr were described as 2 log10 and from 3 to 9 log10 copies per microliter of extracted rna, respectively. in this study, samples were considered positive with a result higher than 2 log10 copies per microliter of extracted rna. all results are given as copy number (cp)/μl of extracted rna expressed in log10 together with the sd 2.4 | exp 1. titration of fr-tcov in 10-day-old spf thirty 10-day-old spf turkeys were separated in 5 groups of 6 birds, and housed for 3 days in negative pressure isolators allowing ad lib feeding and drinking. each isolator had a cardboard floor with a metal grid platform underneath and a surface area of 1.4 m 2 . groups 1, 2, 3 and 4 were inoculated via the oral route with 0.25 ml of brown et al. strain fr-tcov 080385d diluted to 10 −1.5 , 10 −3.0 , 10 −4.5 and 10 −6.0 respectively in mem hepes (gibco, france) supplemented with penicillin (200 μ/ml final concentration) and streptomycin (0.2 mg/ml final concentration). control group 5 was inoculated with memh plus antibiotics alone via the same route. at 1-day post-inoculation (dpi), two spf turkey contacts were introduced into groups 1-4 as sentinels to demonstrate horizontal transmission of infectious virus. from 1 to 3 dpi, cloacal swabs were collected from all subjects, sampling the contacts first, followed by those that had been inoculated. rna was extracted from these samples for molecular analysis as described above. the 50% endpoint was calculated using the method of reed and muench (reed & muench, 1938) . thirty-two 10-day-old spf turkeys were separated into groups, one containing 29 subjects and a second containing 3. each group was housed in a separate negative pressure room at a density of seven birds per m² and floors were covered with wood chippings (reproducing common commercial rearing conditions in france). the group of three subjects was inoculated with 0.25 ml of strain fr-tcov 080385d diluted at 10 −4.5 in the same media as used in exp. 1, via the oral route. at 1 dpi, cloacal swabs were collected to confirm their fr-tcov 080385d positive status by qrt-pcr. at 2 dpi, one positive subject was placed as a seeder infected bird among the group of 29 spf subjects (contacts). cloacal swabs were collected from all subjects every 2 hr until 16 hr post-contact (hpc), at 24 hpc and 2 days post-contact (dpc) then weekly until 41 dpc. during the 2-hr-sampling regime, the order in which the subjects were taken was respected throughout. this ensured that each subject was sampled precisely every two hours. sampling staff wore a new pair of sterile gloves for each sampled bird, so as not to transfer the virus through bird-handling. rna was extracted from these samples to perform qrt-pcr, to determine infection and the excretion period for each subject. one representative positive sample selected at 6 dpc of exp 2. (codified t6) was diluted (same media as exp. 1) so as to inoculate via the oral route 10 5.7 rna copies in three 10-day-old spf turkeys. they were housed in a negative pressure room, under the same rearing conditions as in exp 2, with three 11-day-old spf turkeys introduced as contact-birds at 1 dpi to demonstrate horizontal transmission. cloacal swabs were collected daily for qrt-pcr analyses from all birds until 3 dpi, when the birds were humanely euthanized and duodenum, jejunum, ileocaecal junction and bursa of fabricius were collected. these samples were fixed for 24 hr in 4% formaldehyde then transferred to 70% ethanol and finally embedded in paraffin wax for histopathology and anti-tcov immunohistochemistry (see section histopathology). this process was repeated using one representative positive sample from 13, 21, 27, 34 and 41 dpc of exp 2. (codified t13, t21, t27, t34 and t41, respectively) to make a total of six experiments. airborne transmission was evaluated in each of these experiments by using six 10-day-old spf turkeys housed in a park in the same containment cell but separated from the other animals, at a distance of 3 meters. the sampling programme was as described above. housing, circulation of personal, change of boots, clothes and gloves was organized to minimize physical contamination. fr-tcov was detected with qrt-pcr at 1 dpi in all six inoculated subjects of group 1 (dilution 10 −1.5 , mean ± sd 5.19 ± 0.94 log 10 cp/μl), in 5 out of 6 subjects of group 2 (10 −3 , 4.46 ± 1.81 log 10 cp/ μl) and in 3 out of 6 subjects in group 3 (10 −4.5 , 3.59 ± 1.37 log 10 cp/μl). at 2 and 3 dpi, all subjects of these groups, including contactbirds, were positive, demonstrating horizontal transmission. no viral rna was detected throughout the experiment in groups 4 (10 −6 ) and 5 (memh). the result obtained at 1 dpi (before horizontal transmission) gave a virus titre of 10 4,88 id 50 /ml. the following data are shown graphically in figure 1 . an inoculated subject with a viral rna load of 5.28 log 10 cp/μl at 1 dpi that had been placed among 29 contacts, transmitted the virus to one contact between 8 and 10 hpc, though the level of viral rna detected at 10 hpc in this newly infected bird (2.05 log 10 cp/μl) was almost at the lod. however, between 10 and 12 hpc the level of viral rna detected in the same bird increased to 3.39 log 10 cp/μl and a second contact was positive at 2.19 log 10 cp/μl. the data are shown graphically in figure 2 . in three out of six exp 2. samples (t6, t27 and t41), the number of positive inoculated birds and the level of viral rna detection increased over time during the sampling period, culminating at 3 dpi with rna detected in all birds including contacts (mean ± sd = 4.89 ± 0.69, 5.75 ± 0.32 and 4.55 ± 0.70 cp/μl, respectively). no viral rna was detected throughout the period, neither in inoculated or contact subjects exposed to t13, t21 and t34, nor in subjects assigned to the assessment of airborne transmission. intestinal samples taken from infected subjects at 3 dpi from exp. 3 showed well-preserved characteristic architectural features. except for some very mild hyperemia and rare epithelial desquamation, no clear histopathological changes were seen in any of the samples (figure 3a) . immunohistochemical staining showed an abundance of viral protein expressed in the ileum, caeca and bursa of all inoculated or contact subjects exposed to t6, t27 and t41 (expression in the caecum and bursa is shown for t41 in figure 3 ). as shown in figure 4 histograms, antigen detection in the other regions of the intestine (duodenum or jejunum) was inconsistent in both the inoculated and contact-birds exposed to the same samples, as illustrated by the fact that no viral protein was detected in the duodenum of any contact subject exposed to t6, t27 and t41. no viral protein expression was seen in any of the intestines taken from the inoculated and contact subjects exposed to t13, t21 and t34. to be taken into consideration. similarly, at one id 50 /ml, viral rna levels were also beyond the limit of detection of a well characterized qrt-pcr (maurel et al., 2011) , so that 10-day-old turkeys might be also more sensitive than qrt-pcr to detect infectious tcov. such a high susceptibility of young hosts was also reported in a recent paper where an enteric coronavirus of pigs (porcine epidemic diarrhea virus, pedv) was shown to infect more efficiently 5-day-old piglets than tissue culture (minimal infectious dose = 0.056 tcid50) (thomas et al., 2015) . rna levels at the pedv mid have also been reported to be beyond the limit of detection by qrt-pcr (goyal, 2014) . the effects of turkey age on the id 50 of fr-tcovwere not investigated in the current study however, the fact that tcov associated enteric disorders such as pec or poult enteritis mortality syndrome (pems) are predominantly diseases of younger subjects lends support to more resistance in older birds. was also difficult to justify on an ethical level in respect to the principals of the 3 rs (russell & burch, 1959) . although the "duration of excretion" for every individual could not be obtained, the experiments performed in the current study (in which one sample from each date was re-inoculated) revealed that some subjects continued to shed infectious virus for at least six weeks when others ceased at two. at this time the authors have no data on why some subjects stopped excreting infectious virus four weeks in advance of others or if, in fact, excretion detected at six weeks was representative of subjects with intermittent excretion profiles as has been observed in cats experimentally infected with fecv (kipar, meli, baptiste, bowker, & lutz, 2010) . in cats, this intermittent excretion has been suggested to be linked with persistent infection in the colon (lower intestine) from which viruses then have the potential to re-infect the small intestine at any time. the presence of virus in both regions of the gut can then result in renewed excretion (kipar et al., 2010) . concerning fr-tcov these questions should be assigned to specifically designed trials and histopathological examination, however, the present study seems to indicate a clear tropism of fr-tcov for lower intestine, as described for fecv (kipar et al., 2010) . indeed, in the current study, fr-tcov's ability to infect the turkey intestinal tract was successfully demon(reddy et al., 2016) . however, although the stellate morphology and the localization of the depicted cell (see inset figure 3c ) suggest that it likely belongs to one of the mentioned apc families, additional assays characterizing these cell types (for example double immunohistochemical staining for both viral protein expression and cell-characterizing protein epitopes) are needed to confirm a apc tropism for tcov. fr-tcov viral protein expression was not correlated with histopathologic changes in the sampled tissues collected here, contrary to previous observations following inoculation with a tcov of us lineage that did induce lesions, albeit without associated clinical signs . this discrepancy could be explained by the f i g u r e 4 detection of viral antigen in intestinal tissues. immunohistochemistry of: intestinal tissues d = duodenum, j = jejunum, i = ileum, c = caeca b = bursa of fabricius, taken 3 dpi from subjects inoculated with samples t6, t13, t21, t27, t34 ort41 of exp 2 (a) and from their corresponding contacts (b) difference in age of the birds inoculated as in the us tcov study, birds had been inoculated at 6 days of age; alternatively, like the distribution pattern discussed above, this discrepancy might be a time-dependent feature, with 3 dpi in our study being too early for morphologic changesresulting from epithelial damage, local tissue reactions and influx of immune cells to manifest. furthermore the specific date postinoculation when microscopic lesions were observed in the us tcov study was not given . it is equally possible that under experimental conditions the european lineage of tcov simply has a different pathogenic profile to those of the us lineage. infection studies for eu and us tcovs in turkeys of the same age and under the same controlled conditions are required for comparative analysis into the pathological profiles of these different lineages. in conclusion an extremely low dose of european isolate fr-tcov strain 080385d is required for infection of 10-day-old turkey poults under experimental conditions. the virus spreads very quickly via the oro-faecal route among susceptible subjects (a new subject at least every 2.5 hr) and infectious virus may continue to be excreted for at least six weeks after the initial infection which may be linked to a preferential tropism for the lower intestines. these results stress the importance of 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chickens transmission kinetics and histopathology induced by european turkey coronavirus during experimental infection of specific pathogen free turkeys the authors wish to thank the département des côtes d'armor/conseil régional de bretagne/conseil régional des pays de loire/france agrimer/office de l'élevage/comité interprofessionnel de la dinde française for their financial support. this research was part of the epicorem anr programme: eco-epidemiology of coronaviruses:from wildlife to human & emergence threat assessment. the authors declare that they have no conflict of interest. http://orcid.org/0000-0002-6697-7688 key: cord-269287-vbuepdm4 authors: ogbu, kenneth ikejiofor; mira, francesco; purpari, giuseppa; nwosuh, chika; loria, guido ruggero; schirò, giorgia; chiaramonte, gabriele; tion, metthew terzungwe; di bella, santina; ventriglia, gianluca; decaro, nicola; anene, boniface maduka; guercio, annalisa title: nearly full‐length genome characterization of canine parvovirus strains circulating in nigeria date: 2019-10-16 journal: transbound emerg dis doi: 10.1111/tbed.13379 sha: doc_id: 269287 cord_uid: vbuepdm4 canine parvovirus type 2 (cpv‐2) emerged suddenly in the late 1970s as pathogen of dogs, causing a severe and often fatal gastroenteric disease. the original cpv‐2 was replaced by three antigenic variants, cpv‐2a, cpv‐2b and cpv‐2c, which to date have gained a worldwide distribution with different relative proportions. all previous studies conducted in africa were based on partial vp2 gene sequences. the aim of this study was to provide a genome analysis to characterize the cpv strains collected in nigeria, africa. rectal swab samples (n = 320) were collected in 2018 and tested by means of an immunochromatographic assay. among the 144 positive samples, 59 were selected for further analyses using different molecular assays. the results revealed a high prevalence of cpv‐2c (91.5%) compared to the cpv‐2a variant (8.5%). the vp2 gene sequences showed a divergence from the strains analysed in 2010 in nigeria and a closer connection with cpv strains of asian origin. the non‐structural gene analysis evidenced amino acid changes never previously reported. the molecular analysis based on genomic sequences evidenced a geographical pattern of distribution of the analysed strains, suggesting a potential common evolutionary origin with cpv of asian origin. this study represents the first cpv molecular characterization including all the encoding gene sequences conducted in the african continent and contributes to define the current geographical spread of the cpv variants worldwide. reading frames (orfs) encoding for two non-structural (ns1 and ns2) and two structural (vp1 and vp2) proteins through alternative splicing of the same mrnas (reed at al., 1988) . soon after its emergence, the original cpv-2 was replaced by two antigenic variants, cpv-2a and cpv-2b (parrish et al., 1991; parrish, o'connell, evermann, & carmichael, 1985) , and in 2000, a third antigenic variant (cpv-2c) was described (decaro & buonavoglia, 2012) . to date, all three cpv variants are worldwide distributed, with different relative proportions according to the year and country of collection (amrani et al., 2016; miranda & thompson, 2016; woolford, crocker, bobrowski, baker, & hemmatzadeh, 2017) . in the african continent, cpv has been described in south africa and namibia (dogonyaro, bosman, sibeko, venter, & vuuren, 2013; steinel, venter, van vuuren, parrish, & truyen, 1998) , zambia (kapiya et al., 2019) , mozambique (figuiredo et al., 2017) , ghana (folitse et al., 2017) , morocco (amrani et al., 2016) , cape verde (costanheira et al., 2014) , nigeria (chollom et al., 2013) and tunisia (touhiri et al., 2009 ). in nigeria, only recently the molecular analyses based on the partial vp2 gene sequence of cpv strains described the circulating cpv variants (apaa, daly, & tarlinton, 2016; dogonyaro et al., 2013; fagbohun & omobowale, 2018) . unfortunately, all previous studies conducted in africa lack a comprehensive sequence analysis including all the viral genome, thus preventing a more in-depth knowledge on the origin and evolution of the circulating cpv strains. the aim of this study was to characterize the cpv strains recently collected in nigeria, analysing the nearly complete cpv genome sequence and comparing the obtained sequences with worldwide related sequences available in genbank. 482 m a.s.l). samples were submitted from eight private veterinary clinics, two from each state, and from kennels/breeders in the same areas. rectal swabs were tested using an in-clinic assay for detection of cpv antigen (senspert ® canine parvovirus test kit, vetall laboratories), according to the manufacturer's instructions. among the positive samples, 59 rectal swabs were selected and submitted for molecular analyses, where they were stored at −80°c until use. details are reported in table s1 . swab homogenates were obtained as previously described . viral dna was extracted from 200 µl of swab homogenate using the dneasy blood & tissue kit (qiagen s.p.a.), according to the manufacturer's instructions. the presence of cpv dna was confirmed using a primer pair (table s2 ) in a pcr protocol amplifying a 700-bp fragment of the vp2 gene (touihri et al., 2009) , using the commercial kit gotaq ® g2 dna polymerase (promega italia s.r.l.), as previously described (mira, dowgier, et al., 2018) . amplicons were checked after electrophoresis on a 3% agarose gel supplemented with ethidium bromide. ten microlitres of each amplicon was digested with 5 units (u) of restriction endonuclease mboii (new england biolabs ® inc.) in a 50-µl reaction mix consisting of 5 µl of nebuffer and 34 µl of nuclease-free water, under the following reaction conditions: incubation at 37°c for 2 hr and inactivation at 65°c for 20 min. the profile was determined by electrophoresis on a 3% agarose gel supplemented with ethidium bromide. specimens from each city of collection (n = 4 from makurdi; n = 11 from jos; n = 8 from abuja and n = 5 from lafia), representing dogs with different age, vaccinal and clinical status, were selected to determine the vp2 gene sequence (table 1 ). the nearly complete vp2 gene sequence was determined using a primer pair (table s2) , which allows for amplification of a 1,745-bp fragment (battilani et al., 2001; mokizuki et al., 1996) , using the commercial kit gotaq ® g2 dna polymerase (promega italia s.r.l.), as previously described with minor modifications (thermal conditions: 1 min for the annealing step). after electrophoresis on agarose gel supplemented with ethidium bromide, amplicons were purified with illustra™ gfx™ pcr dna and gel band purification kit (ge healthcare life sciences) and submitted to bmr genomics srl for direct sanger sequencing with 6.4 pmol of the reverse primer used for amplification and of two additional internal primers (table s2 ). sequences were assembled and analysed using bioedit software (hall, 1999) . by excluding the vp2 gene sequences with complete nt identities, 8 cpv dnas from different cities of collection were further selected for a further sequence analysis by amplifying a long genome sequence encompassing both major orfs, without the 5′ and 3′ flanking sequences (table 1) . sequence analyses were carried out using primer pairs described by pérez et al. (2014) , using the commercial kit gotaq ® g2 dna polymerase (promega italia s.r.l.), as previously described (mira, dowgier, et al., 2018) with minor modifications (mira, canuti, et al., 2019) . after electrophoresis on agarose gel, amplicons were purified and submitted for direct sanger sequencing with a set of primers, as previously described (mira, purpari, lorusso, et al., 2018) . sequences were assembled, analysed and submitted to nblast program (zhang, schwartz, wagner, & miller, 2000) to search related sequences into public domain databases. these sequence data were submitted to the ddbj/embl/ genbank databases under accession numbers mk895483-90. phylogenetic analyses based on the full-length ns1 and vp2 gene sequences and on the whole genome encompassing the two orfs were conducted using the best-fit model of nt substitution with mega version x software (kumar, stecher, li, knyaz, & tamura, 2018) , inferred with the maximum-likelihood method based on the tamura 3-parameter (t92) and hasegawa-kishino-yano (hky) models, with discrete gamma distribution (five rate categories) (g) and invariant sites (i) (bootstrap 1,000 replicates), the best-fitting models after the model test analyses (vp2: t92 + g; ns1: hky + g; whole genome: hky + g+i). rna was extracted from samples using the qiaamp viral rna mini kit (qiagen s.p.a.), according to the manufacturer's instructions. extracted dna/rna was amplified using a set of gel-based or real-time (rt-)pcr assays useful for the detection of canine distemper virus (cdv) , canine adenovirus (cadv) types 1 and 2 (dowgier et al., 2016) , canine coronavirus (ccov) (decaro et al., 2004) and canine rotavirus (crov) (freeman, kerin, hull, mccaustland, & gentsch, 2008) . among the collected 320 samples, 144 rectal swabs tested positive for cpv by in-clinic assay (45%). the prevalence of the positive samples for each city of collection is reported in table 2 . the presence of cpv dna was confirmed in the selected samples (n = 59) using the conventional pcr assay. the same samples tested negative for cdv, cadvs, ccov and crov by gel-based or real-time (rt)-pcr assays. based on the rflp analysis, 54 cpv-positive ta b l e 1 identification code, origin, age, vaccination and clinical status, strain and sequence information of the dogs selected for molecular investigation amino acid change i60v in ns1 also lies at the same residue in the ns2-encoding sequence. additional four amino acid changes in the ns2-encoding sequences were observed: d93e, t94a, d151n and m152v (table 5 ). these changes resulted in silent mutations in the corresponding encoded ns1 protein. the aa change k116r in the vp1 gene sequence is added to the aa changes of the vp2 gene sequence lying in the corresponding en canine parvovirus has still been playing a main role in inducing severe and often fatal gastroenteritis in young or non-immunized dogs. during years, cpv spread and evolution have been well documented in north and south america, europe and asia (miranda & thompson, 2016; zhou, zeng, zhang, & li, 2017) . more recently, data about its spread were also obtained from australia and africa (amrani et al., 2016; castanheira et al., 2014; chollom et al., 2013; dogonyaro et al., 2013; figuiredo et al., 2017; folitse et al., 2017; kapiya et al., 2019; touhiri et al., 2009; woolford et al., 2017) . most of these studies were based on the partial or complete vp2 gene sequence, due to the involvement of the vp2 capsid protein in host switch and to its fast evolutionary rate nelson, palermo, hafenstein, & parrish, 2007; shackelton, parrish, truyen, & holmes, 2005) , with limited information on other cpv encoding fagbohun & omobowale, 2018) . these analyses conducted in nigeria evidenced firstly the circulation of cpv-2a strains (apaa et al., 2016; dogonyaro et al., 2013) and only recently of cpv-2b/2c types (fagbohun & omobowale, 2018 moreover, the vp2 aa change was due to a nt change in the second base of the codon (vp2 c14g), but this change was common only to cpv strains of asian origin mira, purpari, lorusso, et al., 2018; wang et al., 2016; zhuang et al., 2019) . the potential biological relevance of these changes has not been described yet and needs to be assessed in further studies. as most recent asian cpvs, the nigerian strains displayed other three aa substitutions in the vp2 sequence (f267y, y324i and q370r). while change at aa residue 324 is predominant in all three cpv variants in asia (geng et al., 2015; yi, tong, cheng, song, & cheng, 2016; zhao et al., 2017; zhou et al., 2017) , the other changes have been less frequently observed, mainly in china since 2013, and change q370r has been detected only in cpv-2c strains (geng et al., 2015; guo et al., 2013; mira, purpari, lorusso, et al., 2018; wang et al., 2016; zhuang et al., 2019) . these aa substitutions are located in the greatest variable vp2 gh loop, comprised between aa 267 and 498, but while residue 267 is not exposed on the capsid surface (chiang, wu, chiou, chang, & lin, 2016) and may not affect the antigenicity of cpv (xu et al., 2015) , residues 324 and 370 could have immunological implications or biological relevance. indeed, residue 324 is subject to positive selection (hoelzer, shackelton, parrish, & holmes, 2008) and is adjacent to residue 323, which affects binding to the canine transferrin receptor . residue 370 is close to residues associated with the ability of cpv to haemagglutinate, altering the ph dependence of haemagglutination or affecting the canine transferrin receptor (tfr) binding that determines the canine host range (guo et al., 2013; kaelber et al., 2012; tsao et al., 1991) . among the synonymous substitutions observed in the cpv-2a vp2 gene sequences, nt change a1275g has been previously described in cpv-2c strains (amrani et al., 2016; decaro, desario, amorisco, et al., 2013; decaro et al., 2009) . this change, observed in the strains uv1 and uv6, was detected in the binding region of the type-2a and type-2c specific probes of the minor groove binder (mgb) probe assay (decaro et al., 2005 . although this change potentially accounts for the absence of vic fluorescence in the 2a/2b and 2b/2c assays, specific additional studies are necessary to evaluate its real implication in the characterization of this cpv-2a mutant by the mgb probe assay, as previously done for the same substitution in the cpv-2c mutants (decaro, desario, billi, et al., 2013) . the in-clinic assay used in this study was able to detect the cpv-2a showing this substitution, as previously observed for another rapid assay used to test also the cpv-2c mutants (decaro, desario, billi, et al., 2013) . limited studies are available on the cpv non-structural genes (hoelzer et al., 2008; pérez et al., 2014) , and, only recently, the analysis of the ns1 gene sequence was included in the cpv phylogenies from several countries (canuti, rodrigues, whitney, & lang, 2017; grecco et al., 2018; mira, canuti, et al., 2019; zhuang et al., 2019) . in this study, sequence analysis revealed aa changes previously described mainly in ns1/ns2 gene sequences of cpv-2a/2c strains of asian origin. additional changes were also evident, some previously reported in south/north america and others never previously observed. this divergence may suggest the same ancestral origin with the cpv strains of asian origin but a separate evolution, as well as a continuous adaptive process of the virus in separate environments. indeed, some of these changes lay in the potential encoding sequence of functional domains (mira, canuti, et al., 2019) and, particularly, residues 351, 517 and 545 are located between the α5-and α6-helices, between the β5-and α11-helices and just close to the α11-helix of the helicase domain protein sequence, respectively, as illustrated in niskanen, ihalainen, kalliolinna, häkkinen, and vihinen-ranta (2010) . therefore, their role needs to be further evaluated. the molecular analysis based on long genome sequences evidenced the geographical origin of the analysed strains rather than the clustering based only on the cpv antigenic variant. therefore, this study supports further studies aimed to track the viral spread and elucidate the cpv evolution. indeed, the recent evidence of specific aa changes, as well as the divergence from previous circulating strains, does not allow to rule out the possible introduction of these strains from other countries, highlighting the need for further studies on cpv whole genome in different geographical areas. this suggestion is supported by the evidence of genetic signatures typical of cpv or other canine viruses with different origins (decaro, campolo, et al., 2007; martella et al., 2006; mira, purpari, lorusso, et al., 2018) , probably connected with the trading and transport of dogs between countries and continents. in this study, with the aim to investigate the prevalence of the most frequent canine enteric viruses, all collected samples were analysed for selected pathogens. cpv was the only enteric viral pathogen detected in this study and this result confirmed the correlation between cpv infection and development of enteric signs, with a limited role of other viral enteric pathogens (dowgier et al., 2017) . nevertheless, further studies, based on a wider sampling also including the wild potential susceptible species, are necessary to better elucidate the effective spread of the other viruses in nigeria. this study represents the first cpv molecular characterization including all the encoding gene sequences conducted in the african continent and contributes to define the current geographical spread of the cpv variants worldwide. the evidence of mutations that have not been detected before suggests the need for further investigations in order to determine any biological consequences and underlines the continue evolution of cpv. the authors would like to thank dr. ijeoma chekwube chukwudi, dr. pam dachung luka, dr. emmanuel tumininu obishakin and dr. ukamaka uchenna eze for their skilful technical assistance and also practitioners, private veterinary clinics and dog breeders for sample collection. the authors of this manuscript declare that there are no conflicts of interest. molecular epidemiology of canine parvovirus in morocco. infection canine parvovirus (cpv-2) variants circulating in nigerian dogs evaluation of immunity and seropositivity of igg antibodies to canine parvoviruses in vaccinated and unvaccinated dogs in abeokuta analysis of canine parvovirus sequences from wolves and dogs isolated in italy introduction of canine parvovirus 2 into wildlife on the island of newfoundland molecular and serological surveillance of canine enteric viruses in stray dogs from vila do maio, cape verde identification of a novel canine parvovirus type 2c in taiwan molecular detection of canine parvovirus in jos ictv virus taxonomy profile: parvoviridae the family parvoviridae canine parvovirus-a review of 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evolution of canine parvovirus -a new perspective genome sequence characterization of canine parvoviruses prevalent in the sichuan province of china key: cord-274773-3jhka8wl authors: zhang, jialin; chen, jianfei; liu, ye; da, shi; shi, hongyan; zhang, xin; liu, jianbo; cao, liyan; zhu, xiangdong; wang, xiaobo; ji, zhaoyang; feng, li title: pathogenicity of porcine deltacoronavirus (pdcov) strain nh and immunization of pregnant sows with an inactivated pdcov vaccine protects 5‐day‐old neonatal piglets from virulent challenge date: 2019-09-30 journal: transbound emerg dis doi: 10.1111/tbed.13369 sha: doc_id: 274773 cord_uid: 3jhka8wl in this study, the pathogenicity of porcine deltacoronavirus (pdcov) strain nh (passage 10, p10) was evaluated. we found that pdcov strain nh is enteropathogenic in 5‐day‐old pigs. pathogenicity experiments provided a challenge model for studying the protection efficiency of passive immunity. in order to investigate the protective efficacy of passive immunity in newborn piglets, pregnant sows were vaccinated with either a pdcov‐inactivated vaccine at the houhai acupoint (n = 5) or dmem as a negative control (n = 2) using a prime/boost strategy 20 and 40 days before delivery. pdcov spike (s)‐specific igg and neutralizing antibody (na) responses were detected in immunized sows and piglets born to immunized sows. pdcov spike (s)‐specific siga was also detected in the colostrum and milk of immunized sows. five days post‐farrowing, piglets were orally challenged with pdcov strain nh (10(5) tcid(50)/piglet). severe diarrhoea, high levels of viral rna copies and substantial intestinal villus atrophy were detected in piglets born to unimmunized sows. only 4 of 31 piglets (12.9%) born to immunized sows in the challenge group displayed mild to moderate diarrhoea, lower viral rna copies and minor intestinal villi damage compared to piglets born to unimmunized sows post‐challenge. mock piglets exhibited no typical clinical symptoms. the challenge experiment results indicated that the inactivated pdcov vaccine exhibited 87.1% protective efficacy in the piglets. these findings suggest that the inactivated pdcov vaccine has the potential to be an effective vaccine, providing protection against virulent pdcov. states wang, byrum, & zhang, 2014) , followed by subsequent outbreaks in canada (ojkic et al., 2015) , south korea (lee et al., 2016) , thailand (janetanakit et al., 2016; saeng-chuto et al., 2017) and mainland china (dong et al., 2015) , exhibiting a global distribution trend. additionally, clinical reports have indicated that pdcov exhibits enteropathogenicity, causing severe diarrhoea and vomiting in roughly 5-to 10-day-old gnotobiotic and conventional piglets . pathological damage to the intestine, primarily in the jejunum and ileum, was characterized by intestinal villi atrophy and shortening and was confirmed by the pathogenicity experiments wang, hayes, sarver, byrum, & zhang, 2016) . such changes are clinically difficult to distinguish from the pathological changes caused by the porcine epidemic diarrhoea virus (pedv) and transmissible gastroenteritis virus (tgev) (jung et al., 2016; zhang, 2016) . pdcov infections have resulted in great economic losses for the global swine industry. therefore, fast and effective preventive measures are essential for the prevention and control of pdcov. currently, implementing vaccines remain the most effective means of disease control; however, there are no commercial vaccines available for pdcov. due to their immature immune system, neonatal piglets are highly susceptible to viral infection during their first few weeks of life. studies suggest that passive immunity is the most effective approach for protecting piglets from viral infection (langel, paim, lager, vlasova, & saif, 2016; leidenberger et al., 2017) . immunized sows can transfer antibodies against enteroviruses (e.g. pedv, tgev and porcine rotavirus) to neonatal piglets through their colostrum and milk. the protective efficiency of passive immunization has a high positive correlation with antibody levels in the colostrum and milk (sestak, lanza, park, weilnau, & saif, 1996) . therefore, passive immunity of newborn piglets can be achieved by immunizing sows that produce high levels of neutralizing antibodies (na) and transfer these antibodies through the colostrum and milk to the nursing piglets, which may be an effective means of controlling viral infection. in this study, a challenge model was established and the results indicated that pdcov strain nh (p10) is pathogenic to 5-day-old specific pathogen-free (spf) piglets. then, an inactivated pdcov vaccine was prepared and the immune responses and protective efficiency of the inactivated pdcov vaccine in pregnant sows was evaluated. after two doses of the vaccine, pregnant sows produced strong igg and na responses specific to pdcov s proteins. high levels of igg antibodies and na were also detected in the serum of neonatal piglets born to immunized sows, which suggests that the antibodies were successfully transferred through the colostrum and milk. five-day-old piglets were challenged with virulent pdcov to assess the protective efficacy of the vaccine, and the findings indicated that the inactivated pdcov vaccine provided 87.1% protective efficacy. pdcov strain nh was isolated from pdcov-positive specimens with llc-pk cells (atcc ® cl-101™), and plaques were purified twice. pdcov strain nh was continuously passaged in swine testis (st) cells in dmem containing 10 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (tpck)-trypsin (invitrogen). at 36 hr post-infection, both the supernatant and cells were harvested, titrated and stored at −70°c for future analyses. the titre of pdcov strain nh (10th passage, p10) was 10 5 tcid 50 /ml, which was used for the challenge experiment. the inactivated pdcov vaccine was prepared using the 15th generation of viruses due to its low mutation rate and similar antigenicity compared to the parent virus. viral culture supernatants were inactivated with beta-propiolactone containing aluminium hydroxide adjuvant at a 1:1 ratio in order to prepare the inactivated pdcov vaccine. ten 5-day-old spf piglets were confirmed negative for pdcov, pedv, tegv and rpv by virus-specific pcr. pigs were maintained in germ-free isolation units of the animal facility located at the harbin veterinary research institute under standard conditions prescribed by the institutional guidelines. piglets were fed suckling piglet formula every 3 hr. six of the 10 spf piglets (piglets #245, #246, #248, #241, #242 and #249) were assigned to the pdcov-inoculation group, which were orally inoculated with the pdcov strain nh (p10) (10 4 tcid 50 /pig). the remaining 4 piglets (piglets #244, #247, #251 and #252) were orally inoculated with volume-matched virus-negative culture medium and served as the negative control group. the clinical signs of vomiting and diarrhoea were evaluated every 12 hr. the level of diarrhoea severity was scored for each piglet using the following criteria: 0 = no vomiting or diarrhoea; 1 = mild; 2 = moderate; 3 = severe. faecal swabs were collected for detecting viral rna. duplicate tissues for the duodenum, jejunum, ileum, caecum, colon and rectum were collected for viral rna detection and histological examinations. seven sows were confirmed to be negative for pdcov, pedv, tegv and rpv using virus-specific pcr and a serum neutralization test. all of the sows were randomly assigned to 2 experimental groups: (1) the immunization group (pdcov-inactivated vaccine; n = 5); or (2) the control group (dmem; n = 2). sows from the immunization group were immunized with 3 ml inactivated pdcov vaccine (pdcov strain nh p15, equivalent to 10 × 10 5 tcid 50 ) at the houhai acupoint (a concave part between the anus and tail). vaccination in the houhai acupoint was found to be helpful for inducing humoral and mucosal immune responses (li et al., 2018; liu, tan, wan, zuo, & liu, 1998) . all of the sows were immunized 40 days prior to delivery, and a booster was administered 20 days before delivery. sows from the control group were immunized with 3 ml dmem as described above (table 1) . serum was collected from sows 0 (prior to the first immunization), 20, 40, 47, 54 and 61 days post-immunization. serum samples were inactivated at 56°c for 30 min and stored at −70°c for future analyses. milk was collected from the sows 1-5 days post-farrowing. three 5-day-old piglets from each pdcov-vaccinated gilts or mock gilts were permitted to continue drinking breast milk in order to detect the level of serum antibodies in piglets, while the other piglets were separated from the sows and housed in the experimental animal centre. separated piglets were fed suckling piglet formula every 3 hr. newborn piglets (2 or 4 piglets from each sow were set as the control group) derived from pdcov-vaccinated gilts or mock gilts were challenged orally with 10 5 tcid 50 of pdcov strain nh (p10). the clinical signs of vomiting and diarrhoea were evaluated daily. diarrhoea severity was scored for each piglet using the following criteria: 0 = no vomiting or diarrhoea; 1 = mild; 2 = moderate; 3 = severe. faecal swabs were collected for the detection of viral rna. serum was collected from the breastfed piglets 5, 12, 19 and 26 days post-birth. the study protocol was approved by the institutional animal care and use committee of the harbin veterinary research institute. faecal swabs were centrifuged at 5,000×g for 10 min at 4°c, and 140 μl supernatant was collected for viral rna extraction. viral rna was extracted using a qiaamp ® viral rna mini kit (qiagen, hilden, germany) following the manufacturer's instructions. one hundred mg tissue samples from each piglet were ground in liquid nitrogen, and the total rna was extracted using an rnaiso plus kit (takara, kusatsu, japan) following the manufacturer's instructions. rna was then used to perform real-time (rt)-qpcr using specific primers and probes (pdcov-n-f: cgcttaactccgccatcaa; pdcov-n-r: tctggtgtaacgcagccagta; pdcov-n-probe: fam-cccgttgaaaacc-mgb) as previously described with minor modifications (ma et al., 2015) . briefly, 2 μl rna was used in a 20 μl pcr reaction system using a one step primescript™ rt-pcr kit (takara, kusatsu, japan) in a lightcycler 480 (roche applied science, in, usa) under the following conditions: one cycle at 95°c for 5 min and 95°c for 10 s, followed by 40 cycles at 95°c for 5 s and 60°c for 20 s. the duodenum, jejunum, ileum, caecum, colon and rectum tissues were cut into 10-μm-thick sections, mounted onto glass slides and blocked with 5% non-fat dry milk in pbs for 60 min at 37°c. then, slides were incubated for 60 min with a mouse polyclonal anti-pdcov s antibody followed by incubation with an alexa fluor ® 680 donkey anti-mouse igg (sigma-aldrich, mo, usa) for 60 min. nuclei were stained with dapi, and samples were observed with an inverted fluorescence microscope. tissues from intestinal tissues were fixed in formalin for 48 hr and embedded in paraffin wax following standard methods. ihc detection of pdcov antigens was performed using an anti-pdcov-n monoclonal antibody prepared in the laboratory followed by an incubation period with horseradish peroxidase (hrp)-conjugated sheep anti-mouse igg (sigma-aldrich) for 40 min at room temperature. reactions were developed with 3,3'-diaminobenzidine (dab). distilled water was used to finish staining and stained with haematoxylin (he). dehydration, clearing and mounting were conducted with neutral gums. the extracellular domain of the pdcov spike gene was amplified and cloned into the pcagg vector with a c-terminal flag tag. recombinant s protein was expressed in 293t cells, purified using anti-dykddddk g1 affinity resin (genscript: l00432-1) and used as the coating antigens. pdcov s-specific igg, iga and siga antibody responses elicited by immunization with the inactivated vaccine were assessed by an indirect enzyme-linked immunosorbent assay (elisa). optimal assay conditions (i.e. antigen coating concentration, serum and sow milk dilutions, and secondary dilutions) were determined by a checkerboard titration. optimal coating concentrations for igg, iga and siga were 0.28, 0.28 and 0.07 μg/ml, respectively. next, 96-well polystyrene microplates were coated with the optimal antigen in a bicarbonate/carbonate coating buffer overnight. plates were washed three times with pbst (pbs with 0.05% tween 20) and blocked with 5% non-fat dry milk at 37°c for 2 hr. plates were washed 3 times with pbst, diluted in either serum or sow milk (1:100, 100 μl/well) and incubated at 37°c for 1 hr followed by incubation with a streptavidin-hrp-conjugated igg or iga antibody (1:10,000) at 37°c for 1 hr. after washing 3 times with pbst, a mouse anti-fc fragment of siga molecule antibody (1:10,000) ta b l e 1 immune procedure collected then confirmed by an immunofluorescence assay (ifa) in order to determine the cut-off value. the levels of sow and piglet serum neutralizing antibodies were determined using pdcov strain nh (p10) with a virus neutralization test (vnt). in order to perform the assay, serum was heated at 56°c for 30 min for complement inactivation. next, 100 μl of twofold serially diluted serum was mixed with 100 μl dmem containing 100 data were analysed by student's t test. a threshold of p < .05 was considered to be significant. the antibody levels of the piglets are presented as box and whisker plots created with graphpad prism v7. values are reported as the mean ± standard deviation (sd). gilts were immunized twice with the inactivated pdcov vaccine. after eating colostrum for 5 days, piglets were divided into the challenge group, mock group and monitoring group in each sow the passive transfer of antibodies from immunized sows to piglets through their colostrum and milk was detected with an s-specific elisa. a high level of s-specific igg was detected in the serum of piglets born to immunized sows 5 days post-farrowing (figure 3a ). in order to assess the protective effect of the vaccine, 5-day-old piglets (31 total) born to immunized sows and 5-day-old piglets (14 total) born to unimmunized sows were challenged with a high dose (10 5 tcid 50 ) of pdcov strain nh (p10). piglets (12 total) born to immunized sows and 5-day-old piglets born to unimmunized sows (4 total) were used as controls. piglets were monitored, and clinical symptoms were scored according to their level of diarrhoea (table 3) . six piglets exhibited mild to moderate diarrhoea, in which the symptoms of four piglets from the challenge group and 2 piglets from the mock group born to immunized group were due to indigestion. following oral inoculation with pdcov, two piglets born to immunized sows #72 (1/5) and #56 (1/7) exhibited mild diarrhoea 2 days ta b l e 3 pathogenicity of pdcov in piglets no obvious clinical symptoms were observed in the mock piglets from each group. the piglet faecal specimens were collected daily, and rna was extracted in order to detect viral rna copies by rt-qpcr. pdcov rna copies were detected in four piglets born to sows #68, #100 and #56, and low levels of viral rna were detected in 1 piglet ( based on pcr data from the faeces and intestinal tissues, 4 of 31 pigs from the vaccinated sows were evidently infected after virus challenge. therefore, the passive immunity obtained from immunized sows induced 87.1% protection against highly pathogenic pdcov challenge. in the present study, the pathogenicity of pdcov strain nh isolated in 2014 was assessed. the results suggest that the virus was enteropathogenic in 5-day-old spf pigs. a challenge model to study the protective passive immunity in neonatal piglets was conducted. the protective efficacy of passive immunity elicited by the inactivated pdcov vaccine against challenge with a highly pathogenic virulent strain in neonatal piglets born to immunized sows was investigated. results revealed that immunization with an inactivated pdcov vaccine could produce a strong antibody response in pregnant sows after the second vaccination 20 days before delivery. high s-specific igg antibody and na responses were observed in the serum of sows post-farrowing. interestingly, a high level of s-specific siga was detected in colostrum and milk, although it lasted for only a short period of time. previous studies have indicated that siga was produced in the mammary gland by antibody-secreting cells and the recruitment of antibody-secreting cells into the mammary gland contribute to the production of siga (wilson & butcher, 2004) . therefore, it has been suggested that the oral route appears to be the most effective method for eliciting a strong siga response when vaccinating sows (gerdts & zakhartchouk, 2017) . however, the findings of this study suggest that vaccination with an inactivated vaccine by injection at the houhai acupoint in pregnant sows could also induce a strong siga response in the colostrum, which may subsequently provide protection for piglets against virulent challenge. results also confirmed that vaccination at the houhai acupoint could induce a mucosal immune response (liu et al., 1998) . the duration of antibody persistence until the end of the experiment was monitored, and the results indicated that a strong positive correlation existed between the igg antibody and na responses in the serum of immunized sows. the s-specific iga antibody response was also detected with an elisa; however, the results indicated that the serum, colostrum and milk of sows failed to produce an iga response after immunization with an inactivated pdcov vaccine. transferring antibodies (i.e. siga, igg and igm) from the sows to the piglets through the colostrum and milk has been considered to be the primary mechanism of protection mediated by passive immunity (poonsuk et al., 2016; saif & bohl, 1983; salmon, berri, gerdts, & meurens, 2009 ). the igg antibodies were absorbed by the piglets within the first 24-48 hr of life through the colostrum. in this study, a high level of igg antibodies was detected in the serum of piglets and, more importantly, such levels displayed longterm persistence (~10 days) although piglets stopped consuming milk from immunized sows. these results suggest that igg from the serum of sows could be absorbed into the serum of piglets through colostrum. the level of igg antibodies in the serum of piglets (31 piglets, 10 days post-challenge) did not increase, which was due to the existing maternal antibodies in the serum of piglets interfering with an active immune response to infection. no s-specific siga antibodies were observed in the serum of piglets, which indicated that the function of siga was limited to the enteric cavity rather than the serum. the challenge assay suggested that antibodies transferred from sows through their colostrum and milk could provide protection for piglets. four piglets born to immunized sows displayed diarrhoea post-inoculation with pdcov strain nh, and viral rna copies were detected in the faeces. however, compared to the mock group, the four piglets exhibited mild to moderate diarrhoea (no severe diarrhoea) and lower viral rna copies in the faeces and intestinal tissues. low pdcov antigen levels were also detected in the jejunum and ileum by ihc, and minor intestinal villi damage was observed by he staining. collectively, these results suggest that the antibodies from sows could provide the 4 piglets with partial, but not complete, protection. previous studies on the passive immunity of tgev suggest that viral inoculation by different routes induce different immunoglobulin isotypes (bohl & saif, 1975) . the production of iga in the milk of tgev antibodies was associated with an intestinal infection, while the production of igg was associated with parenteral antigenic stimulation. this study also indicated that the igg response in serum, colostrum and milk was predominantly detected after pregnant sows were inoculated with virulent tgev by intramammary injections. moreover, the passive immunity protection was good (0% mortality and morbidity) in 3-and 4-day-old newborn piglets born to immunized sows post-challenge. however, morbidity was 100% 11 days post-farrowing with 0% mortality after tgev challenge. these results suggest that within the first week, igg antibodies in colostrum and milk of immunized sows could provide protection for piglets against tgev virulent challenge. in this study, iga antibodies were not detected in serum, colostrum or milk of immunized sows. the immunoglobulin in the antibodies was predominantly igg, and although the piglets were no longer receiving anti-pdcov igg antibodies from the milk, igg lasted ~10 days in the serum of piglets. because of this, the immune protection response observed in pdcov-challenged piglets born to inactivated pdcov-vaccinated mothers was likely due to circulating anti-pdcov igg antibodies that were passively transferred through colostrum rather than anti-pdcov iga antibodies from milk, as observed previously in pedv research (poonsuk et al., 2016) . the siga and igg in colostrum and milk consumed by piglets for 1-2 days post-birth survived and were maintained until challenged to some extent in the gut. in the intestinal environment of piglets born to immunized sows, vnt antibodies might neutralize pdcov in gut and only a small amount of the virus might remain under the minimal infectious doses that could infect piglets (poonsuk et al., 2016) . another possible reason for this result may be due to the antibody-dependent cell-mediated cytotoxicity (adcc) effect, which promotes cell-mediated immune responses against pdcov, as previously studied in pedv (casadevall & pirofski, 2003) . in summary, the findings of the present study demonstrate that immunizing sows twice with an inactivated pdcov vaccine could induce igg, 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porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution pathogenicity of porcine deltacoronavirus (pdcov) strain nh and immunization of pregnant sows with an inactivated pdcov vaccine protects 5-day-old neonatal piglets from virulent challenge the authors declare that there is no conflict of interest. the study's protocol was approved by the institutional animal care and use committee of the harbin veterinary research institute.this study does not contain research with human participants performed by any of the authors. https://orcid.org/0000-0003-4123-0892 key: cord-010053-kniq2mbw authors: lee, sunhee; lee, dong‐uk; noh, yun‐hee; lee, seung‐chul; choi, hwan‐won; yang, hyoung‐seok; seol, jun‐ho; mun, seong hwan; kang, won‐myoung; yoo, hyekyung; lee, changhee title: molecular characteristics and pathogenic assessment of porcine epidemic diarrhoea virus isolates from the 2018 endemic outbreaks on jeju island, south korea date: 2019-05-20 journal: transbound emerg dis doi: 10.1111/tbed.13219 sha: doc_id: 10053 cord_uid: kniq2mbw since the 2013–2014 incursion of the virulent g2b porcine epidemic diarrhoea virus (pedv) pandemic strains in south korea, frequent moderate‐scale regional outbreaks have recurred. in particular, areas of jeju island with extensive swine production have faced repeated epidemics since the re‐emergence in 2014. the current study reports the complete genome sequences and molecular characterization of the representative pedv strains responsible for the 2018 endemic outbreaks on jeju island. all isolates were determined to belong genetically to the highly pathogenic pandemic g2b group. full‐length genome sizes of four isolates differed from that of the g2b epidemic field strain due to insertion or deletion (del) mutations in the non‐structural protein (nsp)‐ or spike (s) protein‐coding regions. the 2018 jeju isolates shared 96.7%–98.7% and 98.5%–99.4% identity at the s gene and whole‐genome levels, respectively, compared to global g2b pedv strains. genetic and phylogenetic analyses indicated that the 2018 isolates were closest to the 2014 g2b re‐emergent jeju strains, but appeared to have undergone substantial rapid independent evolution. among the isolates, a notable nsp3 del variant strain, kor/knu‐1807/2018, was isolated and propagated by continuous passages in vero cells, and displayed typical pedv‐induced syncytia formation. genomic sequencing identified a unique 8‐nt del in the extreme c‐terminal region of the s gene at the 4th passage (knu‐1807‐p4) compared to its original sample. this del resulted in the premature termination of s by nine amino acid residues (evfekvhvq), which contained a kxhxx motif that is a potential endoplasmic reticulum retrieval signal. in vivo animal studies showed that variant strain knu‐1807 had decreased virulence in suckling piglets. these results advance our knowledge regarding the genetic variation and pathogenicity of the g2b pedv endemic strains prevalent in jeju swine herds in south korea. since its emergence in south korea in 1992, small-to large-scale pedv epizootics have occurred annually throughout the nation, leading to substantial economic losses in domestic pig production (lee, 2015 (lee, , 2019 . the 2013 ped pandemic that ravaged the united states (stevenson et al., 2013) also struck the korean peninsula and decimated more than 40% of the pig farms across the country during 2013-2014 (lee, 2015 (lee, , 2019 . subsequently, in late march 2014, the virus invaded jeju island located 80 km from the south korea mainland at the closest point, which had maintained pedv-naïve status for a decade, causing massive neonatal mortality in provincial herds. genetic and phylogenetic analyses revealed that the re-emergent jeju island pedv isolates were most closely related to the pandemic genogroup 2b (g2b) strains that were responsible for the 2013-2014 global outbreaks, suggesting a direct introduction of the virus from the mainland of south korea via unknown contaminating sources . even with province-wide vaccination or intentional virus-exposure practices being implemented in order to provide herd immunity around the areas that contain dense swine populations, pedv has continued to plague the provincial pork industry. since several pig farms have experienced recurrent pedv outbreaks within a single year, ped has become endemic on jeju island . in this study, we determined the complete genome sequences of field isolates on jeju island to investigate the diversity of the pedvs responsible for the ongoing endemic outbreaks. additionally, we isolated and serially cultured a novel pedv strain, kor/knu-1807/2018, in cell culture and investigated its genotypic and phenotypic characteristics in vitro and in vivo. in early 2018, mild sporadic suspect-pedv outbreaks with low mortality rates in newborn piglets occurred on several farms in the hallim and daejeong areas of jeju province. small intestine (si) specimens were collected at 18 different pig farms located in those districts from january through june 2018 from dead piglets that had acute diarrhoea. intestinal homogenates were prepared as 10% (wt/vol) suspensions in phosphate-buffered saline (pbs) using a magna lyser instrument (roche diagnostics, mannheim, germany) with three rounds of 15 s at a force of 8,000 g. the suspensions were then vortexed and centrifuged for 10 min at 4,500 g (hanil centrifuge fleta5, incheon, south korea). the clarified supernatants were initially subjected to rt-pcr analysis using an i-tge/ped detection kit (intron biotechnology) according to the manufacturer's instructions. pedv-positive samples were filtered through a 0.22-μm-pore syringe filter (millipore) and stored at −80°c until subsequent sequencing analysis and virus isolation were performed. the s glycoprotein gene sequences of the virus isolates were determined by traditional sanger methods. two overlapping cdna fragments spanning the entire s gene of each isolate were amplified by rt-pcr as previously described (lee, park, kim, & lee, 2010) . the individual cdna amplicons were gel-purified, cloned using the pgem-t easy vector system (promega) and sequenced in both directions using two commercial vector-specific t7 and sp6 primers and gene-specific primers. in addition, the complete genomes of representative pedv field strains were also sequenced. ten overlapping cdna fragments spanning the entire genome of each virus strain were rt-pcr-amplified as described previously (lee, kim, & lee, 2015; and each pcr product was sequenced as described above. the 5′ and 3′ ends of the genomes of the individual isolates were determined by rapid amplification of cdna ends (race) as described previously . the full-length s gene or whole-genome sequences of the 2018 viruses have been deposited in the genbank database under the accession numbers shown in figure 2a. the sequences of 66 fully sequenced s genes and 39 complete genomes of global pedv isolates were independently used in sequence alignments and phylogenetic analyses. multiple sequence alignments were generated using the clustalx 2.0 program (thompson, gibson, plewniak, jeanmougin, & higgins, 1997) and the percentages of nucleotide sequence divergences were assessed using the same software. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbourjoining method and subsequently subjected to bootstrap analysis with 1,000 replicates to determine the percentage reliability values of each internal node of the tree (saitou & nei, 1987) . all phylogenetic trees were generated using mega 4.0 software (tamura, dudley, nei, & kumar, 2007) . porcine epidemic diarrhoea virus isolation was performed using vero cells in the presence of trypsin (usb) as described previously . briefly, inocula were prepared by adding trypsin (usb) to intestinal suspensions to a final concentration of 10 μg/ml. confluent vero cells grown in 6-well plates were washed with pbs and inoculated with 400 μl of each trypsincontaining inoculum. after incubating at 37°c for 1 hr to allow for viral adsorption, 2 ml of virus growth medium consisting of alpha minimum essential medium (α-mem; invitrogen) supplemented with antibiotic-antimycotic solutions (100×; invitrogen), 0.3% tryptose phosphate broth (tpb; sigma), 0.02% yeast extract (difco), 10 mm hepes (invitrogen) and 5 μg/ml of trypsin was added to each well. the inoculated cells were maintained at 37°c under 5% co 2 and monitored daily for cytopathic effects (cpe). when ~70% of cells showed cpe, the infected cells were subjected to three rounds of freezing and thawing. the culture supernatants were then collected and centrifuged for 10 min at 400 g and filtered through a 0.22-μm pore filter. the clarified supernatants were aliquoted and stored at −80°c as passage 1 (p1) viral stocks for use in plaque purification and subsequent serial passaging. if cpe and rt-pcr results were negative after five blind passages, virus isolation was considered negative for those samples. vero cells grown on microscope coverslips placed in 6-well tissue culture plates were mock infected or infected with pedv at a multiplicity of infection (moi) of 0.1. the virus-infected cells were cultured until 24 hr, fixed with 4% paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked with 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated for 2 hr with a monoclonal antibody (mab) specific for pedv n protein (choogang vaccine laboratories). after being washed five times with pbs, the cells were incubated for 1 hr at rt with a goat anti-mouse secondary antibody conjugated to alexa fluor 488 (invitrogen) followed by counterstaining with 4′,6-diamidino-2phenylindole (dapi; sigma). the coverslips were mounted onto glass microscope slides using mounting buffer and the stained cells were visualized using a fluorescence leica dm il led microscope (leica). vero cells were infected with each passage of knu-1807 virus stock in the presence of trypsin as described above. the culture supernatants were collected at 24 or 48 hr post-infection (hpi) when 70% cpe had commonly developed. for growth kinetics experiments, supernatants were harvested from cells infected with each selected passage virus at various time points (6, 12, 24, 36 and 48 hpi) and stored at −80°c. virus titres were measured by end-point titration in 96-well plates using 10-fold serial dilutions of the samples in triplicate for each dilution to determine the amount of virus required to produce cpe in 50% of the inoculated vero cells. the 50% tissue culture infectious dose (tcid 50 ) per ml of virus stock was calculated using the reed-muench method (reed & muench, 1938) . viral rna was extracted from virus supernatants from infected vero cells and faecal specimens using an i-tge/ped detection kit according to the manufacturer's protocol. quantitative real-time rt-pcr was performed using a one step primescript rt-pcr kit (takara) and primers (forward primer 5′-acgtccctttactttcaattcaca-3′, reverse primer 5′-tatacttggtacacacatccagagtca-3′) and a probe (5′-fam-tgagttgattactggcacgcctaaaccac-bhq1-3′) described elsewhere (kim et al., 2007; sagong & lee, 2011) . amplification of the reaction mixtures was performed using a thermal cycler dice real time system (takara) and the results were analysed using software as described previously sagong & lee, 2011 ). the in vivo swine studies were performed at the choongang vaccine laboratory animal facility under the guidelines established by its institutional animal care and use committee. a total of nine 3-dayold suckling piglets were obtained from commercial cross-bred sows (great yorkshire × dutch landrace) at a conventional breeding farm with a good health record and either vaccinated against pedv or no known prior ped outbreak. all animals were confirmed negative for pedv, transmissible gastroenteritis virus (tgev), porcine deltacoronavirus and porcine rotaviruses by virus-specific rt-pcr analysis of rectal swabs and determined to be free of antibodies to pedv, tgev and porcine reproductive and respiratory syndrome virus (prrsv) by serum neutralization tests as described previously and a commercial prrsv antibody elisa kit (herdchek prrs x3; idexx laboratories). pigs were randomly assigned to three experimental groups: the highly virulent knu-141112-p5-inoculated group (n = 3) , the knu-1807-p10-inoculated group (n = 4) and the sham-inoculated control group (n = 2). animals were fed commercial milk replacer (3-4 times daily) and had ad libitum access to water for the 5-day duration of the study. following a 2-day acclimation period, piglets (5-day old) in the virus-inoculated groups received an oral 1-ml dose of 10 3.0 tcid 50 /ml of the appropriate virus, which was equivalent to 100 median pig diarrhoea dose pdd 50 of knu-141112 (baek et al., 2016; lee et al., 2015 . the sham-inoculated pigs were administered with cell culture media as a placebo. animals were monitored three times daily throughout the experiment for clinical signs of vomiting and diarrhoea and for mortality. stool samples from the pigs in all groups were collected prior to inoculation and thereon daily using 16-inch cotton-tipped swabs. the pedv faecal shedding titres were determined by real-time rt-qpcr as described above. a pedv isolate with a known infectivity titre was 10-fold serially diluted to generate a standard curve in each pcr at necropsy, small intestine tissue specimens (<3 mm thick) were collected from each piglet, fixed in 10% formalin for 24 hr at rt, and embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut at 5-8-μm thick sections using a microtome (leica), floated in a 40°c water bath containing distilled water, and transferred to glass slides. the tissues were then deparaffinized in xylene for 5 min and rehydrated in decreasing concentrations of ethanol (100%, 95%, 90%, 80% and 70%, respectively) for 3 min each. the deparaffinized intestinal tissue sections were stained with haematoxylin and eosin (sigma) for histopathology or subjected to immunohistochemistry (ihc) using pedv n-specific mab as described previously . villous height and crypt depth were also measured throughout the h&e-stained jejunal sections and the mean ratio of jejunal villous height to crypt depth (vh:cd) was calculated as described previously (jung, kim, ha, choi, & chae, 2006 ). all values are expressed as the means ± standard deviation of the means (sdm). all statistical significances were evaluated by a we in vero cells, reaching a titre >10 6 tcid 50 /ml by 12 hpi (figure 3b ). in addition, we sequenced the coding region of nsp3 and the entire s gene of strain knu-1807 at the first productive passages of p4 and table 4 ). these genetic alterations that occurred in p4 remained unchanged through p10 and no additional variations emerged in the s-coding region during the subsequent serial passages. the partial or complete genome sequences of all the cell culture-passaged viruses were compared to the original knu-1807-si and the results are summarized in table 4 . since the knu-1807 strain with the truncated s cytoplasmic tail (0) 100 (0) nsp10 (405) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 99.2 (1) nsp11 (54) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) 100 (0) nsp12 ( the number of individual differences in the 5′-utr, protein-coding region, and 3′-utr, respectively. f i g u r e 2 phylogenetic analyses based on the nucleotide sequences of the spike (s) genes (a) and full-length genomes (b) of the pedv strains. a region of the s gene and the complete genome sequence of tgev were included as the outgroups in each tree. multiple sequence alignments were performed using clustalx software and phylogenetic trees were constructed from the aligned nucleotide sequences using the neighbour-joining method. numbers at each branch are bootstrap values greater than 50% based on 1,000 replicates. the names of the strains, countries and dates ( in the current study, determined the complete genome sequences similar to other coronavirus replicase-encoded nsps, many pedv nsps (nsp1, nsp3, nsp5, nsp7, nsp14, nsp15 and nsp16) function as interferon (ifn) antagonists that modulate the innate immune response (wang et al., 2016; zhang, shi, & yoo, 2016) . along with variations extensively dispersed throughout the s gene, it would be interesting to identify mutations in these nsp genes, including indels that possibly contribute to the pathogenesis of pedv. a majority of the non-silent point mutations, which appeared to have resulted from their continuous accumulation in the field over the past 3-4 years, occurred in the orf1ab region encoding 16 nsps, particularly in nsp3. these 29-41 variations seem to be significant since the cell culture-attenuated g2b strain contained only 4-aa changes over 100 serial passages when compared to the virulent parental strain . although no indels arose in the attenuated g2b virus s del5/orf3 , the attenuated g1a-derived vaccine strains possess an 8-aa del in nsp3, which overlaps or is located 12 or 18 aa downstream of those found in the recent g2b field ta b l e 4 nucleotide and amino acid changes of knu-1807 during serial passages in cell culture strains, implicating the potential involvement of the nsp3 del in attenuation ( figure 1b) . although nsp3 critically acts as a pl pro that post-transcriptionally cleaves replicase polyproteins into functional nsps, the dels present in the glu-rich acidic region of nsp3 have no effect on its own roles and therefore are dispensable for the replication of coronaviruses, including pedv (lee & lee, 2018; lei, kusov, & hilgenfeld, 2018) . consistent with our previous study (lee & lee, 2018) , this tolerance for the nsp3 del during pedv replication was reproduced in the current study as shown in to contain a homologous 3-aa del in nsp3, consistent with a typical farm-to-farm transmission of pedv via lax biosecurity. in addition to genetic drift under field conditions, an outstanding del in the cytosolic endodomain of s was identified in the novel nsp3-del pedv knu-1807 isolate since the first productive passage in cell culture (p4) and subsequently retained, thereby resulting in a 9-aa del at the end of the knu-1807-p4 s protein. like other coronaviruses, the pedv s glycoprotein can be functionally divided into two subdomains, s1 responsible for binding to cell receptor(s) and s2 involved in direct fusion between the viral and cellular membranes (lee, 2015) . the last 5-aa (kvhvq) of the cytoplasmic tail of s is known to be a potential er retrieval signal with its kxhxx motif and loss of this motif increases cell fusion activity by defecting the er retention of s protein and promoting their transport to the cell surface lin et al., 2017; shirato et al., 2011) . since knu-1807-p4 exhibited a premature termination of the s protein by 9-aa residues (evfekvhvq) that included the er retrieval motif, it would be anticipated that this strain would demonstrate increased fusion activity in vitro. our data revealed that the knu-1807 strain in the s gene, resulting in a stop codon and a 9-aa (evfekvhvq) del. the s c-del9 identified in this study differed from aforementioned strains in that the early termination was caused by a combination of a novel nt del and a −1 frameshift event that occurred in the most primitive cell culture passage p4. it is possible that the s del naturally emerged during the initial culturing in the vero cell via an unknown mechanism. indeed, the occurrence of a large 197-aa s del in the n-terminus of the s protein that was absent in the original sample has been reported during the first cell culture passage of the us g2b pc177 strain (oka et al., 2014) . we were consistently unable to detect the same s c-del9 in the original knu-1807-si sample by deep genome sequencing (data not shown). however, since coronaviruses are known to innately exist as quasispecies or as mixed populations of several strains (zhang et al., 2007) , we cannot exclude the possibility that this variant was initially present as a minor proportion in the clinical sample and was able to replicate more competently in cell culture instead of the emergence of the extraordinarily del during the earliest stages of cell adaption. similarly, a chinese field g2b strain, fl2013, that naturally contains a unique 21-nt del in the extreme c-terminus of its s protein, leading to a 7-aa (fekvhvq ) s c-del pattern, has been found to have reduced virulence to newborn piglets (zhang et al., 2015) . thus, it is important to further surveil whether s c-del variant strains naturally circulate in korean pig populations. since a growing body of evidence proposes the early termination of the s protein and the extinction of its er retention signal as being a marker for pedv attenuation, we investigated whether the virulence of the knu-1807 virus would be markedly less than that previously reported for pedv pathogenesis. our data revealed that despite similarities of macroscopic and microscopic small intestine lesions, the knu-1807 virus possessed weakened pathogenicity in experimentally inoculated piglets compared to the virulent knu-141112 strain in terms of disease severity of clinical presentation, including the mortality rate and onset of virus shedding, indicating the involvement of the extreme c-terminal region of the s protein in virulence. however, we were unable to observe a fully attenuated phenotypes for knu-1807 in inoculated animals compared with those seen in our previous study . this suggested that del mutations in several parts of the s protein in conclusion, pedv continues to endemically affect pig farms on jeju island, giving rise to moderate-to-severe clinical disease associated with ped in infected piglets. however, the loss of neonates to death varies among litters and between farms and is less than that reported for severe g2b epidemics, which approach 100% mortality in newborn piglets. this mild-to-moderate outbreak scenario seen on jeju island may be due to herd immunity developed from vaccination and intentional infection since the re-emergence of pedv in the area. another possibility is that genetic variation and/or del in nsps and s protein have arisen under field circumstances leading to the evasion of host immune defenses, such as ifn and neutralizing antibodies, and consequently alter viral pathogenicity, leading to endemic and low-pathogenic outbreaks in the field. thus, cutting-edge research using reverse genetics will be necessary to provide fundamental insights into the specific role of nsp and s gene mutations in pedv pathogenesis. more importantly, the current study confirmed that the contemporary field g2b isolates have nearly 4% amino acid sequence divergence in the s protein compared to the 2013-2014 domestic g2b strains. this mutation rate is approximately twice as high as that in recent mainland strains of pedv (lee & lee, 2018) . furthermore, the evolutionary rate estimated for the s gene of pedv g2b jeju island strains was 14.80 × 10 -4 substitutions/site/ year, whereas that for the g2b mainland strains was 7.18 × 10 -4 substitutions/site/year (lee & lee, 2018) . these results indicated that ongoing genetic drift appears to be faster on jeju island than the authors declare that they have no conflict of interest. changhee lee https://orcid.org/0000-0002-5930-5461 efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets nidovirales: evolving the largest rna virus genome the effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets multiplex real-time 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2015-01-27 journal: transbound emerg dis doi: 10.1111/tbed.12318 sha: doc_id: 291026 cord_uid: 99cit4ig classical swine fever (csf) is an oie‐listed disease that can have a severe impact on the swine industry. user‐friendly, sensitive, rapid diagnostic tests that utilize low‐cost field‐deployable instruments for csf diagnosis can be useful for disease surveillance and outbreak monitoring. in this study, we describe validation of a new probe‐based insulated isothermal reverse transcriptase pcr (iirt‐pcr) assay for rapid detection of classical swine fever virus (csfv) on a compact, user‐friendly device (pockit (™) nucleic acid analyzer) that does not need data interpretation by the user. the assay accurately detected csfv rna from a diverse panel of 33 csfv strains representing all three genotypes plus an additional in vitro‐transcribed rna from cloned sequences representing a vaccine strain. no cross‐reactivity was observed with a panel of 18 viruses associated with livestock including eight other pestivirus strains (bovine viral diarrhoea virus type 1 and type 2, border disease virus, hobi atypical pestivirus), african swine fever virus, swine vesicular disease virus, swine influenza virus, porcine respiratory and reproductive syndrome virus, porcine circovirus 1, porcine circovirus 2, porcine respiratory coronavirus, vesicular exanthema of swine virus, bovine herpes virus type 1 and vesicular stomatitis virus. the iirt‐pcr assay accurately detected csfv as early as 2 days post‐inoculation in rna extracted from serum samples of experimentally infected pigs, before appearance of clinical signs. the limit of detection (lod (95%)) calculated by probit regression analysis was 23 copies per reaction. the assay has a sample to answer turnaround time of less than an hour using extracted rna or diluted or low volume of neat serum. the user‐friendly, compact device that automatically analyses and displays results could potentially be a useful tool for surveillance and monitoring of csf in a disease outbreak. classical swine fever virus (csfv) is a member of the genus pestivirus of the family flaviviridae (wengler 1991) . the virus causes disease that ranges from mild to severe in its natural hosts, domestic pigs and wild boars. classical swine fever virus is an enveloped virus with a positive-sense, single-stranded rna genome of approximately 12 300 nucleotides. the genome encodes a single open reading frame that is flanked by two untranslated regions (utrs) at the 5 0 and 3 0 ends. classical swine fever virus is currently categorized into three genotypes: 1, 2 and 3 that can be further divided into 11 subtypes: 1.1, 1.2, 1.3, 1.4, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3 and 3.4 (lowings et al., 1996; paton et al., 2000; postel et al., 2013) . other pestiviruses that infect livestock are bovine viral diarrhoea virus type 1 (bvdv-1) and type 2 (bvdv-2), border disease virus (bdv) of sheep, the recently described atypical pestiviruses (schirrmeier et al., 2004; st ahl et al., 2010) and bungowannah virus (kirkland et al., 2007) . classical swine fever (csf) is an oie-listed notifiable disease that can have an enormous effect on the livestock industry and on trade. for instance, the direct costs of the 1997-1998 epizootic in the netherlands, excluding loss of exports, amounted to $2 billion (u.s.) and the slaughter of approximately 10 million pigs (terpstra and de smit, 2000) . while csfv has been eradicated from regions such as north america, australasia and parts of northern europe, it remains widespread in regions that include south america, eastern europe and south-east asia (vargas et al., 2004) . pestiviruses are closely related genetically and antigenically. while infections in swine with pestiviruses other than csfv are usually benign, they do present diagnostic problems: they cause serological cross-reactions in antibody tests (colijn et al., 1997; langedijk et al., 2001) . virus isolation is the method of choice for discovering infected herds at the early stage of infection (terpstra and de smit, 2000) . however, rapid and sensitive tests for early detection of csfv are desirable for containing outbreaks as conventional virus isolation procedures are time-consuming and require cell culture infrastructure and expertise. several conventional rt-pcr and real-time rt-pcr assays have been described for detection of csfv and other pestiviruses (handel et al., 2004; hoffmann et al., 2005 hoffmann et al., , 2011 risatti et al., 2005; wernike et al., 2013) . however, the high cost of current real-time pcr devices, and the need for postamplification processing with conventional pcr may be a barrier to the widespread use of these technologies in field applications or in developing countries. thus, low-cost, sensitive and user-friendly methods that can detect csfv rapidly are still needed. insulated isothermal pcr (iipcr) utilizes a single heat source below the reaction vessel to generate temperature gradients by rayleigh-benard convection (krishnan et al., 2002) . the denaturation, annealing and extension steps of a pcr occur in different zones in the reaction vessel (krishnan et al., 2002) . the iirt-pcr assay used in this study utilizes a simple, compact pockit tm nucleic acid analyzer that detects optical signals from a target-specific fluorescent-labelled probe for highly sensitive and specific detection of the target virus. rapid assays using this platform have been used for the detection of pathogens such as white spot syndrome virus (tsai et al., 2014) and salmonella in chicken meat (tsen et al., 2013) . in this study, the validation of a simple iirt-pcr assay for detection of csfv is described. the assay uses lyophilized reagents and can be performed on a simple portable instrument that provides automated data analysis and results readout within 1 h of addition of purified rna or a low volume of unextracted serum. samples and nucleic acid extraction rna from a panel of 30 csfv strains and isolates (table 1 , strains without the superscript 'a') used in this study was obtained from dr. irene greiser-wilke (eu reference laboratory for fmd, hanover, germany). archived viral nucleic acid from 18 laboratory-amplified non-csf viruses including eight other pestiviruses (bvdv 1-hastings, singer and ny1 strains; bvdv 2-ames 125c, 890, 24515 strains; bdv-coos bay; hobi atypical pestivirus), african swine fever virus-lisbon, swine vesicular disease virus-itl 19/92, porcine respiratory and reproductive syndrome virus-ynl, swine influenza virus (h3n2), porcine circovirus 1 (pcv1, derived from infectious clone based on genbank accession no. ay184287), porcine circovirus 2 (pcv2, derived from infectious clone based on genbank accession no. ef394779), porcine respiratory coronavirus-isu, vesicular exanthema of swine virus, bovine herpesvirus 1-edmonton 5 and vesicular stomatitis virus-ind 1 strain were used to determine the specificity of the iirt-pcr. viral rna for pcv1 and 2 was provided by dr. marcus czub (university of calgary). rna was extracted from a total of 91 sera samples obtained from 12 healthy pigs and 12 pigs inoculated with four csfv strains. briefly, 7 to 8-week-old pigs were inoculated oronasally with the peru ll28/2008 la libertad-trujillo (n = 4), peru l8/2008 lima-villa el salvador (n = 4), honduras 1997 (n = 2) and diepholz 1/han94 (n = 2) strains. serum samples that were tested fresh without rna extraction were from pigs inoculated with the koslov strain using the same method. animals were anesthetized with isoflurane by face mask and 1.5-2.0 ml of inoculum containing 10 6 50% tissue culture infectious doses (tcid 50 ) of each virus was administered per animal. sera were collected before inoculation and also on various days post-infection (dpi) for 4 or 5 weeks, or until the animal died or was euthanized. viral rna was extracted from serum samples using the magmax-96 ai/nd viral isolation kit (life technologies, carlsbad, ca, usa) according to the manufacturer's instructions using the magmax express 96 processor (life technologies) and eluted with 50 ll of elution buffer. all laboratory-amplified csfv strains, bvdv 1-singer, bdv-coos bay, asfv-lisbon, svdv-itl 19/92 and vsv-ind 1 were assayed at 1/50 dilutions of nucleic acid extracted from cell culture supernatant, while the remaining non-csf viruses were assayed at neat quantities. extracted nucleic acid from all serum samples were also assayed at neat quantities. all procedures dealing with animal inoculation and care complied with the guidelines of the canadian council on animal care and were approved by the institutional animal care committee. in vitro-transcribed rna was generated from a plasmid containing a fragment of the 5 0 utr of csfv strain hclv (genbank accession no. af531433) using the maxiscript t7 kit (ambion, austin, tx, usa). residual dna was removed using the ambion turbo dna-free kit (life technologies). the concentration of rna was measured by a nanodrop 1000 spectrophotometer (thermo fisher scientific, waltham, ma, usa). serial dilutions of in vitrotranscribed rna were made in 40 ng/ll yeast trna to determine the limit of detection. single-use aliquots were stored at à80°c until use. the csfv iirt-pcr assay was designed and manufactured by genereach usa (lexington, ma, usa) on the basis of the probe hydrolysis-based pockit tm method described previously (tsai et al., 2012) . briefly, 763 nucleotide sequences of the csfv 5 0 utr region were collected from the csf database, eu and oie reference laboratory for csf (http://viro60.tiho-hannover.de/eg/csf/ index.php) and aligned to identify conserved regions that can be used for the design of iirt-pcr primers and probes. classical swine fever virus 5 0 utr-specific primer and probe set were designed according to the recommended principles for iipcr (http://www.iipcr.com/eweb/ uploadfile/20130522114104277.pdf). the recommended length of the primers was between 18 and 30 bases, have a gc content of 45-60% and a t m of 56-60°c. repeat sequences, four or more consecutive gs or cs and hairpins and primer dimers were avoided. the probe had a length of <30 bases, a gc content between 30% and 80% and a t m that was 10°c above the t m of the primers. runs of a single nucleotide and four or more consecutive gs or a g at the 5 0 end of the probe were avoided. the amplicon was approximately 100 bp, within the recommended range of 70-150 bp. no major secondary structures were found in the amplicon, based on prediction made by the mfold program (http://mfold.rna.albany.edu/ ?q=mfold). lyophilized iirt-pcr reagent was rehydrated with 50 ll of premix buffer (genereach usa), and 5 ll of sample was added to the mixture. for laboratorycultured virus samples, 4 ll of dh 2 o was added to 1 ll of rna or dna, while 5 ll of neat extracted rna, various volumes of neat unextracted serum and unextracted serum diluted 1 : 2, 1 : 5 or 1 : 10 in dh 2 o were used for serum samples. both fresh and archived serum from csfv infected pigs were used as indicated. subsequently, 50 ll of the final mixture was transferred to an r-tube tm (gene-reach usa), which was spun briefly in a cubee tm mini centrifuge (genereach usa). the r-tubes tm were placed into the reaction chamber of the pockit tm nucleic acid analyzer and a run was initiated. the pockit tm nucleic acid analyzer collected optical signals through an integrated circuits controller-regulated (cmos) sensor, and signal-to-noise (s/n) ratios were automatically calculated by dividing light signals collected after iirt-pcr by those from before iirt-pcr (tsai et al., 2012) . according to default s/n thresholds, results were converted automatically to '+', 'à', or '?' and displayed on the screen at the end of the run. all samples tested gave either '+' or 'à' result, with the exception of 5 ll of undiluted and unextracted serum, which all gave '?' results. statistical probit analysis, a nonlinear regression model, was performed using commercial software spss 14.0 (spss inc., chicago, il, usa) to determine limit of detection with 95% confidence (lod 95% ). real-time rt-pcr for csf was performed with the cepheid smart cycler ii (cepheid, sunnyvale, ca, usa) for samples from pigs inoculated with the peru l8/2008 and ll28/2008 strains, or the abi 7900 ht (life technologies) for samples from pigs inoculated with the diepholz 1/han 94 and honduras 97 strains as per the standard operating procedure at the canadian reference laboratory for csf (arainga et al., 2010) . each reaction had a total volume of 25 ll and consisted of 9 ll of rna added to 16 ll of master mix. testing of the iirt-pcr assay with laboratory-amplified samples nucleic acid from a panel of laboratory-amplified csfv strains (n = 30, table 1 , strains without superscript 'a') and non-csfv viruses that affect livestock (n = 18) were used to determine the specificity of the iirt-pcr assay. the iirt-pcr utilized a pair of 23-mer primers and a 19-mer probe ( table 2 ). the iirt-pcr assay accurately detected all csfv rna samples which represented all three genotypes, and eight of 11 subgenotypes that were available for testing and gave negative results for three bvdv type 1 strains, three bvdv type 2 strains, bdv, hobi atypical pestivirus, asfv and nine other viruses that affect livestock. data from replicate assays of strains representing each of the three csfv genotypes and two non-target pestiviruses performed on two different poc-kit tm instruments were analysed to evaluate assay reproducibility. all four replicates conducted with each of the three csfv strains and triplicates of the two non-target viruses gave consistent and expected results. the average and standard deviation of s/n values observed for alfort (genotype 1), diepholz (genotype 2), and kanagawa (genotype 3) were 4.14 ae 0.11, 4.01 ae 0.09 and 4.08 ae 0.07, respectively. for the non-target pestiviruses, the average and standard deviation of the s/n ratios were 0.99 ae 0.03 for bvdv type 1-hastings and 0.97 ae 0.02 for bvdv type 2-890. the iirt-pcr assay was tested with rna from 12 serum samples of healthy pigs and 79 serum samples from pigs infected with four different csf strains: honduras 1997, diepholz 1/han94, peru ll28/2008 and peru l8/2008 (table 3 ). all serum samples taken from the healthy pigs as well as all 12 dpi 0 samples taken before pigs were experimentally infected with csfv gave negative iirt-pcr results, indicating 100% specificity for these samples. the earliest dpi samples that generated positive iirt-pcr results were dpi 2 samples from pigs infected with the peru ll28 strain (one of two pigs), dpi 3 samples from peru l8/2008 and 1997 honduras strain infected pigs (four out of four pigs) and dpi 5 samples from pigs infected with the diepholz strain (two of two pigs) ( table 3 ). the diepholz 1/han94 strain was also tested with laboratoryamplified material (table 1) ; however, the honduras 1997, peru ll28/2008 and peru l8/2008 strains were new strains that were only tested with clinical material. thus, the total number of csfv strains detectable by the iirt-pcr assay is 34 (33 virus strains plus in vitro-transcribed rna from synthetic csfv-hclv sequence used in the limit of detection analysis). all samples from the 12 experimentally infected pigs from time points later than the earliest dpi that gave positive results by iirt-pcr were positive. the last time points tested in this study were dpi 15 for diepholz 1 han/94, 11 for honduras 1997, 30 for peru ll28/ 2008 and 12 for peru l8/2008 (table 3) . rna from a total of 78 serum samples was tested with both the iirt-pcr and real-time rt-pcr (table 3 ). all 12 dpi 0 samples taken before the experimental inoculations were negative by both iirt-pcr and real-time rt-pcr. of the remaining 66 samples, 61 gave identical results by both methods. one dpi 2 sample from a pig inoculated with the peru ll28 strain and two dpi 3 samples from pigs inoculated with the honduras 1997 strain were positive by iirt-pcr, but negative by real-time rt-pcr. in addition, two dpi 3 and one dpi 4 sample from pigs inoculated with peru l8/2008 strain were positive by iirt-pcr, but had realtime rt-pcr c t values that were greater than the cut-off for positivity (table 3) . samples taken at all later time points were positive by both assays. these results suggest that iirt-pcr may be more sensitive than the real-time rt-pcr assay for certain strains. serial dilutions of in vitro-transcribed rna were used to evaluate the analytical sensitivity of the optimized csfv iirt-pcr and real-time rt-pcr. the percentages of positive results were 100.0% (10/10), 100.0% (20/20), 85% (17/ 20) and 0% (0/15) for 100, 50, 20 and 0 copies of the in vitro-transcribed rna, respectively. the lod 95% calculated by probit regression analysis was 23 copies per reaction. these results were comparable to the results obtained with the real-time rt-pcr using both smart cycler ii and abi 7900ht instrument platforms. the lowest copy detected by the smart cycler ii was 28 copies (average c t value for three replicates was 33.57), while the lowest copy detectable by the abi 7900ht instrument was 31.4 copies (average c t value for three replicates was 37.006). the ability to directly detect virus in clinical samples without nucleic acid extraction can simplify an assay and shorten the turnaround time needed to obtain results. the iirt-pcr successfully detected csfv rna when up to 4 ll of neat, fresh (unfrozen), unextracted dpi 10 serum from pigs inoculated with the koslov strain was used (table 4) . positive results were also obtained with fresh unextracted serum samples that were diluted 1 : 2 and 1 : 5, and with archived (frozen) serum samples that were thawed and diluted 1 : 2, 1 : 5 and 1 : 10 (table 4 ). in this report, a highly sensitive and specific iirt-pcr for the rapid detection of csfv is described. the assay detected rna from a broad range of csfv strains (n = 34) representing all three genotypes and did not show crossreactivity with other pestiviruses and other livestock-associated viruses. for the testing of serum samples from experimentally infected pigs, less rna (5 ll) was used for the iirt-pcr than the real-time rt-pcr (9 ll) routinely 1, 3, 5, 7, 9, 11, 15 5 (2/2) 5 (2/2) honduras 97 14 0, 1, 3, 5, 7, 9, 11 3 (2/2) 3 (0/2) 5 (2/2) 5 (2/2) peru l8/2008 22 0, 1, 2, 3, 4, 5, 9, 10, 11, 12 3 (2/2) 3 (0/2) a 4 (2/2) 4 (1/2) a peru ll28/2008 27 0, 1, 2, 3, 4, 5, 6, 7, 12, 14, 21, 28 2 (1/2) 2 (0/2) 30 b 3 (2/2) 3 (2/2) iipcr, insulated isothermal pcr. the dpi 30 sample was only tested with iirt-pcr, but not tested by real-time rt-pcr. employed at the canadian reference laboratory for csf, and the iirt-pcr detected honduras 1997, peru l8/2008 and ll28/2008 rna in serum samples that were negative by the 40 cycle real-time rt-pcr assay. these results suggest that the iirt-pcr may be slightly more sensitive than the real-time rt-pcr at least for certain strains. the sensitivity of the real-time rt-pcr may be improved by increasing the cycle number; however, this will also increase the time required to complete the assay. real-time pcr will allow simultaneous amplification and quantitative realtime detection of amplification, while iirt-pcr is an endpoint assay. the manufacturer set threshold for positivity was used in this study for the iirt-pcr assay, but it is possible for the user to adjust the cut-off for positivity with custom programs. not including the time needed to extract rna, the iirt-pcr took less than an hour to complete, much less than the approximately 2-2.5 h needed for the real-time rt-pcr used in this study. the observation that iirt-pcr can detect csfv rna in up to 4 ll of fresh unextracted serum suggests this assay may be used for direct detection of csfv in serum with a total turnaround time of approximately an hour. an inverse correlation was observed between the s/n value and the volume and concentration of unextracted serum used in the iirt-pcr. these results suggest that 4 ll of neat unprocessed serum and 5 ll of 1 : 2 diluted unprocessed serum may be close to the maximum amount of unextracted material that can be used in the csf iirt-pcr assay. whether other types of unextracted clinical material and the minimum amount of processing that can be successfully used in iipcrs remain to be determined. loop-mediated isothermal amplification (lamp) methods that do not require expensive instrumentation have been suggested as suitable platforms for field applications (notomi et al., 2000) . several in-house lamp assays have been developed for the detection of csfv nucleic acids and tested with regional isolates or strains representing a small number of csfv subgenotypes, and few if any non-csfv pestiviruses (chen et al., 2009; yin et al., 2010; zhang et al., 2010) . in contrast to earlier csfv-specific lamp assays which utilized agarose gel electrophoresis or visualization with the naked eye for interpretation of results, a lamp assay combined with a lateral flow dipstick (lfd) that was validated with a large panel of pestivirus strains was recently described (chowdry et al., 2014) . in this study, seven (1.1, 1.2, 1.3, 2.1, 2.2, 2.3 and 3.1) of eight subgenotypes tested were detected, but the assay failed to detect the genotype 3.4 kanagawa strain, perhaps due to mismatches with the primers used in the assay (chowdry et al., 2014) . the lamp-lfd assay had an analytical sensitivity of approximately 100 copies per reaction on the two genotypes tested. in contrast, the iirt-pcr described in this study detected strains representing all eight subgenotypes tested, including the kanagawa strain, and had a limit of detection of about 23 copies, suggesting the iirt-pcr assay has broader coverage and may be more sensitive than the lamp-lfd assay. both the rt-lamp and iirt-pcrs are simple to perform and can be completed in approximately an hour. rt-lamp assays can be performed with just a water bath or heat block and results can be visualized without instrumentation. thus, lamp assays that do not require a signal detector may be less costly to perform than iirt-pcr if cost, and not sensitivity is the primary concern. however, genetic variations of target viruses can have a big impact on the sensitivity and reliability of a lamp assay due to the use of 4-6 primers. thus, use of lamp assays where broader genetic variation of csfv may be encountered will require further validation with more genetically diverse csfv and non-csfv samples. the iirt-pcr assay for csfv reported here is commercially available and utilizes lyophilized reagents, while the lamp assays for csfv detection reported to date are in-house assays using wet reagents. thus, further development and validation of the lamp assays using lyophilized reagents are desirable for on-farm detection. the compact pockit tm nucleic acid analyzer instrument with its lyophilized reagents and automated data analysis and result readout is easy to use. the requirement for a separate rna extraction step requiring additional instrumentation and the maximum throughput of eight samples per run for the instrument used in this study may hinder its use as an instrument for high-throughput applications in the laboratory and at the point-of-need. however, an instrument with a throughput of 24 samples per run is available and with the high sensitivity of the iirt-pcr assay, pooling of samples should be feasible. in combination with existing commercial portable automated nucleic acid extraction systems, the pockit tm nucleic acid analyzer, may have potential as a low-throughput automated two-step method for on-site detection. the observation that low volume of serum can be used directly in iirt-pcr without nucleic acid extraction can potentially simplify the diagnostic workflow considerably in the laboratory and in the field, and shorten the time needed to obtain results from serum samples to <1 h. phylogenetic analysis of classical swine fever virus isolates from peru rapid pre-clinical detection of classical swine fever by reverse transcription loop-mediated isothermal amplification development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the field an improved elisa for the detection of serum antibodies directed against classical swine fever virus comparison of reverse transcriptase-polymerase chain reaction, virus isolation, and immunoperoxidase assays for detecting pigs infected with low, moderate, and high virulent strains of classical swine fever virus validation of a real-time rt-pcr assay for sensitive and specific detection of classical swine fever classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the european network of excellence for epizootic disease diagnosis and control identification of a novel virus in pigs-bungowannah virus: a possible new species of pestivirus pcr in a rayleigh-benard convection cell enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein e rns for serologic diagnosis of pestivirus infections in swine classical swine fever virus diversity and evolution loop-mediated isothermal amplification of dna genetic typing of classical swine fever virus classical swine fever virus isolates from cuba form a new subgenotype 1.4 diagnostic evaluation of a real-time reverse transcriptase pcr assay for detection of classical swine fever virus genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species atypical 'hobi'-like pestivirusesrecent findings and implications thereof the 1997/1998 epizootic of swine fever in the netherlands: control strategies under a non-vaccination regimen development of taqman probe-based insulated isothermal pcr (ii-pcr) for sensitive and specific on-site pathogen detection validation of a commercial insulated isothermal pcr based pockit test for rapid and easy detection of white spot syndrome virus infection in litopenaeus vannamei detection of salmonella in chicken meat by insulated isothermal pcr situation of classical swine fever and the epidemiologic and ecologic aspects affecting its distribution in the american continent classification and nomenclature of viruses. fifth report of the international committee on taxonomy of viruses rapid detection of foot-and-mouth disease virus, influenza a virus and classical swine fever virus by high speed real-time rt-pcr development and rapid detection of classical swine fever virus by reverse transcription loop-mediated isothermal amplification (rt-lamp) validation of a loop-mediated isothermal amplification assay for visualized detection of wild-type classical swine fever virus the canadian safety and security program provided partial support for m.f., c.b. and a.e. the authors are grateful for the technical advice and information regarding the iirt-pcr assay provided by drs. thomas hwa tang wang and alison lee (genereach usa), the technical support provided by janice koziuk, tara furukawa-stoffer and alena babenko and viral nucleic acids provided by drs. irene greiser-wilke (eu reference laboratory for csf), marcus czub (university of calgary) and susan detmer (university of saskatchewan). the authors also acknowledge prof. soren alexandersen and dr. noriko goji for helpful suggestions about experiments and sandor dudas and two anonymous reviewers for suggestions regarding the manuscript. key: cord-011411-hufxjf5p authors: oliveira, thalita evani silva; pelaquim, isadora fernanda; flores, eduardo furtado; massi, rodrigo pelisson; valdiviezo, milton james jiménez; pretto‐giordano, lucienne garcia; alfieri, amauri alcindo; saut, joão paulo elsen; headley, selwyn arlington title: mycoplasma bovis and viral agents associated with the development of bovine respiratory disease in adult dairy cows date: 2019-06-24 journal: transbound emerg dis doi: 10.1111/tbed.13223 sha: doc_id: 11411 cord_uid: hufxjf5p the etiology and pathologic findings of bovine respiratory disease (brd) in adult dairy cows (n = 35) from a commercial dairy herd in southern brazil were investigated. pulmonary samples were examined for histopathologic patterns and specific features within these patterns, while immunohistochemical (ihc) assays were designed to detect the intralesional antigens of viral infectious disease agents and mycoplasma bovis. pneumonia was diagnosed in 91.4% (32/35) of these cases; neither pneumonia nor any of the infectious disease pathogens evaluated occurred in three cows. the presence of multiple respiratory pathogens in 75% (24/32) of these cases indicated the complex origin of pneumonia in cattle. interstitial pneumonia, necrosuppurative bronchopneumonia and suppurative bronchopneumonia were the principal patterns of pulmonary disease identified by histopathology. the most frequent pathogens identified by ihc were bovine viral diarrhea virus (bvdv; n = 18), m. bovis (n = 16) and bovine alphaherpesvirus type 1 (bohv‐1; n = 14), followed by bovine respiratory syncytial virus (brsv; n = 11) and bovine parainfluenza virus type 3 (bpiv‐3; n = 5). obliterative bronchiolitis and peribronchial lymphocytic cuffings were the characteristic histopathologic features associated with m. bovis. necrohemorrhagic bronchitis with bronchial angiogenesis was associated with bohv‐1. necrotizing bronchitis and bronchiolitis were associated with bvdv, bohv‐1 and brsv. ballooning degeneration of the bronchial and bronchiolar epithelia was associated with brsv and bohv‐1. this is the first report from brazil that correlated the histopathologic findings of brd with the associated infectious disease agents by immunohistochemistry. m. bovis was frequently detected in the tissues of cows with fatal pulmonary disease during this study and may be a possible primary disease pathogen associated with the development of brd in dairy cows. additionally, the histopathologic features identified within patterns of pulmonary disease during this investigation may be an efficient diagnostic tool to associate histopathologic findings with specific agents of brd in dairy cows. the bovine respiratory disease (brd) is a multifactorial and multiaetiological disease associated with several infectious disease agents panciera & confer, 2010) . data relative to the occurrence of brd in brazil are scarce and incipient. most of these investigations used polymerase chain reaction (pcr) and identified infectious agents of brd such as histophilus somni (headley, alfieri, oliveira, beuttemmuller, & alfieri, 2014; headley et al., 2018) , bovine alphaherpesvirus type 1, bohv-1 (suarez heinlein et al., 1993) , bovine respiratory syncytial virus, brsv (arns et al., 2003; headley et al., 2017) , bovine viral diarrhea virus, bvdv (cortez et al., 2006; flores, ridpath, weiblen, vogel, & gil, 2002; otonel et al., 2014; silveira et al., 2017) , bovine coronavirus, bcov (headley et al., 2018) , pasteurella multocida headley et al., 2018) , mannheimia haemolytica headley et al., 2018) and mycoplasma bovis (tortorelli et al., 2017) . furthermore, studies done in brazil using serology identified seropositivity to infectious disease agents including bohv-1 (barbosa, brito, & alfaia, 2005; fernandes, pimenta, pituco, brasil, & azevedo, 2016) , bovine parainfluenza virus type 3, bpiv-3 (gonçalves et al., 2003) , brsv (driemeier et al., 1997) , and bvdv (flores et al., 2005; wageck canal, strasser, hertig, masuda, & peterhans, 1998) and m. bovis (pretto et al., 2001) . additionally, there is the isolation of m. bovis (pretto et al., 2001) , while few studies from brazil have investigated only brsv by immunohistochemistry, ihc (brasil et al., 2013; peixoto et al., 2000) . the detection of antigen-coding sequences in tissues by pcr in the absence of histopathologic findings does not necessarily indicate that the identified agent is associated with a specific lesion or disease (maes et al., 2013) . disease due to infectious agents associated with brd is confirmed by the simultaneous presence of pathogens within the affected tissues (fulton & confer, 2012) . the ihc assay is a sensitive diagnostic technique that can be used to identify the intralesional presence of specific protein of infectious disease agents associated with histopathologic lesions in formalin-fixed paraffin embedded (ffpe) tissues sections (fulton & confer, 2012; maes et al., 2013) , and the results obtained are strong evidence of an associated disease process within the affected tissues (fulton & confer, 2012) . the disease pathogens associated with brd have been evaluated extensively mainly in north america (fulton, 2009; fulton et al., 2009; panciera & confer, 2010; wolfger, timsit, white, & orsel, 2015) and australia (cusack, mcmeniman, & lean, 2003; hay, morton, mahony, clements, & barnes, 2016) . however, only a few studies have used histopathologic diagnosis with related ihc assays to confirm the participation of infectious disease agents associated with brd (gershwin et al., 2015; haines, martin, clark, jim, & janzen, 2001; haines et al., 2004; rodríguez, bryson, ball, & forster, 1996) . this study describes the histopathologic patterns with associated histologic features and the ihc findings associated with m. bovis and four viral agents of brd in a commercial dairy herd from southern brazil. this study investigated the occurrence of infectious disease agents of brd and the pathologic findings in holstein cows (n = 35) from a commercial dairy establishment in eastern central paraná, southern brazil. this establishment consisted of 1,500 holstein dairy cows with milk production of 45,000 l/day and an average of 29.2 l/cow/ day. due to the purchase of heifer and cows from different neighbouring herds, as well as from farms from neighbouring cities, this farm is considered as an open dairy cattle herd. between january and september 2017, there were reports of recurrent respiratory distress of the affected dairy cows that demonstrated clinical signs of inappetence, reluctance to walk and pulmonary distress (dyspnea, extended head and neck, and audible noise when breathing) associated with brd, and eventually died spontaneously. respiratory diseases were predominant in recently calved cows that demonstrated low morbidity (10%; 150/1,500) and mortality (2.3%; 35/1,500). autopsies were performed by on-site veterinarians during this seven-month period as mortality occurred; pulmonary samples were collected and submitted for laboratory diagnosis after autopsy. the clinical course of the respiratory disease and possible antibiotic therapies are not known. refrigerated pulmonary sections were submitted for pathologic diagnostics; tissues sections were fixed by immersion in 10% buffered formalin solution for 24 hr and then routinely processed for histopathologic evaluation with the haematoxylin and eosin (h&e) stain. histopathologic patterns were classified (teso, sah) and recorded according to the presence/absence of bronchopneumonia (suppurative, necrosuppurative, fibrinous or fibrinosuppurative) and interstitial pneumonia. additionally, the occurrence of specific histopathologic features associated with these patterns of pulmonary disease such as obliterative bronchiolitis, syncytial formation, necrotizing bronchitis/bronchiolitis/alveolitis, necrohemorrhagic bohv-1, bovine pulmonary mycoplasmosis, bovine respiratory syncytial virus, bovine viral diarrhea virus, diagnostic immunohistochemistry, interstitial pneumonia, respiratory pathogens bronchiolitis, abscesses and intralesional bacterial accumulations was identified and recorded. furthermore, whenever necessary new histologic slides were evaluated with the brown-brenn modified staining technique to detect gram-positive or gram-negative bacteria, the giemsa stain was used to detect intralesional accumulations of mycoplasma spp. immunohistochemistry assays were performed on lung sections to investigative the presence of five pathogens associated with brd: bohv-1, brsv, bvdv, bpiv-3 and m. bovis. selected ffpe tissue sections from the lung of each cow were prepared on silanized slides with polyl-lysine 0,1% (sigma-aldrich, st. louis, mo, usa), deparaffinized, hydrated in alcohol baths and subjected to antigen retrieval. the dilutions of the monoclonal antibodies used during this investigation are shown in table 1 . antigen retrieval (table 1 ) was achieved by using citrate buffer (ph 6.0) or tris-edta buffer with 0.05% tween (ph 9.0). both solutions were utilized with the pressure cooker system (electrolux pressure cooker pcc10, são paulo, brazil) for 5 min. endogenous peroxidase was blocked with distilled water and hydrogen peroxide (6%) for 30 min in a dark chamber. the primary incubation was achieved with the monoclonal antibodies shown in table 1 (oliveira, lorenzetti, alfieri, & lisbôa, 2015) , bvdv (lunardi, headley, lisboa, amude, & alfieri, 2008) and m. bovis (pretto et al., 2001) ; positive controls for bpiv-3 were obtained from tissue culture maintained within our laboratory. negative control consisted of using the same tissue, with substitution of the primary antibody by its diluent. positive and negative controls were included in each ihc assay. an overview of the principal histopathologic patterns of pulmonary disease observed in adult dairy cows during this study and their associated histologic features are given in table 2 . pneumonia was diagnosed in 91.4% (32/35) of the affected cows, and at least one infectious disease agent was identified in each animal by ihc (table 3) ; however, agents associated with brd were not identified in 8.6% (3/35) of cows without pneumonia. interstitial pneumonia (46.8%; 15/32) was the most predominant pattern of pulmonary disease observed, followed by necrosuppurative bronchopneumonia (28.1%; 9/32; figure 1a ) with peribronchial lymphocytic cuffings (21.9%; 7/32), and suppurative bronchopneumonia (18.7%; 6/32). additionally, two cows without histopathologic evidence of pneumonia had necrotizing bronchitis. accumulations of intralesional, giemsa-stained, coccoid bacteria were associated with necrosuppurative and suppurative bronchopneumonia; gram-positive or gram-negative bacteria were not detected by the modified brown-brenn stain. the histopathologic features associated with the patterns of pneumonia are summarized in table 3 ; these lesions included obliterative bronchiolitis (28.6%; 10/35; figure 1b) , necrotizing bronchitis (18.7%; 6/32) and bronchiolitis (12.5%; 4/32; figure 1c ta b l e 1 list of antibodies, dilutions, method of antigen retrieval and source manufactures of the immunohistochemical assays epithelia, hyperplasia of type ii pneumocytes (6.2%; 2/32) and necrohemorrhagic bronchitis with angiogenesis at the lamina propria of the bronchus (6.2%; 2/32; figure 1f ). during this investigation, singular and mixed infections were identified (table 4 ). however, dual (31.3%; 10/32) and triple (18.7%; 6/32) associations were the most frequent forms at this outbreak, followed by quadruple (6.3%; 2/32) and quintuple (3.1%; 1/32, cow #34) simultaneous association of infectious disease agents associated with brd. singular infections were caused by bvdv (15.6%; 5/32), m. bovis (12.5%; 4/32), brsv (9.4%; 3/32) and bohv-1 (3.1%; 1/32); singular infections were not associated with bpiv-3. all antibodies demonstrated cytoplasmic immunolabelling within epithelial cells of bronchi and/or bronchioles of the infected cows. when the histopathologic patterns were correlated with the ta b l e 3 principal histopathologic diagnosis/features and immunohistochemical findings of bovine respiratory disease in 32 dairy cows obliterative bronchiolitis m. bovis (10/10) primary pulmonary infections due to bvdv were demonstrated experimentally (fulton et al., 2016; gershwin et al., 2015; rodríguez et al., 1996) and observed in field outbreaks of brd (fulton et al., 2000 (fulton et al., , 2004 szeredi, janosi, & palfi, 2010 in coinfections (ridpath, 2010) . this aspect is relevant not only for the interpretation of experimental data, but because bvdv may interact with m. bovis in field conditions (bürgi et al., 2018) . additionally, some strains of bvdv are immunosuppressive in cattle, due to the loss of alveolar macrophage functionality (bürgi et al., 2018; welsh, adair, & foster, 1995) and severe and extensive apoptosis of lymphocytes (chase, 2013) . consequently, immunosuppression due to bvdv associated with bohv-1, brsv and m. bovis may have resulted in an increased severity of disease in this dairy herd, due to incapacity of the innate immune system to control these infections. in our study, several chondrocytes of the hyaline cartilage of the bronchus demonstrated positive immunoreactivity for brsv, bohv-1 and bvdv, with diffused immunoreactivity to bvdv occurring within the chondrocytes of two cows. similar positive immunoreactivity to bvdv was described in 60% (6/10) of persistently infected (pi) calves, where it was proposed that the presence of bvdv antigen in respiratory cartilage is an indication that this viral disease pathogen predisposes cows to secondary bacterial infection (confer, fulton, step, johnson, & ridpath, 2005) . in the cases herein described, we were unable to confirm these cows as being pi animals since two biological samples were not available for testing. however, there was positive immunoreactivity to bvdv (using mab 15c5) within the chondrocytes of the hyaline cartilage of the bronchus of the fetus of one of these cows. collectively, these findings may suggest that the fetus of the gravid cow and probably the cow itself were pi animals; moreover, positive immunoreactivity to bvdv within the pulmonary cartilage of these two cows can correlate with the occurrence of simultaneous infections in these animals (confer et al., 2005) . additionally, it must be highlighted that the mab used during this study to identify intralesional antigens of bvdv has elevated specificity and sensitivity for the diagnosis of this infectious disease pathogen (haines, clark, & dubovi, 1992) . the first identified m. bovis-associated pulmonary disease in 26.1% (12/46) of calves due to a combination of bacterial culture and direct immunofluorescence using lung sections (pretto et al., 2001) . while the other amplified nucleic acids of m. bovis from 5.6% (1/18) of nasal swabs from dairy cows (tortorelli et al., 2017) . the marked difference between these and the current study was the confirmation of disease (fulton & confer, 2012) , in our investigation due to the innecrosuppurative bronchopneumonia and peribronchial lymphocytic cuffings are the hallmarks of chronic pulmonary mycoplasmosis in cattle (nicholas & ayling, 2003) and calves (hermeyer et al., 2012; khodakaram-tafti & lópez, 2004) , and were the main findings observed in the affected dairy cows during this study. these findings were previously described (caswell & archambault, 2007; gagea et al., 2006; haines et al., 2004) and demonstrate the ability of m. bovis to invade the pulmonary parenchyma, as was observed in experimental (gershwin et al., 2015; hermeyer et al., 2012; rodríguez et al., 1996; thomas, howard, stott, & parsons, 1986 ) and spontaneous (gagea et al., 2006) cases of pulmonary disease in cattle. furthermore, m. bovis may induce cytotoxicity and induction of apoptosis of the alveolar macrophage (bürgi et al., 2018) , and neutrophils, inhibit the production of nitric oxide in cows (jimbo et al., 2017) and infects a wide range of epithelial and immune cells (bürki, frey, & pilo, 2015) . these mechanisms then favour the dissemination of m. bovis and would have facilitated simultaneous infections in this herd with bvdv, bohv-1 and brsv. the silent immunomodulatory and immunosuppressive effects of m. bovis predisposes the respiratory tract of calves to other bacterial infections (margineda et al., 2017; nicholas, 2011; poumarat et al., 2001) . these effects have been demonstrated experimentally (poumarat et al., 2001; rodríguez et al., 1996; vanden bush & rosenbusch, 2003) and were described in spontaneous cases (haines et al., 2004; rodríguez et al., 1996; yi̇lmaz et al., 2016) , and there are reports of pneumonic diseases in which m. bovis was the only pathogen identified (gershwin et al., 2015; nicholas, 2011) . these findings may suggest that m. bovis can act as a primary disease pathogen and produce brd. however, the possibility of m. bovis being a primary disease agent is still controversial, since this agent may colonize and f i g u r e 3 immunohistochemical findings observed in dairy cattle with bovine respiratory disease. (a) there is positive immunoreactivity to antigens of bohv-1 ballooning degeneration of the bronchiole (black arrows) and endothelial staining in alveolar capillaries (white arrows). (b) there is positive immunoreactivity for bvdv at the endothelium of alveolar venule (black arrows), within degenerated endothelial cells (white arrows), and (c) necrotizing bronchiolitis (arrow). (d) observe positive immunoreactivity for antigens of brsv at the bronchial epithelium, within detached bronchial epithelial cells (arrow), in bronchial hyaline cartilage (asterisk). (e) observe positive immunolabelling for antigens of bvdv at the bronchial epithelium and within chondrocytes of the bronchial hyaline cartilage (asterisk). (f) there is positive immunoreactivity associated with bohv-1 at the bronchial epithelium of a cow with diffused ulcerative bronchitis and epithelial necrotizing bronchitis (arrows heads), at the newly formed capillaries (arrows) and mixed peribronchial glands (asterisks). immunoperoxidase counterstained with haematoxylin. bar, a-c, e 50 μm; d, f 200 μm as far as the authors are aware, this manuscript may represent the first description of m. bovis-related brd in dairy cows. this pathogen was frequently associated with several disease syndromes in dairy calves (mahmood et al., 2017) and feedlot cattle (caswell & archambault, 2007; haines et al., 2001) , including descriptions of m. bovis-related pneumonia (gagea et al., 2006; mahmood et al., 2017 ), but we did not locate reports of m. bovis-induced brd in adult dairy cows on searching major databases. furthermore, m. bovis was not identified in recent studies done by our group to identify this pathogen associated with brd in feedlot cattle from different geographical regions of brazil headley et al., 2014 headley et al., , 2018 . additionally, there are only two reports (pretto et al., 2001; tortorelli et al., 2017) of m. bovis-associated pulmonary disease in cattle from this country. although the exact reasons for the low detection rate of m. bovis-associated brd in brazil are unknown, we believe that the reduced identification of this agent may be related to the diagnostic strategy used and the type of cattle evaluated, that is beef against dairy. all previous cases of m. bovis-associated brd were diagnosed in dairy cows (pretto et al., 2001; tortorelli et al., 2017) , while this bacterial pathogen was not identified in studies done with beef cattle (headley et al., , 2017 (headley et al., , 2018 . it must be highlighted that nasal swabs were tested by pcr in the beef cattle surveys done by our group which resulted in negative results; however, these negative results could have been associated with the sporadic elimination of m. bovis so that pcr testing would not be an efficient method to detect this pathogen (nicholas, 2011) . this sporadic shedding of m. bovis may also explain the low results (5.6%; 1/18) obtained in a study done by another group from brazil that used the same molecular testing strategy in dairy cattle (tortorelli et al., 2017) , as compared with the elevated results (26.1%; 12/46) obtained by bacterial culture and immunofluorescence assay using pulmonary tissue with characteristic lesions (pretto et al., 2001) . since pulmonary mycoplasmosis is a chronic disease (caswell & archambault, 2007) , dairy cows that are maintained for longer dura(nicholas & ayling, 2003) , the sporadic shedding of m. bovis (nicholas, 2011) in affected cows, and the molecular testing frequently used to identify this pathogen in association with brd. consequently, this pathogen should be suspected in dairy cows with clinical manifestations of respiratory distress and be included in the differential diagnosis of infectious disease agents associated with brd in adult dairy cattle from brazil. moreover, pulmonary tissue rather than nasal swabs may be more efficient to diagnose m. bovis in dairy cattle. necrohemorrhagic bronchitis with bronchial angiogenesis was exclusively associated with infections induced by bohv-1 in this study. we have not seen this lesion previously described in the deby bohv-1 is not fully elucidated, these cytokines may be associated with the development of this lesion due to inflammatory reaction, and necrosis; in addition, il-8 has angiogenic and repair qualities (koch et al., 1992) . consequently, further studies are needed to understand the pathophysiology and the importance of this unique lesion. few studies have demonstrated synergism of brsv with bvdv (liu et al., 1999) , h. somni (agnes et al., 2013; headley et al., 2017) and m. bovis (thomas et al., 1986) . experimentally it was demonstrated that this synergism induced alveolar cell retraction and increased degradation of collagen by tnfα (agnes et al., 2013) . both mechanisms may facilitate pulmonary damage resulting in subsequent pneumonia and the dissemination of the infectious pathogen (agnes et al., 2013) . additionally, brsv infects type i and type ii pneumocytes (bryson, mcconnell, mcaliskey, & mcnulty, 1991) , with consequent apoptosis to these cells (viuff et al., 2002) , and produces necrosis of alveoli and small airways (bryson et al., 1991) , due to alveolar neutrophilic exudation (agnes et al., 2013) , resulting in suppurative alveolitis/bronchiolitis and necrotizing bronchiolitis/bronchitis (andrews & kennedy, 1997; brasil et al., 2013; gershwin et al., 2015; peixoto et al., 2000) . moreover, during this investigation, hyperplasia of type ii pneumocytes was observed in association only with positive immunoreactivity to brsv; similar findings were previously associated with chronic epithelial injury in the proximal alveolar region (barry, miller, & crapo, 1985) and identified in beef cattle with chronic pulmonary disease (driemeier et al., 1997) . the low number of cases with syncytial formation (4/11) in the present study may be due to the fact that the manifestations were chronic (driemeier et al., 1997) . these histopathologic findings were observed in cattle infected by brsv during this investigation and may have contributed to the increased severity of pulmonary lesions observed in the dairy cows herein described. this is the first report from brazil that demonstrated and correlated the histopathologic findings of bovine respiratory disease by immunohistochemistry. necrosuppurative bronchopneumonia, obliterative bronchiolitis and peribronchial lymphocytic cuffings were the characteristic histopathologic features associated with mycoplasma pneumonia. necrotizing bronchitis and bronchiolitis were associated with bvdv, bohv-1 and brsv. necrohemorrhagic bronchitis with bronchial angiogenesis was associated with infection by bohv-1. m. bovis was commonly detected in the tissues of fatal pulmonary disease during this outbreak and may be a possible primary disease pathogen associated with the development of bovine respiratory disease. moreover, the histopathologic features observed within the patterns of pulmonary disease may be an excellent diagnostic tool to identify some infectious disease agents of brd in dairy cattle. the authors are grateful for the monoclonal antibody against bvdv massi, rp and valdiviezo, mjj are recipients of capes fellowships. the authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article. bovine respiratory syncytial virus and histophilus somni interaction at the alveolar barrier respiratory diagnostic pathology. veterinary clinics of north america: food animal practice characterization of bovine respiratory syncytial virus isolated in brazil bovine respiratory disease complex associated mortality and morbidity 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activation pathological and immunohistochemical studies of natural and experimental mycoplasma bovis pneumonia in calves coinfection with bovine viral diarrhea virus and mycoplasma bovis in feedlot cattle with chronic pneumonia genetic diversity of brazilian bovine pestiviruses detected between molecular characterization of south american bovine herpesvirus-1 isolates with monoclonal antibodies and sds-page microbiological and pathological examination of fatal calf pneumonia cases induced by bacterial and viral respiratory pathogens mycoplasma bovis infection in gnotobiotic calves and combined infection with respiratory syncytial virus evaluation of mollicutes microorganisms in respiratory disease of cattle and their relationship to clinical signs characterization of the immune response to mycoplasma bovis lung infection replication and clearance of respiratory syncytial virus: apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus detection of antibodies to bovine viral diarrhoea virus (bvdv) and characterization of genomes of bvdv from brazil effect of bvd virus infection on alveolar macrophage functions a systematic review of bovine respiratory disease diagnosis focused on diagnostic confirmation, early detection, and prediction of unfavorable outcomes in feedlot cattle histopathological, immunohistochemical and bacteriological characterization of mycoplasma bovis pneumonia in cattle additional supporting information may be found online in the supporting information section at the end of the article. key: cord-342766-ndzhlf3k authors: ku, x.; chen, f.; li, p.; wang, y.; yu, x.; fan, s.; qian, p.; wu, m.; he, q. title: identification and genetic characterization of porcine circovirus type 3 in china date: 2017-03-19 journal: transbound emerg dis doi: 10.1111/tbed.12638 sha: doc_id: 342766 cord_uid: ndzhlf3k a novel circovirus called porcine circovirus type 3 (pcv3) was recently reported to exist in the usa. this circovirus is associated with porcine dermatitis, nephropathy syndrome and reproductive failure. this study reports on the first identification, widely epidemic, different phylogenetic clusters, potential role in sow reproductive failure and possible origins of pcv3 in china. since 2010, new circoviruses from porcine samples were only initially characterized in some studies (li et al., 2010; zhang, li, deng, kapusinszky, & delwart, 2014) . in 2016, a novel circovirus, called pcv type 3 (pcv3), was reported to exist in the usa (palinski et al., 2016) . pcv3 is associated with porcine dermatitis, nephropathy syndrome and reproductive failure (palinski et al., 2016) and cardiac and multisystemic inflammation (phan et al., 2016) . a total of 356 sows at three farms in the liaoning and jiangxi provinces and chongqin city have suffered from reproductive failure and acute loss of neonatal piglets since march 2016. the stillborn rate of the delivery sows ranged from 5.2% to 20.1% at these three farms, while sow mortality ranged from 5.4% to 10.5%. the general pig pathogens, such as prv, pcv2, prrsv, pedv, tgev, rv and hcv, were excluded by reverse transcription polymerase chain reaction (pcr) or simple pcr methods (li, li, yan, chen, & he, 2009; yu et al., 2016; zhang & he, 2010) . pcv3 was the only detected † these authors contributed equally to this article. pathogen in these cases. to better understand the infection status, epidemic status, geographical distribution, potential pathogenicity and genetic characteristics of pcv3 in china, a total of 222 samples (i.e., stillborn, tissues, semen and serum) were collected from 35 farms in 11 provinces or districts (i.e., anhui, chongqing, fujian, hebei, henan, hunan, jiangsu, jiangxi, liaoning, shenyang and zhejiang) in china. these samples were subjected to pathogen detection in the diagnostic center of animal epidemic diseases of huazhong agricultural university. the aforementioned samples were individually collected, stored, distilled in 2-or 5-ml ep tubes or clear package bags and then transported utilizing ice boxes. the stillborn and tissues samples were homogenized and diluted 10-fold with phosphate-buffered saline (pbs; 0.1 m, ph 7.4). the semen and serum samples were diluted 10-fold with pbs (0.1 m, ph 7.4). all the samples were frozen and thawed twice to release, further subjected to a vortex for 5 min, and centrifuged at 11,000 rpm (eppendorf, germany) for 8 min at 4°c. the supernatants were utilized for dna and rna extraction immediately or stored at à80°c refrigerator for further usage. a pair of primers was designed to detect pcv3 ( table 1 ). the detection limit of this method was 20.5 pg nuclear acid. the primers have no cross-reactions with the pr, pcv2, tgev, tgev, rv, prrsv, porcine parvovirus and deltacoronavirus temples stored in our laboratory. two pairs of primers were utilized for the entire genome sequencing ( table 1) . the reaction conditions were as follows: pre-denaturation at 94°c for 5 min, 35 cycles of denaturation at 94°c for 30 s, annealing at 55°c for 30 s, extension at 72°c for 1 min and a final extension at 72°c for 10 min. the amplicons for the genome sequencing were gel-purified, cloned into pmd â 18-t vector (takara, japan) and also plays a role in these cases must be further researched. the singular infection rate of pcv3 in the samples was 18.9% (42/222 belonged to cluster 3a. the sequence similarity of these cv isolates is plotted in figure 2 to study the genetic relationship of the chinese pcv3 isolates with the chinese bat cvs. two recombination regions were identified between the bat cvs and pcv3 isolates in 264-564nt and 714-1148nt ( figure 2 ). these results suggest that pcv3 can be the recombination results of the bat cvs. pcv3 has been poorly understood because it was first identified in the usa in 2010 (li et al., 2010) . pcv3 has been identified as a pathogen agent associated with pig diseases (palinski et al., 2016 isolation of porcine circovirus-like viruses from pigs with a wasting disease in the usa and europe detection of circovirus in foxes with meningoencephalitis porcine circovirus a historical perspective multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces genotype analysis of porcine circovirus 2 in some areas of china circovirus in tissues of dogs with vasculitis and hemorrhage full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination molecular biology of porcine circovirus: analyses of gene expression and viral replication open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein a novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure genetic variability of porcine circovirus 2 (pcv2) field isolates from vaccinated and nonvaccinated pig herds in germany epidemiology and transmission of porcine circovirus type 2 pcv2 genetic and antigenic characterization of a newly emerging nucleotide position similarity plot of the whole genome of 10 pcv3 isolates with 5 bat circovirus isolates. the chinese pcv3 isolates, pcv3/cn/ fujian-5/2016, was set as query strain. the similarity plot was constructed using the two-parameter (kimura) distance model with a sliding window of 200 bp and step size of 20 bp. the vertical and horizontal axes indicate the nucleotide similarity per cent and nucleotide position (bp) in the graph, respectively pcv3/cn/fujian-5/2016 ky075986.1 china complete genome analysis complete genome analysis complete genome analysis complete genome analysis complete genome analysis complete genome analysis complete genome analysis ky075995.1 china:anhui 2016 partial capsid gene analysis 11 pcv3/cn/fujian-5/201608 ky075996.1 china:fujian 2016 partial china:fujian 2016 partial capsid gene analysis ky076006.1 china:liaoning 2016 partial key: cord-278154-oenuy07r authors: gallien, sarah; andraud, mathieu; moro, angélique; lediguerher, gérald; morin, nadège; gauger, phillip c.; bigault, lionel; paboeuf, frédéric; berri, mustapha; rose, nicolas; grasland, béatrice title: better horizontal transmission of a us non‐indel strain compared with a french indel strain of porcine epidemic diarrhoea virus date: 2018-07-02 journal: transbound emerg dis doi: 10.1111/tbed.12945 sha: doc_id: 278154 cord_uid: oenuy07r from the severe porcine epidemic diarrhoea (ped) epidemics that struck in 2013 in the united states of america and other countries of north and south america, two types of porcine epidemic diarrhoea virus (pedv) were isolated, namely the indel and the non‐indel strains. they are differentiated by insertions/deletions in the s1 nucleotide sequence of the s gene, and differences in virulence were observed from the clinical cases. in 2014, a ped outbreak occurred in a pig farm in france, from which an indel strain was isolated. this study aimed at comparing, under experimental conditions, the pathogenicity and the direct and indirect transmissions between a non‐indel strain isolated from a ped‐affected piglet in 2014 in the usa and the french indel strain. all infected pigs showed clinical signs with the non‐indel strain although only the inoculated and direct contact pigs showed clinical signs in the indel strain group. although viral rna was detected in air samples with both strains, the indirect contact pigs remained free from infection with the indel strain in contrast to the non‐indel group in which airborne transmission occurred in the indirect contact pigs. all infected pigs shed virus in faeces regardless of pedv strain with 9 of 30 pigs showing intermittent faecal shedding. the transmission rate by direct contact was found to be 2.17‐fold higher than the non‐indel strain compared with the indel. in conclusion, the indel strain was less pathogenic than the non‐indel strain in our experimental conditions. the transmission route differed between the two strains. direct contact was the main transmission route for the indel strain, although the non‐indel strain was transmitted through direct contact and indirectly through the air. called the porcine epidemic diarrhoea virus (pedv) (jung & saif, 2015) . pedv reference strain, cv777, was isolated in belgium in 1977 (pensaert & de bouk, 1978) . after several outbreaks in the 70s, ped has been persisting in europe with sporadic cases until the late 1990s (jung & saif, 2015) . outbreaks of ped were also described in asia in the 1980s and then between 2010 and 2012 (jung & saif, 2015; wang et al., 2013) . a new type of strain, genetically different from the reference strain, was isolated during those recent outbreaks, inducing a strong mortality despite the frequent uses of vaccines based on cv777 strain in asia (li et al., 2012; wang et al., 2013) . in 2013, swine producers in the united states of america were hit by ped although the country had previously been free from the disease. the ped disease spread rapidly throughout the country and in neighbouring countries of north and south america such as canada and mexico. two genetically distant pedv strains were isolated in the usa, namely the non-indel strains, genetically similar to the aforementioned asian strains, and the indel strains, showing insertions-deletions in the s1 part of the s gene (jung & saif, 2015; vlasova et al., 2014) . from clinical reports, these two strains seemed to behave differently in terms of morbidity and mortality: the non-indel strains were associated with more severe clinical cases and higher case fatality rate compared with the indel strains (vlasova et al., 2014; wang, byrum, & zhang, 2014 ). in contrast with non-indel strains, exclusively reported in america and asia, indel strains were also identified in europe since 2014 (efsa, 2016; stadler et al., 2015 , vlasova et al., 2014 . to date, pedv circulates in america, asia and europe with different patterns in terms of epidemiological behaviour and persistence of the virus in the pig population. several studies focusing on the different types of pedv strains allowed for a better understanding of pedv epidemiology and pathogenicity. because of a very low infectious dose (thomas et al., 2015) , pedv can be transmitted by contact with contaminated equipment, the staff or contaminated vehicles used for animal transport (bowman, krogwold, price, davis, & moeller, 2015; jung & saif, 2015; lowe et al., 2014) , but oro-faecal transmission is the main transmission route. pedv is shed in faeces favouring a rapid transmission to susceptible pigs sharing the same environment as infected animals (jung & saif, 2015) . airborne transmission was evidenced over relatively long distances for non-indel strains (alonso et al., 2014) , but still needs to be investigated as a potential transmission route for indel pedv strains. although rapid transmission is commonly recognized, the trans two inocula were used during the experimental trials. they resulted from the homogenization of a jejunum sample (20%w/v) collected from pigs affected by ped. the pedv indel strain, pedv/fr/001/ 2014 strain (gb no: kr011756), was amplified on a 3-week-old spf pig inoculated with homogenate of jejunum collected from pedaffected pigs belonging to a farm located in the north of france in 2014. the inoculum was prepared from the jejunum of the spf ped-affected pig. for the pedv non-indel strain, pedv/usa/2014/ iowa strain (gb no: mf37363), jejunum samples were collected from pigs from one herd affected by a non-indel pedv strain in the state of iowa (usa) in 2014 and homogenized in phosphate-buffered saline. the two homogenates were centrifuged at 10,000× g for 10 min at 4°c, and supernatants were passed through a 0.45-μm filter. next-generation sequencing (ngs) was performed on each inoculum to obtain the pedv complete genome sequence and to ensure the absence of other rna viral sequences. the inocula were also negative for porcine circovirus 2 (pcv2) and porcine reproductive and respiratory syndrome virus (prrsv). these absences were assessed by the absence of seroconversion against pcv2 and prrsv after inoculation. the experiments were carried out in the level-3 biosecurity, air-fil(referral no. 16-032) . for each strain, twenty largewhite specific pathogen-free (spf) weaned pigs, 28 days old, were equally distributed into two separate rooms (a and b) containing two separate pens (10 pigs per room) ( figure 1 ). three additional spf weaned piglets were housed in a third room c and used as negative control. in each room a and b, a seeder pig was placed in pen a in direct contact with four other pigs. in pen b, five naïve pigs were placed in indirect contact with pen a. the pens a and b were 40 cm apart and separated by a solid partition between the two pens to prevent any direct transfer of faecal material. the same design was used for the american non-indel strain. the control animals were sampled first. strict biosecurity measures were used during this two experiments to avoid any sample contamination. the outside portion of pen a and pen b was washed before every stage of sampling, and a footbath was set up before the entry of each pen. individuals entering pens washed their boots and wading boots before passing through the footbath. the animals in pen b were also sampled before those of pen a. the manipulator also showered and changed clothes between each room of the two repetitions. gallien et al. on day 0 (d0), each seeder pig of the infected groups was orally inoculated with 5 ml of a pedv inoculum titrating 10 8 copies of viral genome/ml (estimated to ≈10 5 tcid 50 /ml) for each strain. the indel and the non-indel trials lasted 49 and 72 days postinoculation (dpi), respectively, when pigs were euthanized including anaesthesia (zoletil ® , virbac, carros, france, 15 mg/kg) followed by bleeding before necropsy. individual body weights were recorded on a weekly basis from d0, prior to inoculation, until the end of the experiment to assess the average daily weight gain (adwg). rectal temperatures were monitored daily for each pig from d0 until the end of the trial. the pigs were also scored daily for faecal consistency using the following criteria: (0) absence of faeces, (1) normal, (2) semi-liquid without a formed consistency and (3) liquid/watery contents. in addition, because of the severe impact of the non-indel strain, the appearance, behaviour, vivacity and respiration of every pig were recorded the first week of the experiment. faecal samples were collected from all the pigs prior to inoculation and then the afternoon of the inoculation (0.5 dpi), twice a day from 1 to 4 dpi, daily at 5 and 6 dpi and three times a week at 7 dpi until the end of the trial (figure 1 ). one gram of faeces (or one ml of liquid faeces in case of diarrhoea) was homogenized with 9 ml of dulbecco's phosphate-buffered saline (sigma, saint louis, united states of america) and centrifuged at 15,000× g for 10 min at 4°c. supernatants were collected and stored at −80°c until use. blood was sampled prior to inoculation (0 dpi), the afternoon after inoculation (0.5 dpi) and then once a day the first week after inoculation (1-4 dpi) and twice a week until the end of the trial. blood samples were centrifuged at 1,200× g for 10 min at 4°c, and sera were stored at −80°c until use. after euthanasia, at 49 and 72 dpi for the indel and the non-indel trial, respectively, necropsies were performed and macroscopic lesions were evaluated. during necropsy, the following organs were collected and stored at −80°c in rna later tissue storage reagent (sigma, saint louis, usa): duodenum, jejunum, ileum, colon, spleen, liver, mesenteric and inguinal lymph nodes, psoas muscle and lungs. air samples were also collected using a coriolis ® μ air sampler (bertin, montigny-le-bretonneux, france) placed between the two pens ( figure 1 ) prior to inoculation (0 dpi) and then the afternoon after inoculation (0.5 dpi), once a day the first week after inoculation (1-6 dpi) and three times a week until the end of the trial. the coriolis ® μ air sampler technology is based on a cyclonic technology, which is combined with a high air flow rate. the collection of the virus was performed in 0.005% triton x-100 milliq water solution with a flow rate of 300 l/min for 10 min. after collection, the samples were concentrated with the amicon ® ultra-2 30k device (merck, darmstadt, germany) and stored at −80°c until use. viral rnas were extracted from the faecal homogenates, the sera and the air samples using the qiagen rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. five μl of eluted rna was used as templates for pedv rt-qpcr. rna extraction controls were performed, every five samples to check for any pedv contamination, by replacing sample with rnase-free water. rna extracted from faeces with the rneasy mini kit was diluted to 1:10 to avoid any pcr inhibition. the number of pedv genome copies was assessed by real-time pcr using power sybr ® green rna-to-ct ™ 1-step kit (thermo fisher scientific, waltham, united states of america) on an applied biosystems ® 7500 real-time pcr system (thermo fisher scientific, waltham, united states of america). the pcr conditions were a holding stage of two steps, one-first step at 48°c for 30 min and a second one at 95°c for 10 min, followed by 40 repeated cycles with two steps, a first step of 15 s at 95°c and a second of 1 min at 60°c. at last, a melt curve stage composed of four steps, a first step of 1 min at 95°c, a second step of 1 min at 60°c, a third step with a gradual increase in temperature with 0.35°c for 0.3 s to obtain a temperature of 95°c and a fourth step of 15 s at 60°c. the primers used were described in gallien et al. (2018) and were designed from the conserved regions of the pedv nucleocapsid gene for universal detection of both strains (forward, 5′-cgcaaagactgaacccac taa-3′; reverse, 5′-ttgcctctgttgttacttggagat-3′). the copy numbers were quantified using a range of pedv n rna from 10 1 to 10 8 copies/5 μl. for each pcr run, a positive control containing pedv rna extract from a pedv cell culture supernatant was included. two negative controls were also included in the plate; the rna samples were replaced by rnase-free water. one negative control was placed close to the positive control and the second one at the end of the plate. all samples were processed in duplicate. sera were tested for pedv antibodies using a commercial elisa test, id screen ® pedv indirect (id vet, grabels, france). the elisa test is validated if the mean value of the positive control optical density (od) is greater than 0.350 and if the ratio of the mean values of the positive and negative controls is greater than 3. then, for each sample, the s/p (sample-to-positive) ratio was calculated. samples with s/p ratio equal or higher than 60% were considered as positive for pedv antibodies. the duration of the shedding period was characterized as the time interval between the first and the last pcr-positive faecal sample for each animal. survival analysis was used to assess whether differences exist between the two challenge strains (cox proportional hazard model; r software: function coxph) and to estimate the distributions of the shedding period (parametric survival model assuming a weibull distribution; r software: function survreg). pedv transmission process was fitted to a seir model. each individual was classified as susceptible, exposed, infectious or recovered on the basis of their virological results. pigs were considered as susceptible (s) during the time window from exposure (day 0 postinoculation) and the time they were effectively infected (t inf ). at that time, the animals turn into the exposed class (e) for a duration δ e , during which they are infected, but do not shed viral particles, being unable to infect other pigs. t sh = t inf + δ e represents the actual first shedding time, from which time the animals are infectious (i), and stands between the times of the last pcr-negative (t neg ) and the first pcr-positive faecal samples (t pos ) for each animal (t neg < t sh < t pos ). furthermore, the infectious process is initiated by the inoculated seeder pig in each room, and all infection events in contact individuals occurred only once the seeders became infectious, that is, for each contact pig. one can notice here that for seeder pigs, inoculated at t 0 , the shedding time is equivalent to the duration of the latency period (d seeder ). at last, pigs were considered as recovered (r) after their last pcr-positive faecal sample, when they did not play any role in the infectious process anymore. two transmission routes were considered to represent the observed infectious process: transmission by direct contact between penmates and by indirect contact with animals in neighbouring pens. let β w and β b denote the within-and between-pen transmission rates. the force of infection exerted on a typical suswhere i w and i b represent the number of infected animals in the same pen as individual i and in the neighbouring pen, respectively; n is the total number of pigs in each pen. with these notations, the probability p i for an individual i to get infected at time t ðiþ inf is given by while having escaped infection on the time interval ½0;t ðiþ inf with the probability inf 0 kðsþds. assuming a gamma distribution for the duration of the latency period δ e , with shape and scale parameters α and κ, the likelihood of the data is expressed by: the first term of the likelihood corresponds to the probability of occurrence of observed infections; the second term represents the probability of observed infection failure whenever some individual would remain susceptible throughout the experiment; and the third term accounts for the distribution of latency period in seeder pigs. bayesian inference was performed using monte carlo markov chains. the individual values for the shedding time and latency duration were, respectively, drawn within their plausible range, that is, the normality of all the data was checked with a shapiro test. if the data followed a normal law, the homogeneity of the variances was checked with a levene test. then, if the variances were homogeneous, an anova test was carried out to compare the mean between groups. if the data did not follow a normal law or if the variances were not homogeneous, a kruskal-wallis test was used to compare the mean between groups. p-values less than 0.05 were considered significant. statistical analyses of data were carried out with the statistical software r version 3.2.5. (team, 2014) . no clinical signs were observed in any pigs prior to the beginning of the trials and in control pigs throughout the experiment. in the groups challenged with the indel strain, only the inoculated and the direct contacts pigs showed clinical signs. however, diarrhoea was observed in only 25% of these direct contact pigs simultaneously. in fact, diarrhoea was occasionally observed between 4 and 25 dpi. vomiting was sporadically observed between 1 and 49 dpi (at 1, 7, 21, 37, 42, 44, 46 and 49 dpi) . the inoculated and direct contact pigs also showed signs of lethargy and anorexia the first week after inoculation. in the non-indel strain groups, all animals involved in the experiment showed clinical signs (including indirect contact pigs), with more severe clinical signs compared to the indel inoculated groups. diarrhoea started on days 1 and 2 postinoculation for the inoculated and contact pigs (direct and indirect contacts), respectively, with 80% of the indirect contact pigs and 75% of the direct contact pigs with diarrhoea on 2 dpi. after 2 dpi, occasional diarrhoea was noticed in the direct contact pigs (5 dpi) and indirect contact pigs (at 3 and 6 dpi). occasional vomiting was also noticed for the inoculated and the direct contact pigs at 1 and 31 dpi and for the indirect contact pigs at 11, 31 and 46 dpi. all the pigs were lethargic from 2.5 to 3 dpi and showed signs of dehydration and anorexia, the first week of the trial. no hyperthermia was observed during the experiments. a reduction in growth performance was also observed in the prior to inoculation, all the pigs were negative for pedv rna in faeces. all the control pigs remained negative for pedv in faeces until the end of the trials (49 and 72 dpi, respectively). in the indel groups, the inoculated pigs began to shed virus in their faeces 1 ± 0.5 days after the non-indel inoculated pigs (table 1) . for direct contact pigs, faecal samples from indel groups were found positive 4 days later at 5 dpi for 2 pigs, compared with the non-indel pigs ( table 1) . none of the indel indirect contact pigs shed pedv in their faeces (table 1 ). in the non-indel groups, pedv rna was first detected in the faeces of the inoculated pigs and one of the contact group pigs at 1 dpi. the two inoculated pigs shed 4.80 × 10 10 and 1.20 × 10 8 genomic copies/ml of faeces, respectively. faecal samples collected from pigs in the direct contact group were rt-qpcr positive at 1.5 and 2 dpi, in two and five pigs, respectively. the faeces of the 10 indirect contact pigs tested positive for pedv at 2 dpi (table 1) . prior to inoculation, no pedv rna was detected in serum and no pig tested seropositive for pedv antibody. for the two trials, viremia was highly transient and barely detectable ( table 2 ). all non-indel inoculated and direct contact pigs seroconverted except one of the direct contact pigs. all non-indel indirect contact pigs showed also a serological response. however, 7 of 19 animals of the non-indel group, which exhibited a serological response, demonstrated pedv antibodies for a short period of time and were seronegative by the end of the experiment. the delays between infection and seroconversion for the two strains were significantly different. the average delay between infection and seroconversion was twice as long for the non-indel strain than for the indel strain (24.8 vs 12.5 days on average) (figure 3 ). no pedv rna was detected in the air before inoculation of pigs either with the indel or the non-indel strain. in the indel groups, pedv genome was detected in the air at low levels at 2 dpi and then from 5 to 35 dpi (figure 4) . (table 3) . the severity of porcine epidemic diarrhoea can be linked to several parameters such as the animal age, the infectious dose, the susceptibility of pigs, the environmental conditions or the viral strain (shibata et al., 2000) . the objective of this study was to compare the pathogenicity and transmissibility of an american non-indel strain considered highly pathogenic and an indel strain isolated in france in 2014 in experimental conditions. in case of infection with a non-indel and an indel strain, 100% of morbidity (except for the indel indirect contact pigs) was observed, but clinical signs were not apparent at the same time among the two types of pedv-infected groups. as early as 1 dpi, the first clinical signs were observed for the non-indel strain, although the indel strain induced the first clinical signs only at 5 dpi. these results are in agreement with previous experimental studies involving the non-indel strain reporting the occurrence of diarrhoea and occasional vomiting from 2 to 10 days after inoculation (crawford et al., 2015; madson et al., 2014; niederwerder et al., 2016; shibata et al., 2000) and also with previous studies involving indel strains reporting the appearance of clinical signs within 1 week after infection mesquita et al., 2015; stadler et al., 2015) . intermittent clinical signs were also noticed with the two strains after the occurrence of the first clinical signs; indel-infected pigs showed clinical signs (occasional diarrhoea, vomiting and despondency) from 8 to 49 dpi, and non-indel-infected pigs showed clinical signs from 3 to 46 dpi. pedv replicates in the enterocytes and leads to a significant reduction in both the intestinal epithelial cells, as well as the digestive enzyme activity (jung, ahn, & chae, 2006) . however, the regeneration of intestinal enterocytes was shown possible between 2 and 4 days after damage (moon, 1971) , which might explain the intermittent clinical signs observed in the indel and non-indel trials. the clinical signs were more pronounced for the non-indel trial pigs in our study. it has already been shown that differences in severity of clinical signs could be linked to the different strains; the indel strain leads to less severe clinical signs compared with the non-indel strain (chen et al., 2016) . the development of the disease was responsible for weight loss, which depended on the strain used. all the non-indel pigs had more severe weight loss during the first week after inoculation compared to the indel pigs. as in our study, madson et al. (2014) detected a significant alteration in adwg during the first week postinoculation for non-indel-infected pigs (madson et al., 2014) . regarding the indel pigs, growth retardation was observed the second and the third week after inoculation. after the first 3 weeks of the trial, no difference in adwg t a b l e 1 (continued) light grey boxes: viral genome load between 104 and 106 copies/ml; medium grey boxes: between 106 and 108 copies/ml; dark grey: between 108 and 1010 copies/ml and black: >1010 copies/ml. † dead. was observed between controls and infected pigs within the non-indel group. a trend between viral shedding and growth impact was observed for all the non-indel trial pigs. as seen previously, all the inoculated, direct and indirect contact pigs showed a significant difference of adwg compared with the control pigs, the first week postinoculation. this period corresponded to the highest levels of virus shedding in this group. after that, the average amount of genomic copies of pedv declined for the inoculated, the direct and indirect contact pigs and the pigs recovered normal growth performance. pedv rna was detected in the faeces of all pigs of the indel and non-indel trial except for the indel indirect contact pigs. the pedv rna was detected earlier in faeces of the non-indel trial pigs at 1 dpi compared to 2 ± 0.5 dpi in faeces of indel inoculated pigs, that is, 1 ± 0.5 days earlier for the non-indel pigs. similar results were observed from faeces of direct contact pigs where pedv rna was detected in faeces of non-indel direct contact pigs between 1 and 2 dpi and between 4.5 and 14 dpi in faeces of indel direct contact pigs, that is, on average 7 days earlier for the non-indel direct contact pigs. these results suggest the non-indel pedv strain replicates more quickly in the target cells and/or demonstrates the ability to propagate to higher levels more quickly in a pig population. the shedding periods were similar for the indel (except for the indirect contact pigs) and non-indel strains. the shedding periods for the non-indel and indel strains were close to previous observations from other studies which reported shedding periods of 17-23 or 18-20 days on average for the non-indel (madson et al., 2014; thomas et al., 2015) and for the indel strain, respectively (leidenberger et al., 2017; lohse et al., 2017) . two others studies on pedv indel strains revealed that pigs inoculated with an indel strain began to shed virus earlier than in our experimental conditions, that is, shedding began at 1 dpi, and the presence of pedv rna in faeces was detected until the pigs were necropsied at 7 dpi (chen et al., 2016; yamamoto, soma, nakanishi, yamaguchi, & niinuma, 2015) . in accordance with the results of other studies (chen et al., 2016) , the amount of genomic copies in faeces was more important in infected pigs by the non-indel strain than for those that were infected by the indel strain whatever the pig status (inoculated, direct contact, indirect contact) in our experimental conditions. two phases of pedv shedding in faeces were noticed for some pigs in our study without the presence of any clinical signs. the same phenomenon had already been described recently (crawford et al., 2015) . during the second phase of pedv shedding in faeces, no clinical signs were observed. the presence of pedv rna in faeces t a b l e 2 average genome load (number of copies/ml of serum) and number of viremic pigs during the indel and non-indel trials for the inoculated, direct contact and indirect contact pigs for the non-indel strain under our experimental conditions. the amount of pedv genomic copies in air was higher for the non-indel than for the indel strain. thus, we could hypothesize that the amount of pedv genomic copies in air with the indel strain was too low to infect pigs by airborne transmission (10 4 genome copies/l). the infectious capacity of pedv by the air could also be linked to the strain. in fact, in a recent study using a different non-indel strain from ours, airborne transmission to pigs in indirect contact was not effective even when pedv rna was detected in nasal swabs of infected pigs but at low levels (niederwerder et al., 2016) . the transmission characteristics of the two strains were significantly different in our experimental conditions. indeed, the direct transmission rate quantified in our experimental conditions was more than twofold higher for the non-indel strain than for the indel strain. an indirect transmission rate could only be calculated for the non-indel strain (β w = 0.5). a lower duration of latency for the nonnumbers between brackets correspond to the confidence interval of 95%. β w : direct transmission rate; β b : indirect transmission rate. parameters calculated for the two pedv strains with those estimated for other porcine viruses revealed a transmission rate sevenfold higher than those obtained for porcine reproductive and respiratory syndrome virus (β w prrs = 0.24) (rose et al., 2015) or the porcine circovirus type 2b (β w pcv2b = 0.28) (andraud et al., 2009; rose et al., 2015) . however, these parameters were comparable to those obtained recently for swine influenza viruses (cador, andraud, willem, & rose, 2017) . considering the high transmission rate for both strains and the duration of shedding of approximately 20 days, those viruses are expected to persist easily within the population. differences in terms of indirect transmission suggest that indel strains of pedv could be more easily controlled by strict segregation of infected animals. all pigs seroconverted during the trials except some pigs infected by the non-indel strain. the seroconversion appeared for the first time at 14 dpi for both strains. a seroconversion at 14 dpi was already obtained with non-indel strain in other studies (de arriba, carvajal, pozo, & rubio, 2002; thomas et al., 2015) . the absence of seroconversion for some pigs could be linked to the sensitivity of the id vet elisa test which could be too low (tignon et al., 2017) . the elisa test used in our study is based on the pedv n protein as an antigen, and the pedv n antibodies might not persist as long as the antibodies directed against the pedv s protein. to conclude, in our experimental settings, the indel strain was less pathogenic than the non-indel strain. the indel strain could only be transmitted to direct contact pigs although the non-indel strain could be transmitted to direct contact and indirect contact pigs with a faster direct transmission. these data should be considered when developing control strategies for indel or non-indel pedv to reduce the probability of introduction in a pig population. evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral rna at long distances from infected herds concentration, size distribution, and infectivity of airborne particles carrying swine viruses influence of husbandry and control measures on 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doses on infection outcomes in naïve conventional neonatal and weaned pigs comparative study of serological methods for diagnosis of porcine epidemic diarrhea virus (pedv) infection. european symposium of porcine health management distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus porcine epidemic diarrhea virus variants with high pathogenicity isolation and experimental inoculation of an s indel strain of porcine epidemic diarrhea virus in japan better horizontal transmission of a us non-indel strain compared with a french indel strain of porcine epidemic diarrhoea virus the authors are grateful to anses, inra and inaporc for their financial support. we also would like to thank phillip gauger from iowa state university who provided the us non-indel strain, all the team members at the experimental laboratory for their contribution and the members of the unit of viral genetic of anses. the authors declare that they have noncompeting interests. http://orcid.org/0000-0002-5634-1360 key: cord-331740-yjt3q9ph authors: jones, r. m.; ellis, r. j.; cox, w. j.; errington, j.; fuller, c.; irvine, r. m.; wakeley, p. r. title: development and validation of rt‐pcr tests for the detection and s1 genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples date: 2011-04-07 journal: transbound emerg dis doi: 10.1111/j.1865-1682.2011.01222.x sha: doc_id: 331740 cord_uid: yjt3q9ph two tests were developed that allow the detection and genotyping of infectious bronchitis virus (ibv) and other closely related gammacoronaviruses. the first test employs a one‐step, reverse transcription‐polymerase chain reaction (rt‐pcr) assay in which the amplification is monitored in real time using a taqman(®) probe. this real‐time rt‐pcr test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls’ eggs. a total of 323 field samples were tested; 176 samples were positive using the real‐time rt‐pcr method, but only three were positive by virus isolation. sequencing was used to confirm the positive real‐time rt‐pcr results for a subset of samples. the test is suitable for swabs and post‐mortem samples and has been shown to be highly sensitive and specific. the second test, a genotyping method, was developed for identification of the strain of ibv present in field samples based on nucleotide variations within the gene encoding the s1 subunit of the surface spike (s) glycoprotein. this method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of ibv within the uk. the performance of the test was evaluated using laboratory isolates of ibv and field samples. both tests are suitable for use in a high‐throughput diagnostic laboratory. gammacoronaviruses are known to infect many species of birds, including chickens, turkeys, pheasants, greylag geese, mallard ducks and pigeons, often with different tissue tropisms (cavanagh et al., 2001 (cavanagh et al., , 2002 cavanagh, 2005; jonassen et al., 2005) . more specifically, turkey coronavirus (tcov) causes a highly contagious enteric infection, which can lead to mortality and growth retardation (guy, 2000; cavanagh, 2005) , while, in contrast, pheasant coronavirus infection (phcov) typically affects renal and respiratory tissues with consequent disease signs related to those body systems (cavanagh, 2005) . infectious bronchitis virus (ibv), recognized as the prototypic gammacoronavirus, primarily causes disease in chickens that is characterized by upper respiratory tract signs, including nasal discharge, 'snicking', 'râles', watery eyes and lethargy (ignjatovic and sapats, 2000; cavanagh, 2007) . infectious bronchitis virus is of huge economic importance to the poultry industry worldwide, and the economic impact of disease is exacerbated in part by the existence of multiple serotypes of the virus, a property which complicates the detection and prevention of the disease (ignjatovic and sapats, 2000) . multiple serotypes are known to circulate within a location , while new variants frequently spread to different geographical regions. for example, the qx strain, which was initially identified in china, was first detected in western europe in late 2003, but was not identified in the uk until 2007 (landman et al., 2005; summary two tests were developed that allow the detection and genotyping of infectious bronchitis virus (ibv) and other closely related gammacoronaviruses. the first test employs a one-step, reverse transcription-polymerase chain reaction (rt-pcr) assay in which the amplification is monitored in real time using a taqman ò probe. this real-time rt-pcr test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls' eggs. a total of 323 field samples were tested; 176 samples were positive using the real-time rt-pcr method, but only three were positive by virus isolation. sequencing was used to confirm the positive real-time rt-pcr results for a subset of samples. the test is suitable for swabs and post-mortem samples and has been shown to be highly sensitive and specific. the second test, a genotyping method, was developed for identification of the strain of ibv present in field samples based on nucleotide variations within the gene encoding the s1 subunit of the surface spike (s) glycoprotein. this method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of ibv within the uk. the performance of the test was evaluated using laboratory isolates of ibv and field samples. both tests are suitable for use in a high-throughput diagnostic laboratory. gough et al., 2008; worthington et al., 2008) . different serotypes of the virus derive from variation in the s1 subunit of the spike (s) glycoprotein. the s1 subunit induces neutralizing antibodies in the host, and differences of only a few amino acids may exist between the s1 subunits of different ibv serotypes (cavanagh, 2003 (cavanagh, , 2005 . the poultry industry has adopted vaccination as a disease control strategy to limit the economic impacts of ibv infections, although the situation is complicated as different serotypes may not offer cross-protection. consequently, birds may have to be vaccinated with multiple serotypes of the virus, including those contemporaneously circulating in the field (cavanagh, 2003) . this makes constant surveillance and testing necessary to identify cases of ibv and to determine the strains of ibv currently circulating within a location to ensure that the most suitable and cost-effective strategy is implemented. detection of ibv infection has traditionally been carried out using virus isolation, the current gold standard test (world organisation for animal health (oie) (2008). however, this method is expensive and timeconsuming and is unable to deliver a rapid diagnosis in a cost-effective manner (de wit, 2000) . more recently, nucleic acid detection methods such as rt-pcr have been shown to be rapid, cost-effective and suitable for use in a high-throughput diagnostic environment. real-time rt-pcr has been used as a diagnostic method for ibv (jackwood et al., 2003; callison et al., 2006; escutenaire et al., 2007) , and the use of a taqman ò probe to follow the pcr provides an additional level of specificity. nucleic acid amplification methods can also be used for ibv strain identification, as the amplicons generated by rt-pcr can be sequenced to provide phylogenetic data (de wit, 2000; ignjatovic and sapats, 2000) . we describe the development and validation of a one-step real-time rt-pcr for the detection of ibv-like gammacoronaviruses from a range of sample types. the diagnostic test amplifies a 175-bp region within the 3¢ untranslated region (utr) of the viral genome. this diagnostic test can be used in conjunction with amplification and sequencing of the s1 viral gene to identify the strain of ibv present within a sample. these assays have been designed specifically to be used routinely, in a highthroughput diagnostic laboratory, for the rapid detection and strain identification of ibv from clinical samples. transport and initial processing of field samples tissue samples (kidney, brain, liver, oviduct, bursa and respiratory and intestinal tracts) were collected from a variety of bird species (chicken, turkey, pheasant and pigeon) at post-mortem from cases with respiratory disease or suspected coronavirus infection based on clinical observations. the samples were transported in eagle's minimum essential virus transport medium (vtm). tissue samples were homogenized, ground or cut into small pieces before making a 10-20% (w/v) suspension in phosphate-buffered saline (pbs) containing antibiotics (0.1 m pbs solution, ph 7.2 containing 50 mg/l gentamycin, 1 · 10 6 units/l penicillin g, 10 g/l streptomycin sulphate and 5 · 10 6 units/l of nystatin). the suspension was left for a minimum of 60 min at room temperature before centrifugation to deposit debris and the supernatant decanted for testing by virus isolation and real-time rt-pcr. wire-stemmed ent swabs (mw&e, corsham, wiltshire, uk) were used for cloacal or tracheal sampling and were transported either 'dry' (no transport medium) or 'wet' in eagle's minimum essential medium (vtm). for wet swabs, the eagle's minimum essential vtm was diluted 1 : 2 with pbs with antibiotics (as previously described), and the resulting liquid was tested by virus isolation and real-time rt-pcr. dry swabs were processed by being introduced into 1 ml of brain heart infusion broth (bhib) with antibiotics (1000 iu/ml penicillin g, 10 lg/ml amphotericin b and 1 mg/ml gentamycin) and agitated briefly. this liquid was used for testing by virus isolation and real-time rt-pcr. for virus isolation, 0.2 ml of processed sample was inoculated into either the allantoic or amniotic cavity of 9-to 11-day-old embryonated specified pathogen-free (spf) fowls' eggs. eggs were examined daily by 'candling', using a bright light shone on the egg to examine the development of the embryo, which is compromised or altered if virus replication has occurred. samples underwent a minimum of two passages, while three passages were used when testing samples from cases where the clinical signs strongly suggested the involvement of coronaviruses. samples were initially processed as described above, although for 'wet' swabs, the undiluted eagle's minimum essential vtm was tested. for tissue samples and wet swabs, an automated nucleic acid extraction was carried out using the magna pure lc extraction robot, with dedicated buffers contained in the magna pure lc total nucleic acid isolation kit (roche, burgess hill, west sussex, uk). nucleic acid extractions were prepared from 200 ll of sample following the manufacturers' instructions. for material eluted from dry swab samples, an automated extraction was made from 140 ll of the bhib (see transport and initial processing of field samples) using the biorobot universal extraction robot (qiagen, crawley, west sussex, uk) according to the manufacturers' instructions using the 'single-plate swab extraction' protocol. briefly, 420 ll lysis buffer avl (qiagen) was added to the bhib. this solution was then applied to a silica filter, the filter was washed to remove impurities, and the rna was eluted from the filter in elution buffer ave (qiagen). this real-time rt-pcr assay used the following primer and probe sequences: ibvrt1 forward primer cta tcg cca ggg aaa tgt c, ibvrt2 reverse primer gcg tcc tag tgc tgt acc c, ibvrt3 taqman ò probe fam -cct gga aac gaa cgg tag acc ct -tamra. the primer sequences are truncated versions of those described by cavanagh et al. (2002) and were previously shown to detect phcov (cavanagh et al., 2002) . a novel taqman ò real-time pcr probe that recognizes ibv, turkey coronavirus and pheasant coronavirus sequences was designed and incorporated into the test allowing amplification to be followed in real time. one-step rt-pcr reactions were performed using the qiagen onestep rt-pcr kit. each 25 ll reaction contained the following components: 5 ll one-step reaction buffer, 1 ll 25 mm mgcl 2 , 1 ll 10 mm dntp mix, 0.8 lm ibvrt1 primer, 0.8 lm ibvrt2 primer, 0.2 lm ibvrt3 taqman probe, 0.25 ll rnasin ribonuclease inhibitor (between 5 and 10 units of enzyme), 1 ll one-step enzyme mix, 11.75 ll nuclease-free water and 2 ll extracted nucleic acid. the reactions were run on the following programme using a stratagene mx3000p real-time pcr instrument: 50°c for 30 min, 95°c for 15 min, followed by 50 cycles at 95°c for 20 s and 50°c for 30 s. fluorescence data were collected during the 50°c step. this reaction was performed in triplicate on each nucleic acid extract, and samples were deemed to be positive if amplification was recorded in two or more of the triplicate reactions. the amplicons formed by the diagnostic ibv real-time rt-pcr assay were purified using a multiscreen hts 96 well plate (millipore, watford, uk) according to the manufacturers' instructions. the purified pcr products were sequenced by dye-terminator cycle sequencing using bigdye v3.1 kits (applied biosystems, warrington, cheshire, uk) with either the ibvrt1 or the ibvrt2 primer. the products of these reactions were analysed using an abi prism 3130xl dna analyzer (applied biosystems). the sequence data generated were then used to search the ncbi public sequence database using the blastn search tool (altschul et al., 1990) . determination of the sensitivity of the diagnostic ibv real-time rt-pcr assay using in vitro transcribed rna to determine the analytical sensitivity of the ibv realtime rt-pcr, the amplicon produced by the assay was cloned into the pgem t-easy plasmid vector (invitrogen, paisley, renfrewshire, uk), and the identity of the plasmid was confirmed by sequencing. this plasmid was then used as the template to produce rna transcripts by in vitro transcription using the megascript ò kit (applied biosystems). the dna template was digested using turbo dnase (applied biosystems), and the rna was then purified using trizol ò (invitrogen). the concentration of the rna was then determined using a nanodrop instrument (fisher scientific, loughborough, leicestershire, uk), and the value obtained was used to calculate the number of rna template molecules present in the rna preparation, based on the molecular weight of the transcript and avogadro's number. this was used to set up a 1 in 10 serial dilution of rna template containing a known number of template molecules. this dilution series was then used as the template for duplicate diagnostic ibv real-time rt-pcr reactions, and also for duplicate reactions containing the primers and probes in the absence of reverse transcriptase. amplification and sequencing of the s1 gene of ibv the genotyping rt-pcr uses a panel of ten forward and eighteen reverse primers (table 1) . each primer contains a specific sequence to amplify the s1 gene of certain known ibv strains, together with a generic m13 primer sequence (either an m13 forward or reverse sequence, as appropriate). the primers were designed to amplify the strains of ibv currently in circulation in europe , along with other strains such as vics and v18-91 that are not commonly found in europe. to produce amplicons for sequencing, one-step rt-pcr reactions were performed using the qiagen one-step rt-pcr kit. each 25 ll reaction contained the following components: 5 ll one-step reaction buffer, 1 ll 25 mm mgcl 2 , 1 ll 10 mm dntp mix, 0.4 lm of primer s1for2, 0.4 lm of primer s1rev3, 0.04 lm of each of the remaining primers, 0.25 ll rnasin ribonuclease inhibitor (between 5 and 10 units of enzyme) (promega, southampton, hampshire, uk), 1 ll one-step enzyme mix, 12.75 ll nuclease-free water and 2 ll of extracted nucleic acid. the reactions were run on the following programme using an mx3000p real-time pcr instrument: 50°c for 30 min, 95°c for 15 min, followed by 50 cycles at 94°c for 30 s, 54°c for 30 s and 72°c for 30 s. the amplicons generated were sequenced using the m13f (gtaaaacgacggccagtg) and m13r (cagg-aaacagctatgaccatg) generic primers. the forward and reverse sequences were aligned, and a 140-bp region of the sequence data generated was compared to a library of 36 sequences of well-characterized ibv strains using the abi prism seqscape v2.6 software (applied biosystems). the ibv s1 sequence files were taken from the public access genbank database and included representatives of the major groups of ibv strains that are currently circulating in europe alongside other ibv strains (see fig. 1 ). a neighbour-joining phylogenetic tree was constructed using the molecular evolutionary genetics analysis package (mega v4) with the kimura 2-parameter algorithm (tamura et al., 2007) . design and calibration of ibv real-time rt-pcr to confirm that the modified test was suitable for detecting contemporary uk field strains of ibv, a panel of laboratory isolates of ibv representing the major genotypes currently circulating in the uk was tested . the panel included m41, italy-02, 4/91, d1466, d274 and three uk qx-like strains isolated from field samples originating from poultry flocks in great britain. the modified real-time rt-pcr successfully detected all of the ibv strains tested. determination of the analytical specificity of the realtime rt-pcr assay to detect ibv-like coronaviruses in chicken, turkeys and pheasants a wide range of microorganisms, both bacterial and viral, can be responsible for respiratory disease in avian species and might be present in clinical submissions. to investigate the possibility of cross-reactivity of the assay, nucleic acid extracts from the organisms listed in table 2 were prepared and used as the template for the gammacoronavirus diagnostic test. no cross-reaction with any of the organisms was detected, indicating that the test is specific for ibv-like gammacoronaviruses and false-positive results will not be caused by the presence of these additional organisms in field samples. the importance of this was reinforced following testing of field samples, as table 1 . primers used in the s1 genotyping rt-pcr primer name primer sequence s1 for 1 gtaaaacgacggccagtggtttactactaccagagtgc s1 for 2 gtaaaacgacggccagtggtgtactactaccaaagtgc s1 for 3 gtaaaacgacggccagtgacatactattaccagagtcag s1 for 4 gtaaaacgacggccagtggtttactactaccaaagtgc s1 for 5 gtaaaacgacggccagtgaattattactaccaaagtgc s1 for 6 gtaaaacgacggccagtggtgtactactaccagagtgg s1 for 7 gtaaaacgacggccagtggtgtattactaccagagtgc s1 for 8 gtaaaacgacggccagtggtctactactaccaaagcgc s1 for 9 gtaaaacgacggccagtggtgtactactaccaaagcgc s1 for 10 gtaaaacgacggccagtggtctactactaccaaagtgc s1 rev 1 caggaaacagctatgaccatgacatcttgtgcagtaccattaac s1 rev 2 caggaaacagctatgaccatgacatcttgtgctgtaccattaac s1 rev 3 caggaaacagctatgaccatgacatcttgtgcggtgccattaac s1 rev 4 caggaaacagctatgaccatgacttcaacagcagtgccatttac s1 rev 5 caggaaacagctatgaccatgcttgtgcggtaccattaataaag s1 rev 6 caggaaacagctatgaccatgatatcttgcgcagtaccattttc s1 rev 7 caggaaacagctatgaccatgacatcctgtgcagtaccattaac s1 rev 8 caggaaacagctatgaccatgacatcatgtgcagtaccattgac s1 rev 10 caggaaacagctatgaccatgacgtcttgtgcagtaccattaac s1 rev 11 caggaaacagctatgaccatgacatcttgtgcggtaccattaac s1 rev 12 caggaaacagctatgaccatgacgtcttgtgcggtaccattaac s1 rev 13 caggaaacagctatgaccatgacgtcttgtgcagtaccattacc s1 rev 14 caggaaacagctatgaccatgagaataacatcttgcgcagtacc s1 rev 15 caggaaacagctatgaccatgaaaataacatcttgtgcagtacc s1 rev 16 caggaaacagctatgaccatgacatcatgtgcggtgccattaac s1 rev 17 caggaaacagctatgaccatgcttgtgcggtgccattaataaag s1 rev 18 caggaaacagctatgaccatgaaaataatatcctgtgcagtacc several samples that were negative in the molecular diagnostic test were shown to contain other viruses, i.e. infectious laryngotracheitis virus and adenoviruses, using alternative testing methods (data not shown). two approaches were used to estimate the analytical sensitivity of the diagnostic real-time rt-pcr test. to investigate the analytical sensitivity of the test in toto including the extraction process, its performance was correlated with the current standard test, virus isolation in embryonated spf fowls' eggs (world organisation for animal health (oie) (2008). a tenfold dilution series of the ibv laboratory isolate 793/b was made, and 0.2 ml of each dilution was tested in parallel by both tests. virus isolation had a limit of detection between the 10 )5 and 10 )6 dilutions, and it was calculated that the original 793/b virus pool had a titre of 10 5.5 median egg infectious doses (eid 50 ) in 0.2 ml. the limit of detection of the real-time rt-pcr test was the 10 )6 dilution. to investigate the analytical sensitivity of the real-time rt-pcr alone, in vitro transcription of a cloned target sequence was used to prepare an rna target template. the rna produced was used to introduce a known number of rna molecules into the real-time rt-pcr assay. parallel reactions lacking reverse transcriptase (rt) enzyme were also run. this experiment demonstrated that the limit of detection of the real-time rt-pcr reaction was between 10 and 100 copies of template. the absence of any amplification in the 'no-rt' control wells demonstrated the absence of contaminating plasmid dna in the preparation of the rna transcribed in vitro. to further determine the performance of the diagnostic test, it was necessary to test its ability to identify positive and negative field samples correctly. samples received for routine testing using virus isolation were tested in parallel using the diagnostic ibv real-time rt-pcr. the panel contained field samples from a variety of species of birds (chickens, turkeys, pheasants and pigeons) and also a range of sample types including both swabs and postmortem tissue samples. a total of 323 samples were analysed during the course of this study, and while only three samples were found to be positive using virus isolation, 176 samples were positive by real-time rt-pcr, including in this number the three samples that were positive by virus isolation (table 3) . as the results obtained using virus isolation were not comparable to those obtained using the realtime rt-pcr, some of the amplicons formed using the diagnostic test were sequenced. for 118 of the 176 realtime rt-pcr-positive samples, amplicons were generated using the ibvrt1 and ibvrt2 primers and sequenced using these primers. because of sample degradation, amplicons were no longer generated when retesting the remainder of the samples. good-quality sequence data were generated from 41 of the 118 amplicons, but the sequence data obtained for the other samples were of insufficient quality or length to interpret. for 40 samples, the sequence data generated were shown to be most similar to ibv, while for one sample, the most similar sequence within the genbank database was shown to be turkey coronavirus. the original samples for which the real-time rt-pcr results could be confirmed by sequencing included cloacal swabs (16 samples), oropharyngeal and respiratory tract swabs (four samples), intestines and intestinal contents (eight samples), caecal tonsils and caecal contents (five swabs), and trachea and tracheal contents (three samples). real-time rt-pcr-positive results were generated for several poultry species (turkeys, pheasants and chickens), but the ten pigeon samples tested were all negative (table 3) . it was also demonstrated that gammacoronaviruses could be detected from a range of sample types using the molecular ibv real-time rt-pcr (table 4) . positive results were obtained from chickens using oropharyngeal and cloacal swabs (both dry and in vtm). positive results were also obtained from a variety of postmortem sample types, including kidney, small intestine, trachea, caecal tonsils, mixed kidney and oviduct, and caecal contents from chickens. positive post-mortem sample types from turkeys included intestine and intestinal contents, while coronavirus was detected in mixed lung and trachea samples from pheasants. the diagnostic ibv real-time rt-pcr described here has been demonstrated to be a highly sensitive and specific test, which is eminently suitable for demonstrating the presence of gammacoronavirus nucleic acid in field samples. however, because of its design, it will detect both field and vaccine strains of ibv. to allow determination of the strain of ibv present within a field sample, an s1 genotyping method was designed for routine use within a diagnostic laboratory setting. samples of live attenuated ibv vaccines currently in use in the uk were sequenced using this method, and the sequence data generated used to populate the sequence library used during interpretation of the sequence data generated by the s1 genotyping test. analysis of the sequences present in the sequence library demonstrated the sequences clustered in several main ibv serotypes including the so-called massachusetts, italy 02, 4/91 (also known as 793/b and cr88), arkansas and qx-like serotypes (fig. 1) . the variation within sequences in these different groups has been shown to be <5%, equivalent to seven base changes in the 140 bp region used for sequence analysis. in contrast, greater variation is seen between the different serotypes, with the amount of variation between serotypes exceeding 15% of the nucleotides within the region sequenced. initially, to demonstrate that this method was performing well, it was used to test a panel of laboratory isolates representing ibv strains currently circulating in the uk . the panel included m41, italy 02, d1466, d274 and 4/91 along with three qx-like isolates derived from poultry in great britain. gel electrophoresis was used to demonstrate that the s1 rt-pcr was able to amplify the s1 gene of these ibv strains. the amplicons generated were also sequenced, and when the data generated were compared to the ibv sequence library, the ibv strain present within each sample was correctly identified. to determine whether the ibv genotyping method was capable of identifying the strain of ibv present in a field sample, the method was used to test a panel of ten field samples, which had previously tested positive using the diagnostic ibv real-time rt-pcr. the panel included both swabs and post-mortem tissue samples from chickens. the results from this testing are shown in table 5 . five of the field strains of ibv were most closely matched at the sequence level to strains of the 4/91 serotype, three of the field samples were identified as being qx-like strains, and the remaining two field samples were identified as having closest sequence matches within the d274 and italy 02 serotypes, respectively. this paper describes the development and validation of a taqman ò real-time rt-pcr for the detection of ibv, turkey coronavirus and pheasant coronavirus, and an ibv s1 genotyping method to enable the subsequent identification of the strain of ibv present. unfortunately, it was not possible within the scope of this study to determine whether the diagnostic ibv real-time rt-pcr assay can detect pigeon coronaviruses. although clinical samples from pigeons with suspected disease were tested, these were not positive using the diagnostic real-time rt-pcr. comparison of the sequence of the pigeon coronavirus 3¢ utr to the sequence of the primers and probe used within this study shows several differences at the nucleotide level, suggesting that although amplification may occur, this could be inefficient and it may be preferable to use an alternative rt-pcr to detect pigeon coronavirus. the real-time rt-pcr assay targets the 3¢ utr of the ibv genome, adjacent to the poly a tail, which, unlike some other regions of the genome, is highly conserved in all gammacoronaviruses (williams et al., 1993; sapats et al., 1996; dalton et al., 2001) . because of the transcriptional strategy employed by coronaviruses in which sets of 3¢ coterminal nested rna molecules are produced, the 3¢ utr of the virus is present not only in the genomic rna but also in the mrna molecules produced by the virus. this means that there may be more copies of the viral 3¢ utr present within a field sample than other regions of the viral genome, making this region an ideal target for a diagnostic rt-pcr where a high level of sensitivity is required. during this work, for each of the three samples that tested positive by virus isolation, a positive result was also seen using the diagnostic ibv real-time rt-pcr assay, and the presence of ibv within these samples was additionally confirmed by sequencing the amplicon generated by the diagnostic assay. furthermore, a substantial number of field samples were positive using the molecular diagnostic assay, but no virus was isolated following attempted virus isolation in spf embryonated fowls' eggs. unlike virus isolation, real-time rt-pcr does not require the presence of viable virus particles. therefore, the molecular method will be less affected than virus isolation by adverse storage and transport conditions that field submissions may be subjected to prior to receipt for testing which may result in degradation of the viral particles. this could account for the discrepancy between the results obtained with virus isolation and the realtime rt-pcr. the validity of the result obtained for 38 of the 173 real-time rt-pcr positive, virus isolation negative samples could be confirmed by sequencing of the amplicon generated by the diagnostic rt-pcr. how-ever, it was not possible to generate good-quality sequence data from all of the 116 amplicons tested, and we hypothesized that this may be attributed to degradation of the viral rna after the long periods of storage and repeated cycles of freeze-thawing that the rna extracts were subjected to. alternatively, this may be caused by the short length of the amplicon used, as short stretches of sequence information could be difficult to identify using the blastn-based method utilized within this study. infectious bronchitis virus rna has been detected in tracheal swab samples by other real-time rt-pcrs for at least 21 days post-vaccination (callison et al., 2006) and has been isolated from faecal samples in some infected birds as long as 227 days post-infection gough, 1977, 1978) , making it essential to be able to differentiate between vaccine and field strains for diagnostic purposes. s1 genotyping to identify an ibv strain is used widely (kingham et al., 2000; farsang et al., 2002; bochkov et al., 2006; worthington et al., 2008) and has been found to correlate with serotyping of the viral strain (wang and tsai, 1996; keeler et al., 1998) , although it has been demonstrated (bochkov et al., 2007 ) that genotyping ibv strains using either the s or n genes can offer a different perspective on the relationship of ibv strains. genotyping of the hypervariable region (hvr) of the s1 gene has also been reported to yield the same result as genotyping the entire s1 gene sequence (wang and huang, 2000) . the s1 genotyping method described in this paper amplifies a region, including the hvr 2, which is known to show variation at the sequence level between different ibv strains (cavanagh et al., 1988) . the use of a panel of both forward and reverse primers should allow the identification of the majority of ibv strains currently circulating in europe, as well as offering the opportunity to detect novel ibv genotypes as they arise, which is crucial for informing vaccination strategies. it is also recognized that performing such a genotyping assay in a routine testing environment offers an extra challenge in the form of data interpretation, particularly when distinguishing field from vaccine strains. it seems likely, as discussed , that identification of strains with sequences identical to those of vaccine strains is likely to indicate the presence of the vaccine strain within the sampled birds. however, it cannot be discounted that theoretically, these could be separate field strains. in the present study, comparison of the sequence of a 140-bp region of the s1 gene of ibv allowed field strains to be clustered within the main recognized ibv serotypes. the sequence of strains within a serotype was shown to differ with <5% nucleotide identity, which is comparable with previous studies looking at different strains from table 5 . results from testing a panel of field samples using s1 genotyping method. the results show the closest sequence in the s1 sequence database, along with the number of mismatches between the sample sequence and the closest match within the database for the 140-bp region compared caecal tonsils 4/91 pathogenic 2 s1fs10 trachea qx (av2150/107) 2 s1fs84 oropharyngeal swab fr/l-1450t/05 2 s1fs85 oropharyngeal swab nl/l-1449t/04 2 s1fs31 oropharyngeal swab 4/91 pathogenic 0 diagnostic rt-pcr and genotyping methods for gammacoronavirus diagnosis r. m. jones et al. within a particular serotype and comparing the sequence of the entire s1 gene (cavanagh et al., 1988 (cavanagh et al., , 1992 . analysis based on the sequence of a 140-bp region suggested that different groups of serotypes differed at the level of nucleotide identity at >15% in this region, consistent with published data that suggested that different serotypes varied at approximately 70% when comparing the first 560 nucleotides of the s1 gene . one of the drawbacks to this method of ibv strain identification is the possibility of multiple ibv strains being present within a single field sample, as may occur after routine vaccination of birds with a live attenuated ibv vaccine followed by a field challenge with a genetically distinct strain of ibv. while this remains a limitation of the method, evidence exists that more virulent strains of ibv may replicate to higher titres than more attenuated strains in infected birds (cavanagh and gelb, 2008) and should therefore be detected preferentially by this method. the coupling of a generic diagnostic real-time rt-pcr to detect ibv with a strain genotyping method based on the s1 hvr provides a rapid and cost-effective method suitable for the routine detection and identification of ibv and closely related gammacoronaviruses in field samples, and use of such tests within a diagnostic setting enhances existing scanning surveillance capabilities to detect both extant, contemporary ib viruses and new and emerging ibv variants in poultry. isolation of avian infectious bronchitis virus from experimentally infected chickens a long-term study of the pathogenesis of infection of fowls with three strains of avian infectious bronchitis virus lipman, 1990: basic local alignment search tool 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coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys variation in the spike protein of the 793/b type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs 2001: cis-acting sequences required for coronavirus infectious bronchitis virus defective-rna replication and packaging detection of infectious bronchitis virus sybr green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses molecular epizootiology of infectious bronchitis virus in sweden indicating the involvement of a vaccine strain chinese qx strain of infectious bronchitis virus isolated in the uk. vet. rec turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review avian infectious bronchitis virus detection of infectious bronchitis virus by real-time reverse transcriptase-polymerase chain reaction and identification of a quasispecies in the beaudette strain molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) serotype identification of avian infectious bronchitis virus by rt-pcr of the peplomer (s-1) gene identification of avian infectious bronchitis virus by direct automated cycle sequencing of the s-1 gene high incidence of false layers in (re)production hens supposedly attributed to a juvenile infectious bronchitis virus infection novel variation in the n protein of avian infectious bronchitis virus mega4: molecular evolutionary genetics analysis (mega) software version 4.0 relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus genetic grouping for the isolates of avian infectious bronchitis virus in taiwan analysis of a hypervariable region in the 3¢ non-coding end of the infectious bronchitis virus genome avian infectious bronchitis. chapter 2.3.2. manual of diagnostic tests and vaccines for terrestrial animals a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from this work was funded by the veterinary laboratories agency (vla) test development projects, td0022 and td0069. the authors thank staff from the vla regional laboratories and from private veterinary practices who submitted samples for this study, and the vaccine companies who provided vaccines for use in this work. the authors also thank robin sayers for his help with the statistical analysis of the data, jo mayers, vaneesa ceeraz and shital patel for technical assistance, and richard gough, mike collins, jason sawyer and kath webster for their advice and support. key: cord-351584-380s4j70 authors: ward, michael p.; xiao, shuang; zhang, zhijie title: the role of climate during the covid‐19 epidemic in new south wales, australia date: 2020-06-01 journal: transbound emerg dis doi: 10.1111/tbed.13631 sha: doc_id: 351584 cord_uid: 380s4j70 previous research has identified a relationship between climate and occurrence of sars‐cov and mers‐cov cases, information that can be used to reduce the risk of infection. using covid‐19 notification and postcode data from new south wales, australia during the exponential phase of the epidemic in 2020, we used time series analysis to investigate the relationship between 749 cases of locally acquired covid‐19 and daily rainfall, 9 a.m. and 3 p.m. temperature, and 9 a.m. and 3 p.m. relative humidity. lower 9 a.m. relative humidity (but not rainfall or temperature) was associated with increased case occurrence; a reduction in relative humidity of 1% was predicted to be associated with an increase of covid‐19 cases by 6.11%. during periods of low relative humidity, the public health system should anticipate an increased number of covid‐19 cases. survival in the environment and these factors. an inverse relationship between relative humidity and sars cases (cai et al., 2007; tan et al., 2005) , and a positive correlation with temperature (tan et al., 2005) , in china have previously been identified. similarly in a study of middle east respiratory syndrome coronavirus (mers-cov) (gardner et al., 2019) -in which a case-crossover design was used to identify statistically significant temporal associations between 446 cases reported from saudi arabia, 2015-2017 (likely to be spillover transmission from camels) and climatic variables-lower humidity and lower temperature were associated with increased cases 8-10 days later. in another study based on mers-cov case occurrence between 2012 and 2018, high temperature and low relative humidity were identified as contributors to increased mers-cov cases (altamimi & ahmed, 2020) . thus, for both sars and mers-cov, lower relative humidity appears to be associated with case occurrence, however the nature of the relationship between temperature and case occurrence is less clear. in one of the first studies of its type, based on the daily count of covid-19 cases in 30 chinese provinces qi et al. (2020) found significant negative associations between cases and average temperature and relative humidity. in addition, an interaction between temperature and humidity in hubei province was identified. every 1% increase in average relative humidity was predicted to decrease daily confirmed cases by 11%-22%, when average temperature was in the range of 5-8°c. it was suggested that in china in the spring, there should be a focus on monitoring and prevention of covid-19 in northern regions with low temperature and low relative humidity, because of the presence of suitable climatic conditions for sars-cov-2 transmission. in the current study, our aim was to further investigate the relationship between reports of covid-19 cases during the early epidemic phase in nsw, australia and temperature and relative humidity. understanding this relationship in different parts of the world enables the development of better pandemic response plans and surveillance systems, and potentially highlights regions where additional public health inventions might be needed to control covid-19. case reports in nsw, australia from the beginning of the epidemic in january 2020 to the peak of the epidemic at the end of march 2020 were accessed (nsw government, 2020a) . case reports in which the source of infection was determined to be locally acquired, and in which a date of notification and postcode of residence was reported, were selected for inclusion into the study. a time series of case reports was then created. based on the reported postcode, for all reported cases in the time series the closest weather observation station reporting rainfall, temperature and humidity for the period january to march 2020 was identified (nsw government, 2020b). daily observations of the following meteorological recordings were downloaded: rainfall (mm), and temperature (°c) and relative humidity (%) recorded at 9 a.m. and at 3 p.m. (australian government bureau of meteorology, 2020). the median values for each day for all selected weather observation stations were estimated, and time series of median rainfall, and 9 a.m. and 3 p.m. temperature and relative humidity were created. two additional time series were then created by determining the daily difference between 9 a.m. and 3 p.m. temperature, and between 9 a.m. and 3 p.m. relative humidity. thus, 7 predictor time series were available for modelling. a correlation matrix was used to select meteorological variables to avoid multicollinearity in the analysis. variables with a correlation coefficient <0.6 were retained. each remaining variable was included in a univariate generalized additive model (gam) with daily f i g u r e 1 time series (a) of 749 notified cases of covid-19 in new south wales, australia (counts) in which infection was determined to be locally acquired and for which postcode of residence was reported, during the period 12 february (day 43) to 31 march (day 91); and median rainfall and 9 a.m. (green) and 3 p.m. (red) temperature and relative humidity (rh) and (b) heatmap of standardized notified cases (counts), rainfall, temperature and relative humidity, 9 a.m.-3 p.m. temperature difference and 9 a.m.-3 p.m. relative humidity difference recorded at weather observation stations closest to reported case postcode of residence. two periods of high rainfall were observed, and 9 a.m. temperatures tended to decrease throughout the period, but 9 a.m.-3 p.m. temperature differences tended to be less variable than 9 a.m.-3 p.m. humidity differences number of reported cases as the dependent variable. variables with p value < 0.1 in univariate analyses were then included in a multivariate gam, and the best-fitting model based on akaike information criterion (aic) was selected using a backward algorithm. covid-19 cases were assumed to follow a negative binomial distribution given that the variances of the daily cases reported were greater than their means. meteorological variables were analysed using a 14-day exponential moving average (ema), based on the assumed incubation period of sars-cov-2. natural splines of two degrees of freedom were also included to account for additional short-term trend. a sensitivity analysis was performed by modifying the ema from 14 days to 10 and 21 days, respectively. r software (version 3.5.3, http://cran.r-proje ct.org; r foundation for statistical computing, vienna, austria) was used to perform all the statistical analyses and visualization. the first case was reported in nsw on 22 january 2020. for this case and the subsequent 5 cases, infections were determined to have been acquired overseas. the first case in which infection was determined to have been acquired locally was notified on 26 february (nsw government, 2020b . substantial rainfall (28-71 mm daily medians) was reported between 7 and 10 february. the difference in 9 a.m. and 3 p.m. relative humidity difference varied over a wide range, whereas 9 a.m. and 3 p.m. temperature difference remained relatively constant about a median value of 3.95°c ( figure 1b) . collinearity was identified between some of the predictor variables, so univariate gams were fit to cases and rainfall, 9 a.m. temperature, 9 a.m. relative humidity, and the difference between 9 a.m. and 3 p.m. relative humidity. a negative relationship between cases and 9 a.m. relative humidity was found, and a positive relationship between cases and 9a.m. temperature (table 1) . in multivariate analysis, only 9 a.m. relative humidity was found to be significantly (p = .0304) associated with cases (aic 211.8; table 2 ). the interaction between 9 a.m. temperature and 9 a.m. relative humidity was not statistically significant. using either a 10-day or 21-day exponential moving average, the best-fitting model (aic 212.2 and 211.4, respectively) remained 9 a.m. relative humidity only (p = .0408 and .0254, respectively). including 9 a.m. temperature in either of these three models did not improve the model fit. ta b l e 1 association between 749 notified cases of covid-19 in new south wales, australia in which infection was determined to be locally acquired and for which postcode of residence was reported, during the period 12 february (day 43) to 31 march (day 91), and climate recorded at the weather observation station closest to reported case postcode of residence multivariate generalized additive model of the association between 749 notified cases of covid-19 in new south wales, australia in which infection was determined to be locally acquired and for which postcode of residence was reported, during the period 12 february (day 43) to 31 march (day 91), and 9 a.m. relative humidity (10-, 14-and 21-day exponential moving average) recorded at the weather observation station closest to reported case postcode of residence. natural splines were included to control short term trend. there have been few published investigations on the relationship between climate and covid-19, and to our knowledge none in the southern hemisphere or specifically australia. we found a negative relationship between the evolving covid-19 epidemic in nsw, australia and relative humidity, but not rainfall nor temperature; a 1% decrease in 9 a.m. relative humidity could increase the number of covid-19 cases by 6.11%. temperature and relative humidity are known to be important factors in the spread of respiratory diseases. several epidemiological and laboratory studies have found that temperature and relative humidity affect the spread of coronavirus-related diseases, with both low temperature and relative humidity being suitable for the survival and transmission of coronavirus (casanova et al., 2010; chan et al., 2011; guionie et al., 2013) . some statistical modelling studies have found that higher relative humidity could lead to a reduction in covid-19 cases (qi et al., 2020; ma et al., 2020) , which is consistent with our study findings. however, we did not find an association between temperature and covid-19 cases. despite some studies suggesting that lower temperature could increase the incidence of covid-19 (qi et al., 2020) , other studies have not identified such a relationship (yao et al., 2020; zhu & xie, 2020) . these inconsistencies might suggest that the impact of temperature on sars-cov-2 transmission is more complex and heterogeneous across the landscape and in different countries-or even hemispheres. temperature ranged from 16 to 24°c (9 a.m.) and 16 to 34°c (3 p.m.) in our study area during the epidemic period, which is very different from most of the recent studies from china and elsewhere in the northern hemisphere winter. the timing of the covid-19 pandemic-beginning in january-and the differences between climate (especially temperature) between the northern and southern hemispheres could explain a lack of association in our study with temperature and might also partially explain the relatively limited transmission of sars-cov-2 in australia to date (265 cases per million). however, despite the temperatures experienced in the southern hemisphere summer, sars-cov-2 can be transmitted. our study suggests that even with higher temperature, covid-19 could persist through the coming northern summer and ongoing surveillance and prevention will be needed. in addition, our finding that lower relative humidity is associated with cases together with other published studies suggests that any area-regardless of season or location-can be at risk of sars-cov-2 transmission and that this risk increases at lower humidity. in conclusion, under the conditions of high temperature in the southern hemisphere summer, our study provides evidence that lower relative humidity is associated with covid-19 cases. it also suggests that all countries need to maintain vigilance for covid-19, even during the summer months. nsw ministry of health is thanked for freely making available covid-19 case notification data. no conflict of interest is declared. the authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. no ethical approval was required as the case notification data was accessed from the public (nsw government) domain. the data that support the findings of this study are available from nsw.data and the australian bureau of meteorology. these data were derived from the following resources available in the public do climate factors and incidence of middle east respiratory syndrome coronavirus new south wales weather observation stations alian -healt h-secto r-emerg ency-respo nse-plan-fornovel -coron aviru s-covid -19-short -form-the-austr alian -healt h-secto r-emerg ency-respo nse-plan-for-novel -coron aviru s-short -form.pdf australian government department of health (2020b) coronavirus (covid-19) health alert. current status indirect virus transmission in cluster of covid-19 cases influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome effects of air temperature and relative humidity on coronavirus survival on surfaces the effects of temperature and relative humidity on the viability of the sars coronavirus a case-crossover analysis of the impact of weather on primary cases of middle east respiratory syndrome an experimental study of the survival of turkey coronavirus at room temperature and +4 degrees c effects of temperature variation and humidity on the death of covid-19 in wuhan covid-19 transmission in mainland china is associated with temperature and humidity: a time-series analysis an initial investigation of the association between the sars outbreak and weather: with the view of the environmental temperature and its variation nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study no association of covid-19 transmission with temperature or uv radiation in chinese cities enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? association between ambient temperature and covid-19 infection in 122 cities from china the role of climate during the covid-19 epidemic in new south wales key: cord-343949-zmuvq6e3 authors: lu, gang; zhang, xin; luo, jie; sun, yankuo; xu, haibin; huang, ji; ou, jiajun; li, shoujun title: first report and genetic characterization of feline kobuvirus in diarrhoeic cats in china date: 2018-06-06 journal: transbound emerg dis doi: 10.1111/tbed.12916 sha: doc_id: 343949 cord_uid: zmuvq6e3 feline kobuvirus (fekov) is a newly discovered organism, classified under the species aichivirus a of the genus kobuvirus. since it was first reported in 2013, molecular evidence for fekov in the feline population has been restricted to two countries: korea and italy. in this study, we collected faecal samples from cats in southern china and detected the fekov rna in these samples. a prevalence rate of 9.9% (8/81) was identified by rt‐pcr, and all positive samples were obtained from diarrhoeic animals. in addition, fekov was shown positive associated with diarrhoea in cats, with a correlation coefficient of 0.25. next, we designed three primer pairs with degenerate bases, which targeted the conservative overlapping region of the entire published fekov genome, and sequenced the near‐complete genome of the first chinese field fekov strain, whj‐1, using long‐fragment pcr. finally, we analysed whj‐1's homology and phylogeny using the polyprotein gene. the results indicated that fekov has rapidly mutated since it was first discovered. this study will help to better understand fekov's epidemiology, evolutionary pattern and genetic diversity. 1), aichivirus e (rabbit picornavirus) and aichivirus f (bat kobuvirus) (adams, king, & carstens, 2013; adams et al., 2017; pringle, 1999) . other members of the kobuvirus genus have been detected in sheep, goats, rodents and wild boar (lee et al., 2012; phan et al., 2011; reuter, boros, pankovics, & egyed, 2010; reuter et al., 2013) . the species aichivirus a includes six types: aichi virus, canine kobuvirus, murine kobuvirus, kathmandu sewage kobuvirus, roller kobuvirus and feline kobuvirus (fekov) (adams et al., 2017) . kobuviruses are small, nonenveloped, icosahedral particles with linear, positive-sense ssrna genomes of 8.2-8.4 kb (lee et al., 2012; reuter, boldizsar, & pankovics, 2009; yamashita et al., 1998; yu et al., 2011) . their genome is composed of a 5 0 untranslated region (utr), a large open reading frame (orf), and a 3 0 utr. the orf encodes a single polyprotein precursor, which is cleaved into three structural viral proteins (vp0, vp1 and vp3) and eight nonstructural proteins (l, 2a, 2b, 2c, 3a, 3b, 3c and 3d) . kobuviruses have similar genome organizations to those described in other picornaviruses, 5 0 -l-vp0-vp3-vp1-2a-2b-2c-3a-3b-3c-3d-3 0 (choi, lee, lee, & oem, 2015; kapoor et al., 2011; pankovics et al., 2016; reuter et al., 2009 ). the first serological evidence supporting kobuviral infections in cats was reported in the united kingdom by n. carmona-vicente et al. in 2013 (carmona-vicente et al., 2013 . using the aichi virus as an antigen, igg antibody to this organism was detected in 67 of 97 cat serum samples, indicating an aichi virus crossreactive kobuviral infection is common in cats. the first fekov molecular evidence was reported in korea by joon-yee chung et al. in 2013 (chung et al., 2013 . using reverse transcription polymerase chain reaction (rt-pcr), six of 39 collected faecal samples from korean cats were confirmed positive for kobuvirus rna. phylogenetic analysis revealed that fekov (12d240, fk-13) is most closely related to the canine kobuvirus (choi et al., 2015) . in 2015, barbara di martino et al. screened faecal samples obtained from asymptomatic and diarrhoeic cats for fekov rna in italy (di martino, profio, melegari, marsilio, & martella, 2015) . fekov rna was found in five of 37 diarrhoeic cats but was undetected in asymptomatic cats. the full-length genome sequence of one italian fekov strain (fekov/ te/52/it/13) was sequenced, and it displayed a high nucleotide identity (96.0%) to the korean strain, fk-13. until now, molecular reports of the kobuvirus in cats have been restricted to korea and italy, and only three fekov strain genomes have been sequenced (cho et al., 2014; choi et al., 2015; di martino et al., 2015) . therefore, it is important to investigate and genetically characterize fekov infections in other countries to assess the global epidemiology of this emerging virus. our study determined that fekov is present in china. we evaluated its molecular prevalence in cats in china, sequenced the near-complete genome of one chinese fekov strain and analysed its homology and phylogeny based on the sequence. the fekov screening rt-polymerase chain reaction (rt-pcr) was performed using one published primer pair with broad reactivity with various kobuvirus species (reuter et al., 2009) . the primer pair, univ-kobu-f and univ-kobu-r, was designed targeting a 216-bp fragment of the 3d rdrp region of the three prototype kobuviruses (aichi virus, bovine kobuvirus and porcine kobuvirus). fekov rna was detected by pcr using primestar â hs (premix) (takara, dalian, china). the pcr conditions were 35 cycles at 98°c for 10 s, 50°c for 15 s and 72°c for 30 s, followed by 1 cycle at 72°c for 5 min. the pcr product was electrophoresed in a 1% ethidium-bromidestained agarose gel, and the faecal samples yielding pcr products of thirty-eight kobuvirus polyprotein orf sequences were retrieved from the ncbi database (https://www.ncbi.nlm.nih.gov/). their strain names and accession numbers are shown in figure 2 . the whj-1 polyprotein orf sequence was then aligned with the kobuvirus polyprotein gene sequences using bioedit (version 7.0.9.0). the nucleotide and amino acid homologies between these sequences were calculated by the megalign module of the dnastar package (version 7.1.0). finally, a neighbour-joining tree based on maximum composite likelihood was constructed using a 1,000-bootstrap value in mega (version 5.05). faecal samples that were only positive for fekov were processed for viral isolation, which was performed by inoculating 0.5 ml of the filtrate onto confluent cell layers grown in 25-cm 2 flasks at 37°c. six cell types (crfk, mdck, a549, pk-15, df-1, vero) were used and grown in dulbecco's modified eagle's medium (gibco, shanghai, china) supplemented with 100 lg/ml of streptomycin, 100 units/ml of penicillin and 10% foetal calf serum. after inoculating for 1 hr, the inocula were removed and fresh medium was added. negative noninoculated controls were also cultivated. cultures were inspected daily by inverted microscopy for cytopathic effects (cpe) until 4 days postinoculation. the cultures were frozen and used for further passage. culture lysates and supernatant were also collected for fekov rt-pcr. to detect the presence of fekov, rna was extracted from the colone field fekov strain demonstrated here was named whj-1. to elucidate the possibility of a correlation between fekov and feline diarrhoea, the data were processed for calculating the phi coefficient of association. the obtained correlation coefficient was 0.25. in addition, the p value of one-tailed and one-tailed fisher exact probability test was 0.023 (p < 0.05) and 0.046 (p < 0.05), respectively. the result suggested a positive association between fekov and feline diarrhoea. to acquire the whj-1 genome, all fekov genome sequences available in the ncbi database were retrieved and aligned, and three primer pairs were designed based on the fekov genome conservative region (figure 1a,b) . after agarose gel electrophoresis, the pcr products amplified by the three primer pairs had bands of %3,000, 950, and 4,400 bp, respectively (figure 1c) . after sequencing and assembly, the near-complete genome of whj-1 was obtained, including a 647-nucleotide 5 0 utr, a 7,311-nucleotide complete polyprotein the nucleotide and amino acid sequences of the whj-1 polyprotein and cleaved viral proteins were obtained and compared with those of three fekov strains and four representative kobuviruses (table 1) . whj-1 had a higher homology with fekov than with kobuviruses of other origins when analysing each gene at both the nucleotide and the amino acid levels. in addition, except the 3a gene homology between whj-1 and the canine and mouse kobuviruses, higher nucleotide and amino acid identities were found in each gene between whj-1 and the canine kobuvirus compared with mouse, human and bovine kobuviruses. among the polyprotein genes of the three fekov strains, whj-1 had a higher nucleotide identity with the italian strain, fekov/ te/52/it/13 (93.2%), and a higher amino acid homology with the korean strain, fk-13 (98.6%). the highest nucleotide identity was found between the fk-13 3b gene and the 12d240 2a gene, with a value of 95.1%, and a highest amino acid identity was found between the fekov/te/52/it/13 2b protein, the fk-13 3a and 3b proteins, and the 12d240 vp0 and 2b proteins, with a value of 100%. the highest nucleotide divergence was found in the fekov/te/52/it/13 3b gene, with a value of 88.9%, and the highest amino acid divergence was found in the 12d240 l protein, with a value of 83.9%. phylogenetic analysis of the kobuvirus polyprotein orf gene indicated the chinese fekov strain, whj-1, was clustered with the italian and korean fekov strains (figure 2 ). in addition, the phylogenetic tree showed that fekov was most closely related to the canine kobuvirus, as indicated by the nucleotide and amino acid homology analyses. both fekov and the canine kobuvirus were grouped in aichivirus a, the species that comprise the genus kobuvirus together with aichivirus b-f. after serial passages, no cpe were observed in the cultured cells, and no kobuvirus rna was detected in the culture supernatant or cells by rt-pcr. in this study, we provided the first evidence to support fekov circulation in the chinese feline population, demonstrating that fekov is not geographically restricted to korea and italy (cho et al., 2014 (cho et al., , 2015 choi et al., 2015; chung et al., 2013; di martino et al., 2015) . all fekov-positive faecal samples were collected from diarrhoeic cats, with a prevalence rate of 8/52, similar to those identified in korea (6/39) and italy (5/37) (chung et al., 2013; di martino et al., 2015) . no fekov was found in asymptomatic cats in this study, nor in another study in italy. however, a study conducted by yoon(cho et al., 2015) . fekov prevalence studies in china, korea and italy indicate fekov is commonly detected in diarrhoeic cats (di martino et al., 2015) . in addition, our field study to isolate fekov, we used six cell types originating from cats, dogs, humans, pigs, chickens and monkeys but detected no fekov replication in any of these cell lines after serial passages. to date, kobuvirus culturing remains an important problem to be solved. to our knowledge, except for the aichi virus strain, a846/88, and the bovine kobuvirus strain, u-1, other attempts to isolate kobuviruses have failed (reuter, kecskemeti, & pankovics, 2010; yamashita et al., 1991 yamashita et al., , 2003 . the lack of pure viral stocks after culturing makes it difficult to study kobuviral pathogenicity. in conclusion, in this study, we first determined fekov presence in faecal samples from diarrhoeic cats in southern china. we also sequenced the near-complete genome of one field fekov strain using long-fragment pcr. homology analysis indicated fekov's genetic variation. our study indicated fekov was associated with feline diarrhoea. further study is required to isolate this virus and study its pathogenicity in cats. this work was supported by the national natural science foundation of china (31672563) the authors declare no conflict of interest. lu http://orcid.org/0000-0002-4969-1768 yankuo sun http://orcid.org/0000-0001-6283-4172 ratification vote on taxonomic proposals to the international committee on taxonomy of viruses changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses phylogeny and prevalence of kobuviruses in dogs and cats in the uk molecular characterization of the full kobuvirus genome in a cat molecular evolution of kobuviruses in cats genetic characteristics of the complete feline kobuvirus genome detection and genetic characterization of feline kobuviruses detection of feline kobuviruses in diarrhoeic cats characterization of a canine homolog of human aichivirus kobuvirus in south korean black goats novel picornavirus in domestic rabbits (oryctolagus cuniculus var. domestica). infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases the fecal viral flora of wild rodents virus taxonomy-1999. the universal system of virus taxonomy, updated to include the new proposals ratified by the international committee on taxonomy of viruses during 1998 complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus, a member of a new species in the genus kobuvirus, family picornaviridae kobuvirus in domestic sheep evolution of porcine kobuvirus infection porcine kobuvirus in wild boars (sus scrofa) isolation and characterization of a new species of kobuvirus associated with cattle isolation of cytopathic small round viruses with bs-c-1 cells from patients with gastroenteritis complete nucleotide sequence and genetic organization of aichi virus, a distinct member of the picornaviridae associated with acute gastroenteritis in humans analysis and characterization of the complete genome of a member of a new species of kobuvirus associated with swine key: cord-326596-8ux1q9xw authors: chen, yanyu; ding, zhuang; liu, xinxin; chen, jianjun; li, junjiao; fei, yidong; liu, zhe; stoeger, tobias; bi, yuhai; yin, renfu title: biological and phylogenetic characterization of a novel hemagglutination‐negative avian avulavirus 6 isolated from wild waterfowl in china date: 2018-09-08 journal: transbound emerg dis doi: 10.1111/tbed.13005 sha: doc_id: 326596 cord_uid: 8ux1q9xw up to now only nine whole genome sequences of avian avulavirus 6 (aavv‐6) had been documented in the world since the first discovery of aavv‐6 (aavv‐6/duck/hongkong/18/199/77) at a domestic duck in 1977 from hong kong of china. very limited information is known about the regularities of transmission, genetic and biological characteristics of aavv‐6 because of the lower isolation rate and mild losses for poultry industry. to better further explore the relationships among above factors, an aavv‐6 epidemiological surveillance of domestic poultry and wild birds in six provinces of china suspected of sites of inter‐species transmission and being intercontinental flyways during the year 2013–2017 was conducted. therefore, 9,872 faecal samples from wild birds and 1,642 cloacal and tracheal swab samples from clinically healthy poultry of live bird market (lbm) were collected respectively. however, only one novel hemagglutination‐negative aavv‐6 isolate (aavv‐6/mallard/hubei/2015) was isolated from a fresh faecal sample obtained from mallard at a wetland of hubei province. sequencing and phylogenetic analyses of this aavv‐6 isolate (aavv‐6/mallard/hubei/2015) indicated that this isolate grouping to genotype i were epidemiological intercontinentally linked with viruses from the wild birds in europe and america. meanwhile, at least two genotypes (i and ii) are existed within serotype aavv‐6. in additional, this novel hemagglutination‐negative aavv‐6 isolate in chicken embryos restored its hemagglutination when pre‐treated with trypsin. these findings, together with data from other aavv‐6, suggest potential epidemiological intercontinental spreads among aavv‐6 transmission by wild migratory birds, and reveal potential threats to wild birds and domestic poultry worldwide. over the last 40 years, many viruses from the paramyxoviridae family isolated from not only human or animal but also in birds have been newly identified (kolakofsky & roux, 1987; samal, 2011) . paramyxoviruses are enveloped, non-segmented, pleomorphic rna viruses containing a single stranded, negative-sense genome. avian paramyxoviruses that have been isolated from birds; however, due to changes in taxonomy is now referred to as avian avulavirus (aavv) (amarasinghe et al., 2017 ). there are 13 described aavv *equal contributors. serotypes (aavv-1 to -13) based on neuraminidase inhibition tests and hemagglutination inhibition (hi), and eight another putative serotypes have been recently isolated (aavv-14 to -21) (jeong et al., 2017; lee et al., 2017; neira et al., 2017; thampaisarn et al., 2017; thomazelli et al., 2017; yamamoto, ito, & ito, 2016) . while very limited information is known about the biological and molecular characteristics of aavv-2 to -21, extensive study has been mainly conducted on aavv-1 (newcastle disease virus, ndv) (cardenas-garcia et al., 2015; umali, ito, katoh, & ito, 2014) . newcastle disease (nd), caused by the virulent aavv-1, a wellcharacterized aavv serotype, is a highly contagious devastating viral disease to the domestic poultry worldwide because of its high mortality and heavy losses for economy (saif & barnes, 2008) . other serotypes aavv, such as aavv-2, -3 and -7, are also known to cause reproductive and respiratory diseases in turkeys and chickens, sometimes resulting in death of the infected birds (samuel, subbiah, shive, collins, & samal, 2011; warke, stallknecht, williams, pritchard, & mundt, 2008) . meanwhile, some serotypes aavv strains display their specific host restriction, such as aavv-5 cause diarrhoea and high mortality in budgerigars but not in chickens and ducks (briand, henry, massin, & jestin, 2012) . however, was first identified at a domestic duck in 1977 from hong kong (duck/hong kong/18/199/77) and then was found to cause drop in egg production and mild respiratory disease in turkeys, but was avirulent in chickens (chang et al., 2001; tian et al., 2012; xiao et al., 2010) . but recent serosurveillance of commercial chickens in the usa showed the likely prevalence of all serotypes aavv including aavv-6, excepted with aavv-5 (warke, appleby, & mundt, 2008) . the genome size of aavv range from 14.9 to 17.4 kb that is transcribed into at least six genes, which separately encode for up to nine different proteins (saif & barnes, 2008) . however, aavv-6 has an rna genome consists of seven genes in the order of 3′-np (56-1,626)-p(1,634-3,119)-m(3,122-4,526)-f(4,586-6,420 or 4,586-6,416)-sh(6,470-7,061 or 6,464-7,037)-hn(7,072-9,102 or 7,066-9,096)-l(9, 166-16,182 or 9,160-16,176 )-5′ in length with 16,230 or 16,236 nucleotides (xiao et al., 2010; yamamoto, ito, tomioka, & ito, 2015) . six major proteins are encoded, including the nucleocapsid protein (np, 128-1,525, 1,398 nt), phosphoprotein (p, 1,687-2,979, 1,293 nt), matrix protein (m, 3,235-4,335, 1,101 nt), fusion protein (f, 4, 265, 1, 668 nt or 4, 265, 1, 638 nt), 7, 963 or 7, 957, 1, 842 nt) and large polymerase protein (l, 9,278-16,003 or 9,272-15,997, 6,726 nt) . in addition, the small hydrophobic protein (sh, 6, 970 or 6, 964, 429 nt) that aavv-6 has, is not found in the other serotypes (sobolev et al., 2016; ). the few reports on the incidence of aavv-6 in commercial and domestic poultry from different parts of the world have shown a notable presence of several of this virus (chang et al., 2001; . despite this, knowledge about the regularities of transmission, genetic and biological characteristics of aavv-6 viruses in commercial poultry and wild birds in the china recent years remains limited. therefore, in this study, an aavv-6 surveillance of domestic poultry and wild birds in six provinces of china suspected of sites of inter-species transmission and being intercontinental flyways from december 2013 to june 2017 was conducted. all experimental protocols (approval id: 20130113-1, approval date: 15th jan 2013) used in this work were reviewed and approved by the experimental animal council of jilin university, china. presence and identification of aavv-6 in each individual collected specimen was performed through allantoic cavities inoculation of 9 to 10-day-old specific-pathogen-free (spf) chicken embryos (merial, beijing, china) (kim, king, suarez, wong, & afonso, 2007; yin et al., 2017) . the presence of the aavv-6 in allantoic fluid was identified by rt-pcr and sequencing for paramyxoviruses (tong, chern, li, pallansch, & anderson, 2008) . the chicken fibroblast cell line df-1 and the chicken bone marrow macrophages cell line hd11 were grown in dmem containing 10% foetal bovine serum (fbs) (gibco, life technologies) and complete dmem/f12 containing 10% fbs respectively. cells were planted into a 24-well cell culture plate at a viable cell density (determined by trypan blue exclusion, sigma, shanghai, china) of 3 × 10 5 cells per well at 37°c under 5% co 2 for 8 hr. cells then were washed three times with phosphate buffered saline and supernatant was changed into fresh medium supplemented with 100 μg/ml streptomycin and 100 u/ml penicillin without fbs. thereafter, cells were absorbed with virus at 100 μl allantoic fluid containing the hubei isolate for 1 hr in the presence or absence of tpck-trypsin (sigma, shanghai, china) and fresh medium was added into the well and then incubated with 72 hr post infection (hpi). subsequent to infection, virus titre in the supernatants was measured using a micro-hemagglutination assay (ha) method (zhang et al., 2018) . viral rna was isolated from allantoic fluid using the axyprep body fluid viral dna/rna miniprep kit (axygen, shanghai, china) according to the manufacturer's instructions. following extraction, cdna synthesis was performed by using goscript ™ reverse transcription system (promega, shanghai, china) following the manufacturer's instructions using random primer. then samples were measured by seminested pcr for l gene of paramyxoviruses using 2×easytaq pcr kit (transgen biotech, beijing, china) (tong et al., 2008) . the first amplification in the seminested pcr assay consists of 10 μl 2×easytaq pcr supermix, 2 μl cdna, 10 μm par-f1 primer, 10 μm par-r primer and h 2 o to achieve a final volume of 20 μl. the cycling reactions consisted of a cycle of 94°c for 2 min followed by 40 cycles of 94°c for 15 s, 48-50°c for 30 s and 72°c for 30 s. for the second amplification in the seminested pcr assay, we used 2 μl aliquot from the first pcr reaction, 10 μl 2×easytaq pcr supermix,10 μm par-f2 primer, 10 μm par-r primer and h 2 o to achieve a final volume of 20 μl. the cycling conditions consisted of an initial denaturation at 94°c for 2 min followed by 40 cycles of 94°c for 15 s, 48-50°c for 30 s and 72°c for 30 s. after that, the par-f2 and par-r primers were used for pcr amplicons sequencing (sangon biotech, shanghai, china). the blast search identified the relatedness of the isolated viruses with other reported aavv-6 strains and therefore this hubei stain was designated as aavv-6/mallard/hubei/2015. after that this aavv-6 in this study were amplified for the entire genome using 16 primer pairs (table 1 ). the cycling reactions consisted of a cycle of 95°c for 3 min followed by 40 cycles of 95°c for 1 min, 45-57°c for 45 s and 72°c for 150 s. pcr amplicons sequencing was performed by major-bio company (beijing, china). the pathogenicity of the aavv-6 isolate was determined by (a) ha and hi assay were carried out according to the oie guidelines (newcastle disease 2018). in hi tests, anti-sera against avian influenza virus (aiv) h1, h5 and h9 (weike biotechnology, harbin, china) and ndv lasota strain (weike biotechnology, harbin, china), aavv-4 (prepared by our lab) were used as references. nucleotide sequences of aavv-6 in this study were aligned through mega x software with the sequences of representative aavv-6 strains retrieved from genbank database (http://www.ncbi.nlm.nih. gov/genbank). the homology analysis was carried out using the maximum likelihood method through megalign (dnastar a tremendous amount of information about aavv-1 is available on the characteristics and genetic relationships because of the severe disease it causes in poultries worldwide (shittu, joannis, odaibo, & olaleye, 2016; zhang et al., 2015) . by comparison, the pathological phenomenon which aavv-6 causes are relatively weak, just manifested in decreased egg production and mild respiratory disease in turkeys and was avirulent in chickens (alexander, 2000; saif & barnes, 2008; sobolev et al., 2016) . as a low virulence virus for chickens and low separation rate, the potential harm of the aavv-6 is easily overlooked. however, in a recent study of the pathogenicity of two aavv-6 variant isolates, aavv-6/red-necked stint/japan/8ks0813/ 2008 and aavv-6/duck/hong kong/18/199/1977 , as representative isolate of genotype i and ii respectively, could replicate in respiratory tissues of infected mice and induce respiratory disease, sometimes resulting in death of the infected mice . further researches about the virulence and susceptibility of aavv-6 should be include more isolates, since differences of viral propagation properties in same cells were observed between the two variant isolates, owing to the change of host from red-necked stint to duck and sites where the two variant isolates separated at such a distance to some extent (bui et al., 2014) . therefore, the identification and isolation of hubei isolate is beneficial for the further understanding of ha-negative aavv-6 in this study for the high sequence identity (99.1%-99.2%) with two jilin isolates (aavv-6/mallard/jilin/190/2011 and aavv-6/mallard/jilin/127/2011) and the same cleavage site with other aavv-6 isolates. in conclusion, our current data indicate that aavv-6 is distributed sporadically in wild migratory birds, not in domestic birds, in the number of base substitutions per site from averaging over all sequence pairs between groups are shown. standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (500 replicates). analyses were conducted using the maximum composite likelihood model. the rate variation among sites was modelled with a gamma distribution (shape parameter = 1). the analysis involved (a) 24 nucleotide sequences (i, n = 13; ii, n = 11), (b) 13 nucleotide sequences (ia, n = 8; ib, n = 4) and 11 nucleotide sequences (iia, n = 5; iib, n = 6). codon positions included were 1st+2nd+3rd+non-coding. all positions containing gaps and missing data were eliminated. there were a total of 1,638 positions in the final dataset. evolutionary analyses were conducted in mega x. (20160414029gh), and three grants from the national science foundation of china (31402195, 31472195 and 31570026). ry, xl, yc and zd designed and performed the study, drafted the manuscript and analyzed the data. all authors collected clinical samples. ry, xl, zd and yc carried out experiments. yin http://orcid.org/0000-0001-7431-2523 newcastle disease and other avian paramyxoviruses taxonomy of the order mononegavirales: update 2017 complete genome sequence of a novel avian paramyxovirus characterization of a genetic and antigenic variant of avian paramyxovirus 6 isolated from a migratory wild bird, the red-necked stint (calidris ruficollis) evaluation of the replication and pathogenicity of a variant avian paramyxovirus serotype 6 in mice development of an improved vaccine evaluation protocol to compare the efficacy of newcastle disease vaccines complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks genetic diversity of avian paramyxovirus type 6 isolated from wild ducks in the republic of genetic diversity of avian paramyxovirus type 1: proposal for a unified nomenclature and classification system of newcastle disease virus genotypes a virological survey in migrating waders and other waterfowl in one of the most important resting sites of germany complete genome sequence of a novel avian paramyxovirus isolated from wild birds in south korea characterization of class i newcastle disease virus isolates from hong kong live bird markets and detection using real-time reverse transcription-pcr the molecular biology of paramyxoviruses mega x: molecular evolutionary genetics analysis across computing platforms a novel avian paramyxovirus (putative serotype 15) isolated from wild birds novel avulaviruses in penguins oie, the world organisation for animal health newcastle disease, pneumovirus infection and other paramyxoviruses the biology of paramyxoviruses experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9 newcastle disease in nigeria: epizootiology and current knowledge of circulating genotypes isolation and properties of viruses from poultry in hong kong which represent a new (sixth) distinct group of avian paramyxoviruses characterization of avian paramyxovirus type 6 isolated from a eurasian teal in the intersection of migratory flyways in russia characterization of avian paramyxovirus serotype 14, a novel serotype, isolated from a duck fecal sample in japan novel avian paramyxovirus (apmv-15) isolated from a migratory bird in south america complete nucleotide sequence of avian paramyxovirus type 6 strain jl isolated from mallard ducks in china sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses surveillance of avian paramyxovirus in migratory waterfowls in the san-in region of western japan from prevalence of antibodies to different avian paramyxoviruses in commercial poultry in the united states comparative study on the pathogenicity and immunogenicity of wild bird isolates of avian paramyxovirus 2, 4, and 6 in chickens complete genome sequences of avian paramyxovirus serotype 6 prototype strain hong kong and a recent novel strain from italy: evidence for the existence of subgroups within the serotype genetic diversity of the genotype vii newcastle disease virus: identification of a novel viij sub-genotype identification and pathotypical analysis of a novel vik sub-genotype newcastle disease virus obtained from pigeon in china completion of full length genome sequence of novel avian paramyxovirus strain apmv/shimane67 isolated from migratory wild geese in japan characterization of novel avian paramyxovirus strain apmv/shimane67 isolated from migratory wild geese in japan dispersal and transmission of avian paramyxovirus serotype 4 among wild birds and domestic poultry enhanced replication of virulent newcastle disease virus in chicken macrophages is due to polarized activation of cells by inhibition of tlr7 high genetic diversity of newcastle disease virus in wild and domestic birds in northeastern china from 2013 to 2015 reveals potential epidemic trends biological and phylogenetic characterization of a novel hemagglutination-negative avian avulavirus 6 isolated from wild waterfowl in china key: cord-284398-rhfwbyav authors: aboubakr, hamada a.; sharafeldin, tamer a.; goyal, sagar m. title: stability of sars‐cov‐2 and other coronaviruses in the environment and on common touch surfaces and the influence of climatic conditions: a review date: 2020-07-14 journal: transbound emerg dis doi: 10.1111/tbed.13707 sha: doc_id: 284398 cord_uid: rhfwbyav although the unprecedented efforts the world has been taking to control the spread of the human coronavirus disease (covid‐19) and its causative aetiology [severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2)], the number of confirmed cases has been increasing drastically. therefore, there is an urgent need for devising more efficient preventive measures, to limit the spread of the infection until an effective treatment or vaccine is available. the preventive measures depend mainly on the understanding of the transmission routes of this virus, its environmental stability, and its persistence on common touch surfaces. due to the very limited knowledge about sars‐cov‐2, we can speculate its stability in the light of previous studies conducted on other human and animal coronaviruses. in this review, we present the available data on the stability of coronaviruses (covs), including sars‐cov‐2, from previous reports to help understand its environmental survival. according to available data, possible airborne transmission of sars‐cov‐2 has been suggested. sars‐cov‐2 and other human and animal covs have remarkably short persistence on copper, latex and surfaces with low porosity as compared to other surfaces like stainless steel, plastics, glass and highly porous fabrics. it has also been reported that sars‐cov‐2 is associated with diarrhoea and that it is shed in the faeces of covid‐19 patients. some covs show persistence in human excrement, sewage and waters for a few days. these findings suggest a possible risk of faecal–oral, foodborne and waterborne transmission of sars‐cov‐2 in developing countries that often use sewage‐polluted waters in irrigation and have poor water treatment systems. covs survive longer in the environment at lower temperatures and lower relative humidity. it has been suggested that large numbers of covid‐19 cases are associated with cold and dry climates in temperate regions of the world and that seasonality of the virus spread is suspected. nidovirales, which encompasses positive-sense, single-stranded rna viruses that replicate using a nested ('nido') set of mrnas (peiris, 2016) . according to the international committee on taxonomy of viruses (ictv), the family coronaviridae is classified into two subfamilies, letovirinae and orthocoronavirinae (king et al., 2018) . the latter includes human and animal coronaviruses ( figure 1 ) and is classified into four genera: alpha-, beta-, gamma-and deltacoronaviruses ( figure 1 ). most of human coronaviruses (hcovs) are betacoronaviruses including hcov-oc43, hcov-hku1, severe acute respiratory syndrome coronavirus 1 (sars-cov-1), sars-cov-2 and middle east respiratory syndrome coronavirus (mers-cov) (cdc, 2020; dilcher, werno, & jennings, 2020) . a few human coronaviruses, such as hcov-229e and hcov-nl63, are alphacoronaviruses. all bat coronaviruses are either alpha-or betacoronaviruses. three swine coronaviruses that are of significant concern to the porcine industry are transmissible gastroenteritis virus (tgev), (ashour, elkhatib, rahman, & elshabrawy, 2020) . before 2002, human covs were thought of as nuisance viruses causing common cold and were never perceived as serious public health threats (ashour et al., 2020) . this perception changed in [2002] [2003] after the emergence of the sars-cov-1, which was the first lethal form of covs to infect humans (drosten et al., 2003) . the epidemic of sars-cov-1 caused 8,422 illnesses and 916 deaths in 29 countries (cdc, 2017; who, 2020a) . after its containment in 2004, the number of cases of sars-cov-1 is approaching zero (cdc, 2017) . in 2012, another novel zoonotic coronavirus (named middle east respiratory syndrome coronavirus [mers-cov] ) caused an epidemic claiming the lives of 866 people in 27 countries (who, 2020b) . city, hubei province, china, causing severe acute respiratory disease, and this disease is referred to as the coronavirus disease-2019 . due to the drastic increase in the number of reported covid-19 cases worldwide, it was declared as a pandemic by the who on 11 march 2020. on the basis of preliminary genetic studies, this new virus was tentatively named as 2019-new coronavirus (2019-ncov). later, it was renamed as 'severe acute respiratory syndrome coronavirus 2' (sars-cov-2) after the coronaviridae study group of the ictv determined that the virus belongs to the existing virus species, severe acute respiratory syndrome-related coronavirus (gorbalenya, baker, & baric, 2020; who, 2020c) . as of 21 may 2020 (10:35 a.m.), about 4,858,850 confirmed covid-19 cases including 329,300 deaths have been reported in 188 countries. in the united states alone, 1,556,749 cases have been reported resulting in more than 93,606 deaths (jhu, 2020). the overall fatality rate of sars-cov-2 is relatively low (~6.8%) as compared to that of sars-cov-1 and mers-cov (10.9% and 34.4%, respectively) but it is still in flux and very well could be lower than 6.8%. however, it is obvious that sars-cov-2 is much more contagious as evidenced by its spread to 185 countries across the globe within a very short time. this has led to an increased concern of possible collapse of the medical care systems, as they will not be able to accommodate a large number of cases simultaneously. (decaprio, gartner, burgess, kothari, & sayed, 2020; specht, 2020) . for that reason, the governments and public health sectors are racing against time to contain this pandemic before the occurrence of f i g u r e 1 the most recent classification of coronaviruses within the family coronaviridae, subfamily orthocoronavirinae, and the respective genera: alpha -, beta -, gamma -and deltacoronaviruses. the sars-cov-2 is classified as a betacoronavirus. covs that are presented in red colour are human-infectious this catastrophic scenario. because an effective and safe vaccine or antiviral drug for sars-cov-2 does not exist, infection control is the only available method to limit the spread of the virus (lai, shih, ko, tang, & hsueh, 2020) . the infection control and preventive measures depend mainly on our primary understanding of the routes of transmission of this virus. a reported familial cluster of pneumonia associated with covid-19 in hospital and family settings has confirmed the direct person-to-person transmission route for this virus . other indirect transmission routes are postulated and/or indicated such as faecal-oral, nosocomial, airborne and contact with contaminated surfaces and fomites (cai et al., 2020; han, lin, ni, & you, 2020) . the impact of the environmental conditions such as temperature, relative and absolute humidity, and sunlight on the virus stability and spread is largely unknown. this review has collected all available data on the stability of sars-cov-2 and other coronaviruses from previously published reports. we believe that the data provided herein should help establish a solid long-term protocol to interrupt indirect environmental transmission of sars-cov-2, limit its spread, and mitigate its risks. the association of sars-cov-2, sars-cov-1 and mers-cov with acute respiratory diseases and their high loads detected in throat, sputum and lower respiratory tract of infected persons indicate that viral particles of sars-cov-2 are shed in aerosols during coughing and sneezing (calvet et al., 2016; guery et al., 2013; nhcprc, 2020; pan et al., 2020) . the rna of sars-cov-1 has been detected in air samples collected from hospitals in china (xiao et al., 2004) . the detection of rna of an animal cov (such as pedv) in air at 16.1 km distance from an infected farm in the united states indicates possible airborne transmission (alonso et al., 2014) . although the detection of sars-cov-2 or its rna in aerosols has not yet been reported, confirmed aerosol transmission of other coronaviruses suggests possible aerosol transmission of sars-cov-2 (ge, yang, xia, fu, & zhang, 2020) . therefore, understanding the persistence of sars-cov-2 is important to develop effective infection control measures of the virus in aerosols. the persistence of various covs in aerosols at different environmental conditions has been studied. the results of these studies are summarized in table 1 . only two studies on aerosolized sars-cov-2 are available. the first study compared the decay rates of sars-cov-2 and sars-cov-1 within 3h aerosolization time at room temperature (21°c-23°c) and a fixed relative humidity (rh) of 65%; both viruses were detectable after 3 hr of aerosolization. the median half-lives were 1.09 and 1.18 hr for sars-cov-2 and sars-cov-1, respectively (van doremalen et al., 2020) . in another study, aerosolized sars-cov-2 retained its infectivity for a period of 16h at room temperature and the authors concluded that the virus can be considered as an airborne pathogen (fears et al., 2020 and was infectious after 72 hr of aerosolization (ijaz, brunner, sattar, nair, & johnson-lussenburg, 1985) . another study reported that infectious mers-cov was detectable after 1h of aerosolization despite a reduction in virus titre over the aerosolization time (pyankov, bodnev, pyankova, & agranovski, 2018) . in general, the persistence of a given virus in the environment outside its host is essential to allow its spread. however, the characteristic of cov-1 and mers-cov as discussed below and summarized in table 2 . persistence of sars-cov-2 on plastic surface has been reported in two recent studies. in the first study, sars-cov-2 retained its infectivity for 4 days but was completely decayed after 7 days on plastic surface at room temperature and 65% rh (chin et al., 2020) . the second study demonstrated that sars-cov-2 retained its infectivity for 3 days on plastic surface at room temperature. the same study found no difference between the persistence of sars-cov-2 and sars-cov-1 on plastic surface and both viruses completely lost their infectivity after 4 days (van doremalen et al., 2020) . duan et al. (2003) reported longer persistence (4 days with complete decay after 5 days) of sars-cov-1 on plastic surface. on polystyrene petri dish, sars-cov-1 survived for at least 6 days at room temperature and completely decayed after 9 days (rabenau et al., 2005) . in another study, sars-cov-1 retained its infectivity on plastic surface for 28 days at room temperature and 40%-50% rh (chan et al., 2011) . although this study reported longer virus survival, it has been shown that the survivability of sars-cov-1 on plastic surface is drastically affected by increases in temperature and rh as described below. as compared to sars-cov-1 and sars-cov-2, a little shorter survivability on plastic has been shown for mers-cov and hcov-229e at room temperature. both retained their infectivity for up to 2 days only and were completely inactivated after 3 days (rabenau et al., 2005; van doremalen, bushmaker, & munster, 2013) . in another study, however, hcov-229e showed longer persistence (5 days) on polyvinyl chloride (pvc) and polytetrafluoroethylene (teflon) at 21°c and 30%-40% rh (warnes, little, & keevil, 2015) . one study reported that sars-cov-2 (initial load = 3.6 log tcid 50 ) persisted for 3 days on stainless steel surface and that it became undetectable after 4 days (van doremalen et al., 2020) . in another study, a this virus with a higher initial load (5.5 log tcid 50 ) retained its infectivity for 4 days and was completely inactivated after 7 days on stainless steel at room temperature and rh of 65% (chin et al., 2020) . the available data demonstrated that the survivability of coronaviruses on metal surfaces differs according to the type of metal. in general, coronaviruses survive for shorter periods on copper, copper nickel and brass than on stainless steel and zinc surfaces. for instance, sars-cov-1 persisted on copper for 8 hr while it remained infectious for 2 days on stainless steel with complete decay after 3 days (van doremalen et al., 2020) . similarly, hcov-229e showed lower persistence on brass (ranging from 10 min to 2 hr) and copper nickel (from 20 min to 1 hr) than on stainless steel (5 days) at room temperature and 30%-40% rh. the reduction in virus persistence was found proportional to an increase in the copper content in brass and nickel ( recently, sars-cov-2 survivability on glass was studied at room temperature and rh of 65%. the virus stayed infectious for 2 days and became completely undetectable after 4 days (chin et al., 2020) . sars-cov-1 retained its infectivity for a longer time (4 days) on glass at room temperature and completely decayed after 5 days (duan et al., 2003) . the stability of sars-cov-1 on mosaic at room temperature was similar to its stability on glass (survived for 3 days and decayed after 4 days) (duan et al., 2003) . similarly, hcov-229e survived for 5 days on either glass or ceramic surfaces at room temperature. the time required for complete inactivation of this virus on both surfaces was not reported (warnes et al., 2015) . on surgical latex gloves, hcov-229e survived for 3 hr and decayed after 6 hr while hcov-oc43 survived for less than an hour and completely decayed after 1 hr (sizun et al., 2000) . another study found that infectivity of hcov-229e was detectable on silicon rubber at room temperature and 30%-40% rh for 3 days and that the virus became undetectable after 5 days (warnes et al., 2015) . the survivability of sars-cov-2 on cardboard was studied in comparison with sars-cov-1. sars-cov-2 survived for a longer time (1 day) than sars-cov-1, which survived for only 8 hr under the same conditions (van doremalen et al., 2020) . in a comparative study ( (sizun et al., 2000) . oral and upper respiratory tract fluids of the covid-19 patients are key factors in sars-cov-2 transmission as the current data indicate that the major routes of transmission are droplet, contact and aerosols (lu & shi, 2020) . faecal-oral transmission is also postulated since rna of sars-cov-2 was detected in anal swab samples collected from covid-19 patients in china zhang et al., 2020a) . in addition, infectious sars-cov-2 particles were isolated from stool specimens of covid-19 patients . furthermore, nucleic acid of sars-cov-2 was detected in urine samples from covid-19 cases . therefore, it is very important to know how stable sars-cov-2 is in oral fluids and excrements of humans to help us project the roles that these items can play in transmitting this virus. to date, no data are available on the survival of sars-cov-2 in human excrements. however, this can be extrapolated from the available data on other covs (table 3) . it has been found that coronaviruses can survive in stools for 1 hr to 4 days depending on the type and ph of the stool samples. for instance, sars-cov-1 survived in stool specimens from baby (ph = 6-7), normal adult (ph = 7-8), another normal adult (ph = 8), and adult with diarrhoea (ph = 9) for 1 hr, 3 hr, 6 hr and 4 days, respectively. the same virus was completely decayed in the same samples after 3 hr, 6 hr, 1 day and 5 days, respectively (lai et al., 2005) . similarly, wang et al. (2005) two studies reported different persistence patterns of coronaviruses in urine. duan et al. (2003) detected the infectivity of sars-cov-1 in urine for up to 5 days. however, the infectivity of the same virus was detected in urine for up to 17 days at room temperature in another study (wang et al., 2005) . none of the two studies reported the period for complete decay of the virus. in human sputum, sars-cov-1 persisted for 5 days while in human blood serum, it persisted for 4 days and decayed completely after 5 days (duan et al., 2003) . in light of these results, possible faecal-oral transmission of sars-cov-2 is suggested. furthermore, human coronaviruses such as sars-cov-1 and mers-cov have been considered as having potential for foodborne transmission (greening & cannon, 2016) . this is because several studies reported the association of gastroenteritis symptoms and infection by sars-cov-1 and mers-cov (chan et al., 2015; cheng et al., 2004) . some studies revealed that up to 10.6% of patients with sars-cov-1 and 30% of patients with mers-cov had diarrhoea . likewise, diarrhoea and gastroenteritis symptoms have been reported in some cases of sars-cov-2 infection song et al., 2020) . this indicates that sars-cov-2 may also have the potential for foodborne the stability of coronaviruses has been studied in several types of waters (table 4) . at room temperature, sars-cov-1 suspended in water stayed detectable for 3 days and was undetectable after 5 days (duan et al., 2003) . another study reported only 2 days persistence and 3 days for complete decay of this virus in both chlorinated and dechlorinated tap water at room temperature (wang the fragile structure of viruses, particularly enveloped viruses like covs, and the way they infect their host cells make them susceptible to heat. virus inactivation by heat is due to denaturation of the secondary structures of viral capsid proteins thereby altering the conformation of virion proteins involved in attachment and replication within a host cell (lelie, reesink, & lucas, 1987; schlegel, immelmann, & kempf, 2001) . the inactivation of viruses at low temperature is due to a random degradation in the nucleic acid; but at high temperature, a greater change in the conformation of the virus structural proteins occurs and leads to virus inactivation (laude, 1981) . in addition, other environmental parameters such as relative humidity can play a role in virus persistence in the environment, particularly in aerosols. therefore, understanding the possible effect of heat and rh on the persistence of sars-cov-2 is of significant value to develop proper infection control measures. many studies have reported higher persistence of several covs in water and liquids at lower temperatures as compared to higher temperatures (table 4 ). for instance, the infectivity of sars-cov-1 in dechlorinated tap water was detectable for 14 days at 4°c but for only 2 days at 20°c (wang et al., 2005) . similarly, hcov-229e decayed completely after 10 days in dechlorinated water at 23°c, but was detectable at least for 25 days at 4°c in the same type of water (gundy et al., 2009) . another study showed that after 49 days, the tgev completely decayed in reagent-grade water at room temperature (25°c) while stayed infectious in the same type of water when stored at 4°c for the same period. the mcov (mhv) showed similar results in the same study (casanova et al., 2009) . after 2 weeks, the titres of lake water-suspended tgev and mhv were reduced by 2.5 log when stored at 25°c but only 1.2 and <1 log of virus titres, respectively, were reduced at 4°c in the same type of water (casanova et al., 2009 ). sars-cov-2 persisted for 14, 7 and 1 day in dulbecco's modified eagle medium (dmem), at 4°c, 22°c and 37°c, respectively. when the temperature was increased to 56°c and 70°c, the persistence time was dramatically reduced to 10 min and 1 min, respectively (chin et al., 2020) . sars-cov-1 stayed detectable in dmem for 2 hr at 4°c, 20°c and 37°c. however, when storage temperature was increased to 56°c, 67°c and 75°c, the virus decayed completely after 1.5 hr, 1 hr and 30 min, respectively (duan et al., 2003) . another study on sars-cov-1 in dmem found that the virus stayed detectable after 1 hr at 56°c and 65°c but decayed completely after 45 min at 75°c (darnell, subbarao, feinstone, & taylor, 2004) . likewise, mers-cov stability in dmem decreased with an increase in temperature (leclercq, batejat, burguière, & manuguerra, 2014) . sars-cov-1 was detectable in minimal essential medium (mem) for 30 min at 4°c, while at 56°c and 60°c, it became completely undetectable after 30 min (rabenau et al., 2005) . the decrease in virus infectivity due to an increase in temperature was also reported for animal coronaviruses such as mouse coronavirus (mcov or mhv) and canine coronavirus (ccov) in mem, and for tgev in hepes buffer (laude, 1981; saknimit, inatsuki, sugiyama, & yagami, 1988 ). several studies have found that the survivability of coronaviruses in aerosols is affected by environmental conditions, particularly temperature and relative humidity. for instance, the survival of hcov-229e in aerosols was studied at two temperatures (6°c and 20°c) and three rh levels (low, 30%; medium, 50%; high, 80%) (ijaz et al., 1985) . (ijaz et al., 1985) . drastically decreased (4.7% survival) in the hot and dry air common to the summer season (pyankov et al., 2018 ). many studies have shown that the persistence of coronaviruses on surfaces and fomites is affected by temperature and relative humidity. in general, the available data show that coronaviruses survive longer at low temperatures and low rh (table 2) . for instance, under 80%-90%rh and >95% rh, sars-cov-1 lost 0.75log and 1 log of its titre, respectively, on plastic surface at 33°c after 1 day, while at 38°c, 2 and 3.5 log reduction in virus titre was seen (chan et al., 2011) . similar results were observed for mers-cov on plastic and stainless steel surfaces at 30°c; the virus decayed completely after 2 days and 1day when the samples were stored at 30% and 80% rh, respectively. however, at 40% rh, lower temperature (20°c) in temperature has also been shown to influence the persistence of coronaviruses in sewage. the infectivity of sars-cov-1 was detectable for 14 days in domestic sewage when it was stored at 4°c but for only 2 days at 20°c (wang et al., 2005) . in pasteurized settled sewage, the infectivity of both porcine coronavirus (tgev) and mouse coronavirus (mcov or mhv) was detectable for up to 35 days at 4°c but for 21 days only at 25°c (casanova et al., 2009) . conformational changes in the spike proteins of covs are essential to enable the fusion of the virion with the host cell. weismiller, sturman, buchmeier, fleming, and holmes (1990) found that this process is induced in mcov (mhv) at a ph of 8.0. on the contrary, neutral ph mediated the spike protein's fusion of sars-cov-1 with the host cell (xiao, chakraborti, dimitrov, gramatikoff, & dimitrov, 2003) . procock and garwes (1975) also demonstrated that adsorption, penetration, uncoating and rna replication of tgev in the host cell was determined by ph. in general, it has been found that covs are more stable at near-neutral ph as compared to the extreme acidic or alkaline ph. sars-cov-1 suspended in mem completely lost its infectivity after 1 hr exposure to extreme acidic ph (1 and 3) and extreme alkaline ph (12 and 14) regardless of the temperature (4°c, 25°c and 37°c). however, the virus retained its infectivity when stored at ph 5, 7 and 9 for 1 hr (darnell et al., 2004) . similarly, hcov 229e, mhv, tgev and ccov showed more stability at slightly acidic to neutral ph (6-7.5) than at highly acidic or highly alkaline ph (8) at both low and high temperatures. however, low temperature (4°c) increases the stability of these viruses at extreme ph values than at higher temperatures (25°c and 37°c) (daniel & talbot, 1987; lamarre & talbot, 1989; pocock et al., 1975; pratelli, 2008; sturman, ricard, & holmes, 1990) . in contrast to other covs, sars-cov-2 showed higher stability when incubated at room temperature in the transport medium for 1 hr at a wide range of ph values (ph 3-10) (chin et al., 2020) . this finding may help explain the high spread rate of sars-cov-2 as compared to other human coronaviruses such as sars-cov-1 and mers-cov. the results presented in the previous sections clearly show that the ability of coronaviruses to survive in aerosols, on surfaces and fomites, and in suspensions and liquids is affected by temperature and relative humidity. in general, human and animal covs including sars-cov-2 showed more persistence under low temperature and low rh. these results indicate that the spread of sars-cov-2 might be seasonally associated with winter and that it might be easier to control the virus spread during the summer months because of the high temperature and high humidity during those months. this assumption is supported by the fact that annual epidemics of influenza virus and hcov in temperate climates are usually activated by a sudden drop in outdoor temperatures (sundell, andersson, brittain-long, lindh, & westin, 2016) . this is attributed to the lower amount of water vapour that a unit of air can hold at low temperature; which means that the air is very dry in terms of the absolute humidity (ah) and this leads to a reduction in the size of aerosol droplets due to evaporation. this prolongs the time when the infectious droplets remain airborne thereby increasing the chance of infecting new hosts (harper, 1961) . on the same principle, maintaining high humidity along with indoor heating during winter months might reduce the transmission of these viruses. this is attributed to the indoor heating during wintertime, which causes a sharp decrease in the rh of the indoor environment and subsequently reduces the size of aerosol particles through evaporation (yang & marr, 2011) . recent epidemiological studies consistently report a strong relationship between climatic conditions and the spread of sars-cov-2. wang, jiang, et al. (2020) studied the relationship between daily means of temperatures and cumulative numbers of confirmed covid-19 cases in the world from january 20 to february 4, 2020. they found that temperature can alter the transmission of sars-cov-2 and suggested that countries and regions with a lower temperature should adopt the strictest control measures to prevent future reversal. another study examined the effect of temperature and humidity on the global patterns of early outbreak dynamics of covid-19 (between january and march 2020). they found a strong impact of temperature and the humidity on the growth rate of covid-19 cases across the world. the growth rate of covid-19 cases peaked at a temperature of ~5°c and a humidity of 0.6-1 kpa in the temperate regions of the northern hemisphere during the outbreak month, while it decreased in regions that had warmer or colder temperatures (ficetola & rubolini, 2020) . a similar study found that the high covid-19 community transmission areas across the world are located along the 30-50ᵒ n' corridor at similar weather patterns of 5°c -11°c average temperatures with low specific (3-6 g/kg) and absolute humidity (4-7 g/m 3 ) (sajadi et al., 2020) . likewise, average pressure, average temperature, minimum temperature and average water vapour pressure were found to be significantly correlated with the incidence of covid-19 . another epidemiological modelling study projected recurrent wintertime outbreaks of sars-cov-2 within the next five years (kissler, tedijanto, goldstein, grad, & lipsitch, 2020 the international commission on illumination (cie) classified the ultraviolet radiation into three bands: uvc (100-280 nm), uvb (280-315 nm) and uva (315-400 nm). visible light is the region between 400 nm and 780 nm. the uvc is known as germicidal uv as it is absorbed by rna and dna bases of the virus thereby causing photochemical fusion of two adjacent pyrimidines and forming covalently linked dimers, which then become non-pairing bases (perdiz et al., 2000) . the potential of uvb inducing the formation of pyrimidine dimers is 20-100-fold lower than that of uvc (perdiz et al., 2000) . dna and rna absorb uva weakly and, therefore, its effect is much lower than uvc and uvb in the formation of pyrimidine dimers. however, uva may cause other genetic damage such as oxidation of the bases and strand cleavage through the production of reactive oxygen species (ravanat, douki, & cadet, 2001) . a few studies have investigated the effect of artificial ultraviolet radiation (uvr) on coronaviruses (table 5) . sars-cov-1 was completely inactivated (~6 log reduction) in mem following 1-hr exposure to uvc (260nm) irradiance of >90 µw/cm 2 at 83 cm exposure distance (duan et al., 2003) . in a comparative study, 6 min exposure to uvc (254nm) irradiance of 4,016 µw/cm 2 completely inactivated 5.5 log of sars-cov-1. however, 15 min exposure to uva (365nm) irradiance of 2,133 µw/cm 2 did not show any virucidal efficacy against this virus (darnell et al., 2004) . a third study demonstrated 5.3 and 6.3 log reduction of sars-cov-1 following exposure to 134 µw/cm 2 of uvc (254) for 15 and 60 min, respectively, without complete inactivation of the virus (kariwa, fujii, & takashima, 2006) . another study showed a 4.8 log reduction in ccov after 3 days of exposure to a very weak irradiance (7.1 µw/cm 2 ) of uvc at 4 cm exposure distance (pratelli, 2008) . it is known that optical radiation from the sun is the only natural source of uvr that reaches the earth through the atmosphere. however, only two-thirds of the energy from the sun that impinges on the atmosphere penetrates to the ground level. the uvr comprises ~5% of the total radiation received at the surface of the earth. this component is extremely important in various biological processes (solar iarc, 1992 norval, 1998; hart, reid, & hart, 1993; karmer, bos, & teunissen, 1995; sagripanti & lytle, 2007) . therefore, studying the efficacy of sunlight and uvb on sars-cov-2 and the spread of covid-19 might provide some explanation on the observed correlation between sun irradiance and covid-19 spread. the the longer survival of covs at low temperatures and low relative humidity explains the observed peaks of covid-19 cases during the cold and dry climates in temperate regions of the world and explains the predicted seasonality of the virus spread by epidemiological models. additionally, a sun irradiance-dependent spread of sars-cov-2 has been suggested in an observational study. although the efficacy of the artificial uvc against sars-cov-1 has been reported, it does not support the suggested influence of sun's irradiance on sars-cov-2 spread because all natural uvc radiated by the sun is blocked by the atmosphere and does not reach the earth. since artificial uva showed no effect on sars-cov-1, it does not support the suggested sun irradiance-dependent sars-cov-2 spread because the major type of natural solar uv radiation that reaches the earth is uva. a few studies have shown virucidal efficacy of uvb on viruses other than covs. however, experimental studies on the efficacy of artificial uvb on sars-cov-2 are required to provide an explanation of the observed sun irradiance-depended covid-19 spread. none. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. no ethical approval was required as this is a review article with no original research data. haa conceived the work and discussed the content with tas and smg. haa drafted the manuscript. haa, tas and smg reviewed and edited the final version of the manuscript. data sharing is not applicable to this article as no new data were created or analysed in this study. hamada a. aboubakr https://orcid.org/0000-0002-8233-2353 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(2020f) environmental survival and microbicide inactivation of coronaviruses detection of sars-cov and rna on aerosol samples from sars-patients admitted to hospital. zhonghua liu xing bing xue za zhi= the sars-cov s glycoprotein: expression and functional characterization dynamics of airborne influenza a viruses indoors and dependence on humidity molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes. emerging microbes and infections key: cord-352211-3ps5o8ji authors: mai, k.; feng, j.; chen, g.; li, d.; zhou, l.; bai, y.; wu, q.; ma, j. title: the detection and phylogenetic analysis of porcine deltacoronavirus from guangdong province in southern china date: 2017-03-27 journal: transbound emerg dis doi: 10.1111/tbed.12644 sha: doc_id: 352211 cord_uid: 3ps5o8ji porcine deltacoronavirus (pdcov) is a newly discovered coronavirus that causes diarrhoea, vomiting and dehydration in sucking and nursing piglets. it was first reported in hong kong in 2012 and has since been discovered in the united states, canada, south korea, mainland china, thailand and laos. pdcov has been experimentally proved to lead to diarrhoea in swine and it was detected positive in pigs in guangdong, southern china. in our study, 252 faecal and intestinal samples from sucking piglets and sows with diarrhoea were surveyed for common enteric viruses. we found a prevalence of pdcov (21.8%), porcine epidemic diarrhoea virus (65.5%), transmissible gastroenteritis virus (0%), rotavirus group a (25.0%) and porcine kobuvirus (68.7%). we isolated 13 pdcov strains and discovered that pdcov infections were often co‐infections with kobuvirus rather than the commonly linked porcine epidemic diarrhoea virus. phylogenetic analysis of s gene and n gene revealed that 11 of 13 pdcov strains belonged to chinese lineage. as for the left two strains, one single strain (chn‐gd16‐05) belonged to american and korean lineages while another strain (chn‐gd16‐03) was similar to a thai strain, but only in the s gene. this suggested a possible recombination event between the thai and the newly described chinese strain. industry (chen, gauger et al., 2015; chen, zhu et al., 2015; homwong et al., 2016; song et al., 2015; wang et al., 2014) . rt-pcr detection methods have provided data for the prevalence of pdcov in china (dong et al., 2015; song et al., 2015; zhai et al., 2016) . a previous study (zhai et al., 2016) reported that the current prevalence in southern china was 1.54% (5/390), and three of the five detected strains were from guangdong province, revealing that the information regarding the molecular epidemiology of pdcovs in guangdong was still limited. the current study further investigates the prevalence, epidemiology and genomic properties of pdcov in guangdong. in order to monitor the prevalence and sequence properties of (song et al., 2015) . the thermal cycling (worked on bio-rad t100, forster city, ca) was operated with the following thermal profile: 94°c for 5 min, 30 cycles of 94°c for 45 s, 55°c for 30 s, 72°c for 1 min and a final step of 72°c for 10 min. amplicons were analysed on 1% agarose gels. moreover, on the basis of the four porcine enteric pathogens below, additional rt-pcr detections from the 13 tissue cultured purified pdcov-positive samples targeting porcine bocavirus (pbv), porcine sapelovirus (psv) and porcine astrovirus (pastv) were added. primers specific for pedv, tgev, rotavirus a, pkv, pbv, psv and pastv have been described at table s1. 2.2 | amplification, cloning and sequencing the spike (s) protein and nucleocapsid protein (n) genes the complete s and n genes were amplified using three pairs of primers: pdcov-s1, 5 0 -atgcagagagctctattg-3 0 and 5 0 -tattt caacttcgccatc-3 0 ; pdcov-s2, 5 0 -cgaccatccatagtttca-3 0 and pdcov-s2, 5 0 -ctaccattccttaaactt-3 0 ; and pdcov-n, 5 0 -atggccgcaccagtagtc-3 0 and 5 0 -ctacgctgctgattcctg-3 0 (based on chjxni2/2015/china). pcr amplification was carried out using the la taq polymerase kit (takara, biotechnology, dalian, china) using directions supplied by the manufacturer. pcr products were purified using the gel band purification kit (omega bio-tek, usa) and then cloned into the pmd-19t vector (takara) using an in-fusion pcr cloning kit (takara). the recombinant plasmids were sequenced by the beijing genomics institute (shenzhen, guangdong). nucleotide sequences were submitted to genbank with accession numbers (ky078891-ky078903 for nucleocapsid gene and ky078904-ky078916 for spike gene of chn-gd16-01 to chn-gd16-13, respectively). reference sequences included 33 strains for the s gene and 33 strains of n gene from different farms of global isolates obtained from genbank (table s2 and the secondary structure and surface properties of s1 protein of (table. 1 ). in addition, co-infections with pedv and pkv were the most common (44.4%, 79/178) ( table 2) . pdcov occurrence was examined further by an additional rt-pcr analysis using 13 pdcov isolates we successfully purified in cell culture (table 3) we identified 11 potential and distinct b-cell antigenic epitopes in the s1 protein ( figure s1 ). peptides less than 4 amino acid homology were not computed. these predicted epitopes all contained mostly b structure and high hydrophilicity, strong accessibility, good flexibility and a high, predicted antigenicity. we also identified 17 potential n until now, numerous reports had revealed that pedv was the most common porcine enteric pathogen in pigs due to its high prevalence in diarrhoeal samples. pdcov-pedv co-infection occurred at a rate greater than pdcov-rota a and pdcov-tgev. but in our study, co-infection of pdcov and pkv was greatly higher than pdcov and pedv co-occurrence (45.4%, 25/55 vs. 3.6%, 2/55, table. therefore, evidence is lacking to prove that pkv co-infects more frequently than pedv with pdcov. this warrants further investigation. hku15-44 and hku15-155 were the first two pdcov strains that were sequenced (woo et al., 2012) . the whole s gene deletion (chen, gauger et al., 2015; chen, zhu et al., 2015; dong et al., 2015; song et al., 2015; wang et al., 2015; zhai et al., 2016) . in strain chn/ah/2004 (dong et al., 2015) , a 3-nt (taa) deletion existed in its 3 0 -utr. the n gene, however, lacked any nucleotide deletions or insertions. in our work, pairwise alignment analysis revealed that two s genes from our strains 03 and 05 lacked any deletions or insertions. t a b l e 3 detection of porcine enteric pathogens from 13 tissue cultured purified pdcov-positive samples from 11 swine farms in guangdong two pairs of strains 07 and 08 from farm g and 10 and 11 from farm i were isolated from the same commercial swine farms, but at different times. unlike 07 and 08 that were identical in nucleotide sequence, 10 and 11 showed nucleotide identities of 99.0% and 98.3%, respectively, resulting in 10 amino acid changes. interestingly, these differences were in the s protein and concentrated within the s1 region. according to the two phylogenetic trees established for the s and n genes, 11 of 13 strains clustered within the chinese category and had the highest nucleotide identities with five pubtable s2 ) (zhai et al., 2016) , which were collected from guangdong and nearby guangxi provinces. however, the n gene of this strain revealed a completely reverse conclusion; that it belongs to the chinese group with a low nt identity (96.7%) with the thai strains s5011 and 5015l (janetanakit et al., 2016) ( fig. 2 and table s3 ). therefore, we inferred that a recombination event might occur in pdcov strain 03. recombination events are often reported in pedv studies and the majority are intrarecombinants with different strain lineages of the same kinds of enteric coronaviruses (jarvis et al., 2015; tian et al., 2014) . a recent study (boniotti et al., 2016; valerij et al., 2016) identified a swine enteric coronavirus (secov) from italy as more closely related to pedv in the s gene, while the remainder of the genome shared highest identity with tgev. recombination events have not been reported in pdcov strains. since we were lack of the full-length genome of this pdcov strain, the mechanism of the construction of this chimera as well as with the recombination in strain 03 is unknown, something suppose to be further work and explain with it. the spike protein is an important surface glycoprotein of coronavirus and plays a significant role in receptor binding and fusion of the viral and cellular membranes (chakraborti, prabakaran, xiao, & dimitrov, 2005; schwegmann-weßels et al., 2009) . and also, it mediates interspecies transmission (bosch, van, de haan, & rottier, 2003) . all coronavirus spike proteins share the same two functional components: an n-terminal subunit and a membrane-anchored subunit (c-terminal) that are covalently bound. the s protein of pdcov can be similarly subdivided into s1 (1-573 aa) and s2 (574-1161 aa) regions (thachil, gerber, xiao, huang, & opriessnig, 2015) . s1 is a dominant viral antigen and an elisa is available for its detection (thachil et al., 2015) . three different groups of s1 proteins from coviral infections shared less than 12% amino acid sequence identities with each other (wang, deng et al., 2016) . the b-cell response is directed against the spike protein of coronaviruses (cao et al., 2015) and it plays an important role in pathogenesis of virus infection. in our study, 11 potential b-cell antigenic epitopes of pdcov ( figure s1 ) and 16 or 17 n-glycosylation sites were analysed. interestingly, in amino acids 549-561 in strain 05, a t559i mutation greatly reduced the predicted level of its antigenicity, hydrophilicity, surface probability and flexibility ( figure s2 & table s4 ). whether this mutation has altered its antigenicity will be interesting to test in the laboratory. overall, further studies are needed to confirm whether these alterations of b-cell antigenic epitopes and n-glycosylation sites affect the pathogenicity and antigenicity of each pdcov strain. in conclusion, diarrhoeal samples collected from pigs in guangdong province were screened to detect the prevalence of pdcov. phylogenetic analysis suggested that nearly all of the strains in mainland china were clustered into the chinese lineage except one newly discovered pdcov strain that had a close relationship with us and korea strains. this complements the geographical lineage theory of global pdcov distribution. the presence of another suspected recombinant strain will provide additional data to examine the diversity of the pdcov genome. the study was supported by the national key research and development program (2016yfd0501300) of china. the authors declare no conflicts of interest. porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion 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in southern china the rate of co-infection for piglet diarrhea viruses in china and the genetic characterization of porcine epidemic diarrhea virus and porcine kobuvirus additional supporting information may be found online in the supporting information tab for this article. key: cord-283316-a8jewy2h authors: bianchini, juana; humblet, marie‐france; cargnel, mickaël; van der stede, yves; koenen, frank; de clercq, kris; saegerman, claude title: prioritization of livestock transboundary diseases in belgium using a multicriteria decision analysis tool based on drivers of emergence date: 2019-10-09 journal: transbound emerg dis doi: 10.1111/tbed.13356 sha: doc_id: 283316 cord_uid: a8jewy2h during the past decade, livestock diseases have (re‐)emerged in areas where they had been previously eradicated or never been recorded before. drivers (i.e. factors of (re‐)emergence) have been identified. livestock diseases spread irrespective of borders, and therefore, reliable methods are required to help decision‐makers to identify potential threats and try stopping their (re‐)emergence. ranking methods and multicriteria approaches are cost‐effective tools for such purpose and were applied to prioritize a list of selected diseases (n = 29 including 6 zoonoses) based on the opinion of 62 experts in accordance with 50 drivers‐related criteria. diseases appearing in the upper ranking were porcine epidemic diarrhoea, foot‐and‐mouth disease, low pathogenic avian influenza, african horse sickness and highly pathogenic avian influenza. the tool proposed uses a multicriteria decision analysis approach to prioritize pathogens according to drivers and can be applied to other countries or diseases. the oie from different european member states including belgium (world organisation for animal health, 2018) . another very important recent emerging livestock disease reported specifically in belgium at the end of 2018 was african swine fever, although cases so far have been reported only in wild boars . its emergence is of great concern for the pig industry of the region and being a disease, which until now has been exotic for belgium. it shows how diseases may re-emerge unexpectedly with most likely origin attributable to human activity (saegerman, 2018) . the (re-)emergence of diseases shifts in relation to several underlying set of factors inherent to modern society, that is the so-called 'drivers'. the joint presence of these drivers can create an environment in which infectious disease can (re-)emerge and be maintained in animal and/or human compartments (king, 2004) . many drivers have been identified, such as climate change, global travel, immigration patterns, increase in the human population, environmental degradation and others (altizer, ostfeld, johnson, kutz, & harvell, 2013; daszak, cunningham, & hyatt, 2000; king, 2004) . the threat of (re-)emergence is more likely to increase and past experience has shown that no country, however economically welldeveloped it may be, is capable of ensuring 100% security of its borders, even by imposing measures such as quarantine protocols or import bans on animals and animal products (ben jebara, 2004) . in belgium, the monitoring and reporting of livestock diseases are subjected mostly on self-reporting of suspected clinical cases by the farmers to the federal agency for the safety of the food chain (fasfc), with an established list of mandatory notifiable diseases for livestock and other species (aquatic, exotic) (federal agency for the safety of the food chain, 2019). each suspicion is then confirmed by laboratory analysis (federal agency for the safety of the food chain, 2019). thus, a rational priority setting approach is needed to assist decision-makers in identifying and prioritize diseases that are more likely to (re-)emerge and as such allocating the right resources tailored to a particular disease threat. one such approach used is disease prioritization, which has as main objectives: to optimize financial and human resources for the surveillance, prevention, control and eradication of infectious disease and to target surveillance for early detection of any emerging diseases (humblet et al., 2012) . some studies identified key characteristics of potential emerging infectious diseases and prioritized infectious diseases according to their risk of (re-)emergence or impact in some countries (cardoen et al., 2009; cox, sanchez, & revie, 2013; havelaar et al., 2010; humblet et al., 2012) . hence, these focused on human or zoonotic diseases and the impact they would have in certain countries. in this study, the focus is livestock epidemic diseases and the aim was to identify (re-)emergence drivers' criteria and with it use expert elicitation to prioritize livestock epidemic diseases that may emerge in belgium. a multicriteria decision analysis (mcda) method was chosen because it provides a systematic way to integrate information from a range of sources (cox et al., 2013) and it aims to improve transparency and repeatability (european centre & for disease prevention & control, 2015) . multicriteria decision analysis requires identifying criteria and scoring criteria according to the pathogen/disease. by weighting each criterion and calculating weighted scores from the criteria, an overall score per pathogen/disease was calculated (european centre & for disease prevention & control, 2015; humblet et al., 2012) . this is the first study to prioritize livestock epidemic disease using drivers as criteria. this prioritization list could be an aid to decision-makers to make an informed decision on course of actions to be taken and use the correct resources when there is a threat of a disease (re-)emerging in belgium. we compiled a list of livestock-associated infectious diseases ( figure 1) using a systematic approach. this was done by collating in a single database notifiable terrestrial animal diseases from different governmental official lists from belgium (federal agency for the safety of the food chain, 2015) and neighbouring countries (luxembourg was excluded because of high similarity), that is germany (federal ministry of food & agriculture of germany, 2015), france (légifrance, 2015a (légifrance, , 2015b , the netherlands (ministerie van landbouw, 2015) and great britain (scottish government, 2015) . in order to broaden the spectrum, diseases included in two other lists of official international organizations, that is the world organisation for animal health (oie) (world organisation for animal health, 2015) and the european union (european commission, 2012) , were also added to the database. only diseases that affect cattle, sheep, goats, swine and poultry (livestock) were selected from the official lists and included in database. (azkur et al., 2013; chaintoutis et al., 2014; lievaart-peterson et al., 2012; yilmaz et al., 2014) . thus, the risk of any of these viruses to (re-) emerge may be present, which further prompted the necessity of adding these three viruses to the list of diseases to be prioritized. the main objective was to prioritize the diseases according to drivers of (re-)emergence. a driver was defined as a factor, which has the potential to directly or indirectly precipitate ('drive') or lead to the (re-)emergence of a livestock infectious disease. we identified different criteria considered as drivers through scientific literature and previous disease prioritization exercises, and discussion with experts from academia, government agencies and international bodies. a total of 50 criteria were identified and classified under 8 different domains (table 1) each criterion had a definition of the coefficient, which ranged from 0 to 4 accordingly (appendix a). each domain spreadsheet had a number of criteria. for each criterion, coefficients were clearly defined for a good comprehension and standardization. coefficients were from scores of 0 to 4 or from 1 to 4 (a number of criteria could not be scored with a zero, e.g. current f i g u r e 1 systematic process for selecting the livestock diseases. * livestock diseases were those which affected cattle, sheep, goats, swine and poultry ta b l e 1 list of criteria used to prioritise (re)emerging infectious diseases, according to their likelihood of (re)emergence in belgium in response to different categories of drivers (gore, 1987) . the number of points to be distributed was proportional to the number of criteria per category multiplied by ten. indeed, the criterion with the most points allocated is considered the one that weighs the most in the category. if, on the other hand, all the criteria have the same weight in the category, the distribution is equitable, with 10 points for each criterion. for example, 90 points were to be distributed between the 9 criteria of the 'pathogen characteristics' domain. indeed, the criterion with the most points allocated is considered the one that weighs the most in the pathogen characteristics. such process illustrated the experts' opinion on the relative importance of criteria within one domain. the last spreadsheet was dedicated to the inter-domain weighting. experts were asked to distribute a total of 80 points (n = 8 domains) among the domains to classify the domains according to their opinion. two rounds of expert elicitation were implemented. the first round consisted in the questionnaire assessment; experts were asked to verify whether the questions were in relation with the drivers and whether the scoring systems were correctly defined and identified. the questionnaire and related instructions were sent to 14 experts (appendix b) by e-mail. the experts were asked to complete questionnaire by scoring and additionally to assess and give comments on the criteria and coefficient definitions. the questionnaire was then refined according to experts' comments and suggestions. for the second round, 62 experts were identified (appendix c) via internet searching and recommendations from the project partners and recruited participants. these experts were asked to answer the questionnaire in order to rank the diseases. thus, they had to choose the defined coefficient for each criterion (i.e. criterion scoring), then distribute the points for within each domain (i.e. the intradomain weighting), and lastly distribute the points within the domains (i.e. inter-domain weighting). they were invited to participate via a project summary e-mail and were sent the reviewed questionnaire via e-mail if they agreed to participate. experts were recruited until a minimum of 4 experts per disease was obtained with a maximum of 5 experts. in some cases, one expert could answer several questionnaires (one per disease) if the diseases were within is area of expertise. to obtain the overall score for the ranking, an aggregation method that combined the 2 types of weighting (i.e. the intra-and inter-domain) was used. first, the criterion score (coefficients attributed by experts) had to be standardized. indeed, some criteria were allocated coefficients from 0 to 4 and others from 1 to 4. this standardized score was then multiplied by the intradomain weight as given by the expert. these results were summed to obtain a domain score. in this formula, dsj = domain score, crit = criterion, scj = standardized score of the criterion and wdwj = intradomain weight for each criterion. each domain score was then multiplied by the inter-domain weight. these results were summed and an overall weighted score calculated, per expert and per disease. in this formula, ows = overall weighting score of each expert for a specific disease, cat = category, dsj = domain score and idwj = inter-domain weight. each disease had 4 or 5 ows (since there were 4 or 5 experts per disease), and thus, for each disease, the final score was the average of all disease experts' ows. the final score was then used to rank the diseases, based on drivers, from the highest score to the lowest. the highest score corresponded to the disease with the highest risk of (re-) emerging according to the drivers. in addition, the median and range among the scores of all the disease experts were also obtained. with the median, a ranking was done to observe whether there was any significant difference with the ranking obtained using the mean. the range was used to note which diseases had the highest and lowest level of variation/uncertainty among the final experts' average score. in order to determine which driver(s) was/were considered as the most influential for the (re-) emergence of diseases, the domains were ranked. domain ranking was performed using the inter-domain scores (weights). the sum of each domain weight (∑idwj) per disease and per domain given by each expert was ranked from the high to the low, that is 1 to 8. then, for each domain, the frequency of their rank was used to display in graph. a cluster analysis was implemented using regression tree analysis (salford predictive modeler ® , version 8.2, salford systems, san diego, california, usa). the normalized disease score is a continuous variable, and the aim was to obtain groups in qualitative categories of importance (e.g. very high, high, moderate and low) with minimal within-group variance. two sensitivity analyses were assessed, that is on expert elicitation and influence of a domain. this was achieved by repeating the disease ranking with a 'reduced' version of the model and comparing the new ranking to the complete model. the experts' sensitivity analysis consisted in dividing them into 4 groups. scores were then re-calculated by deleting a group of experts. each reduced ranking model was compared to the full complete model by using the spearman's rank test to establish whether the ranking was correlated between the complete and the reduced models. the sensitivity analysis on the domains was done by deleting one domain and re-calculating the mean scores to rank the diseases. this 'reduced' ranking was then compared with the complete model, and the spearman's rank test was applied. if the ranking position changed to less than three places, then the final score was considered as robust. if it changed to more than two places, then it was considered as a domain of drivers influencing greatly disease (re-) emergence. we compiled a list of 29 diseases ( all 14 experts contacted for the first phase (questionnaire assessment) answered positively (appendix b). there was a general agreement on which criteria and coefficients were clear or not. neither criterion nor coefficient were deleted but only amended according to experts' suggestions. for the second phase of expert elicitation, a total of 62 experts agreed to participate and answered the questionnaires (appendix c). the objective of minimum of 4 experts per disease was reached, and the maximum of 5 experts was reached for 8 diseases. the final disease ranking based on the average final scores is shown in figure 2 . the higher the mean score, the higher the ranking, which means the disease is most likely to (re-)emerge in belgium. the top 5 diseases in decreasing order were porcine epidemic diarrhoea (ped), fmd, low pathogenic avian influenza (lpai), african horse sickness (ahs) and hpai (table 3) . on the other end, the diseases with the lowest mean scores were haemorrhagic septicaemia, japanese encephalitis, wnf, peste des petits ruminants (ppr) and nipah disease. (1) the regression tree analysis determined 4 clusters ( figure 2 ). the clusters distinguished five, eleven, nine and four diseases, and were classified, respectively, as of 'low importance', 'moderate importance', 'high importance' and 'very high importance' (i.e. highly influenced by drivers). the diseases belonging to the node 'highest importance' were ped, fmd, lpai and ahs. the node of the lowest importance included haemorrhagic septicaemia, japanese encephalitis, ppr, nipah disease and wnf. the relative importance of the 8 domains varied depending on the disease. however, when considering all domains for all 29 diseases, 'economy and trade activities' obtained the highest number of points, being ranked first 15 times and zero times last ranked (8th). the opposite can be said about 'characteristics of farm/production system', as it was never ranked 1st nor 2nd ( figure 3 ). the sensitivity analysis done on the groups of experts showed that the ranking of diseases was not affected in the reduced models. indeed, the spearman's rank-order correlation indicated a strong positive association of ranks when using different groups of experts for different reduced models, showing that there was a consistency among the scoring of the experts. ta b l e 2 (continued) as for the domain sensitivity analysis, table 3 the mcda approach allowed the selection of 29 livestock diseases exotic to belgium and their prioritization based on drivers. whilst such an approach was used in previous disease prioritization exercises, this is one of the first to consider livestock epidemic diseases only and to use criteria related to drivers of (re-)emergence. only diseases exotic to belgium were prioritized. the diseases that fitted the eligibility criteria were all of viral origin, except haemorrhagic septicaemia (pasteurella multocida, serotypes 6:b, 6:e), ccpp and cbpp. few zoonoses were included in the list (n = 6) as the prioritization exercise focused on livestock epidemic diseases. therefore, several zoonoses included in other prioritization processes were excluded. regarding prioritization, ped ranked top of the list. although currently not reportable neither in the eu (except in the uk) nor to the oie, it ranked high in all models (high mean score), possibly due to its highly transmissible character and the difficulty to control it; furthermore, the disease mainly concerns intensive production. cases have already been reported in eu member states: for example in may 2014, an outbreak of diarrhoea occurred in fattening pigs on german farms. an outbreak of diarrhoea occurred on a belgian fattening pig farm at the end of january 2015; this was the first confirmed ped case in belgium in decades (theuns et al., 2015) . when the list of diseases was compiled, the outbreak had not occurred yet, but when the experts answered the questionnaire it had, and therefore, this was most likely the reason why it ranked at the top of the prioritized list. low pathogenic avian influenza ranked slightly higher than hpai in this multicriteria analysis on the risk of (re)-emergence (lpai ranked 3rd whilst hpai ranked 5th (1) (2) (2) (monne et al., 2014) . hence, these characteristics of the virus give in this prioritization lpai a higher score than hpai, but hpai is more likely to be detected and notified. african horse sickness surprisingly ranked 4th, although its last know incursion in europe (portugal and spain) was in 1987 and its eradication dates back to 1990. such high position in the ranking could be related to its vector-borne transmission, that is by culicoides biting midges. these vectors are often highly abundant, across most of africa, the middle east, europe and southern asia (carpenter, mellor, fall, garros, & venter, 2017) . additionally, the recent changes in the epidemiology of bluetongue and its latest epidemic in europe and the emergence of schmallenberg disease (afonso et al., 2014; anonimous, 2013; carpenter et al., 2009; highlight the uncertainty about the variables controlling the spread and persistence of culicoides-borne arboviruses. these different factors have raised concerns that ahs may also amount similar incursions, hence explaining such high mean final score in the prioritization process. in this prioritization, most of the diseases were in clusters 2 (high importance, n = 9) and 3 (moderate importance, n = 11 this score can only be compared with the prioritization work done by humblet and collaborators (humblet et al., 2012) as other prioritization works using the mcda method, such as those by cardoen et al. (2009), and havelaar et al. (2010) , only included zoonoses. indeed, in regression tree analysis of prioritized diseases of food-producing animals and zoonoses, asf also fell in the 3th group of importance out of the 4th group (humblet et al., 2012) , just like in this prioritization work. another study, which may be used for comparison as it used mcda approach and had swine diseases, done by brookes, hernandez-jover, cowled, holyoake, and ward (2014), asf ranked higher, but in this study only exotic diseases for the pig industry in australia were ranked using criteria related to impact and the experts were pig producers, which changes the importance in the scores, giving asf a higher ranking. the livestock diseases at the bottom of the list were nipah disease, ppr, wnf, japanese encephalitis and haemorrhagic septicaemia. in other prioritization exercises, nipah, japanese encephalitis and wnf were ranked in a higher category (cox et al., 2013; havelaar et al., 2010; humblet et al., 2012) . the prioritizadrivers are a complex set of factors, and their convergence can cause the (re-)emergence of a disease. several drivers have a stronger impact on diseases compared to others, as shown in the results section. porcine epidemic diarrhoea ranked at the top in all models, except in the reduced models of production system characteristics. porcine epidemic diarrhoea affects mainly intensive production systems; thus, the driver category 'production system characteristics' logically influences a lot. when using the reduced model, the mean score decreases and the disease moved from the 1st place to the 8th place. in comparison, fmd ranked high in the prioritization process (2nd), but lowered to the 12th place in the reduced model, which excluded disease pathogen characteristics. for fmd, the strongest driver was the 'pathogens characteristics'. the virus is highly contagious, spreads via airborne and direct contact and affects different livestock species, giving this driver category a strong weight. all experts considered that 'economy and trade activities' was the most important driver (high weight). it was ranked first more often than others. in the reduced model (without the 'economy and trade activities' domain), all diseases with the exception of 7 moved up or down in the ranking by more than 3 places. this is of no surprise, as economic and trade activity has priority in the age of globalization; increased movement of live animals and animal products crossing oceans and international boundaries increase the risk of spread for animal and zoonotic diseases (domenech, lubroth, eddi, martin, & roger, 2006 furthermore, the sensitivity analysis of experts also showed a high correlation among the ranking of models, which confirms that experts were in agreement in regards to the scores. overall, the importance of validating each generated model is the authors declare no conflict of interest. ethical statement is not applicable to this study as the data were gathered through questionnaire survey without any animal experimentation. number of criteria = 7, hence 70 points to be distributed within this domain for the intra-domain weighing. mono species farms-one single farmed animal (e.g. only bovines) or multi species farms (farms with more than one species, for example goats and bovines in the same farm/land/premises). score 0 score 1 negligible: the type of farm does not influence in any form (re)emergence of the disease among the livestock population. score 2 low: mono or multi species farm has a low effect on the risk of disease to emerge or re-emerge. score 3 moderate: the type or types of farmed animals has a moderate effect on the emergence of the disease in belgium. high: the type of farmed animals has a high influence for the disease to emerge and spread in belgium. farm demography/management: such as type of dairy or beef (cattle) production. for pigs-reproduction, fattening, finishing farm or both. chickens-only laying eggs chickens or solely finishing broilers score 0 score 1 negligible: population demography does not influence in any form the (re)emergence of the disease among the livestock population. score 2 low: the demographic population of the farm is a low influencing factor for disease (re)emergence. for example, disease only clinically affects only one age strata (i.e.) newborns, therefore adults are immune to it. moderate: the demographic of the population has a moderate effect on the (re)emergence of the disease, as it can (re)emerge in more than one type of demography but other conditioning factors have to occur in conjunction. high: the type of demographic of the farm has a high effect on the (re)emergence of the disease as it can (re)emerge in different types of farmed animals and all types of age groups d3 animal density of farms. extensive (small holders with a few animals) v/s intensive farming score 0 score 1 negligible: animal farm density is not a risk factor for the disease to emerge in belgium score 2 low: farm density (extensive or intensive) of animals has a low effect on the pathogen's/disease (re)emergence score 3 moderate: farm density of animals in the farm (extensive v/s intensive) has a moderate effect on the emergence of pathogen/ disease score 4 high: farm density of animals has a high effect on the (re)emergence of pathogen/disease. feeding practices of farms potential roles of zoo's in the (re)emergence of the pathogen score 0 score 1 negligible: the disease can be present in zoo animals but it is not known to have been transmitted from zoo animals to livestock. score 2 low: the disease can enter a zoo (e.g. with introduction of an infected exotic animal) but only accidental transmissions of the disease from zoo animals to livestock have been reported. hence, zoos have a low effect on the (re)emergence of the disease in belgium's livestock score 3 moderate: the disease can enter a zoo and be present in zoo animals but it needs a vector (biological/mechanical) for its transmission into livestock. therefore, zoos have a moderate effect on the (re)emergence of the disease in belgium. high: disease can be introduced to a zoo via an infected imported animal, zoo animals can carry the disease that can easily jump to livestock animals the rural(farm)-wildlife interface score 0 score 1 negligible: the disease has never (re)emerged from the narrowing of the farm-wild interface score 2 low: the disease has a low probability to (re)emerge via the livestock farm-forest interface. the disease has been known to (re)emerge from the wild bush but very rarely score 3 moderate: the disease has a moderate probability of (re)emergence via the farm/wildlife interface. barriers ( natural or artificial) are needed to keep the disease/pathogen (re)emerging in livestock score 4 high: there is a high probability for the disease to (re)emerge via the farm/forest interface. barriers (natural or artificial) separating farms from natural forests are ineffective score 2 low: there is a low probability of the disease (re)emerging and spreading through increased populations of endemic/migrating wild birds. disease has spread from the endemic/migrating wild birds but only accidentally or under exceptional circumstances score 3 moderate: there is a moderate probability of disease being introduced and spread through increased populations of endemic/migrating wild birds. they are hosts and in close contact with domestic livestock (i.e. poultry farms) may spread the disease score 4 high: there is a high probability for a disease to (re)emerge through increased populations of wild/migrating birds. these are hosts or reservoirs of the disease hunting activities: hunted animals can be brought back to where livestock is present score 0 score 1 negligible: the risk of the disease/pathogen of (re)emerging in livestock due to hunting activities is practically null score 2 low: disease is present in hunted wildlife and birds and only accidental cases have been reported in livestock that have (re)emerged because of hunting. the risk of the disease/pathogen of (re)emerging in livestock due to hunting activities is practically null score 3 moderate: disease is present in hunted wildlife and birds but a certain control is established by the hunter score 4 high: disease is present in hunted wildlife and birds and hunting is one of the main modes of transmission of the disease to livestock f6 transboundary movements of terrestrial wildlife from other countries score 0 null: disease is not carried by terrestrial wildlife score 1 negligible: (re)emergence of the disease by terrestrial movements of wildlife has only been suspected but never confirmed. low: there is a low probability for the disease to (re)emerge and spread through transboundary movements of terrestrial wildlife score 3 moderate: there is a moderate probability for the disease to (re)emerge and spread through transboundary movements of terrestrial wildlife score 4 high: there is a high probability for the disease to (re)emerge and spread through transboundary movements of terrestrial wildlife. these are host and may spread/carry the disease along. number of criteria = 6, hence 60 points to be distributed within this domain for the intra-domain weighing. (continued) g1 in-and out-people movements linked to tourism score 0 score 1 negligible: the movement of tourism is a negligible driver on the emergence or re-emergence of the disease score 2 low: tourism increase has a low driver of the (re)emergence of the disease. score 3 moderate: tourism increase has a moderate driver for the (re)emergence of the disease. biosecurity measures are enough to stop the entering of the pathogen. high: tourist movement is a high driver on the (re)emergence of a disease. tourists are highly likely to bring the disease into belgium in their belongings and biosecurity measures are insufficient to stop the pathogen g2 human immigration score 0 score 1 negligible: the immigration movements are a negligible driver of the disease (re)emergence in belgium score 2 low: the immigration movements are a low driver of the disease (re)emergence in belgium score 3 moderate: the disease is currently present in countries where more immigrants come from and pathogen highly likely to enter through, clothes, shoes and or possession, but the current biosecurity measures in place are able to prevent the emergence of the disease in belgium score 4 high: the immigration movement has a high effect as a driver on the emergence or re-emergence of disease in belgium. disease is highly likely to emerge using this route as biosecurity measures are not enough to avoid emergence of the disease g3 transport movements: more specifically commercial flights, commercial transport by ships, cars or military (excluding transport vehicles of live animals). score 0 score 1 negligible: the role of commercial movements as a driver on the (re)emergence of the disease in belgium is negligible. score 2 low: the role of commercial movements as a driver on the (re)emergence of the disease in belgium is low. it is easily preventable by implementing biosecurity measures score 3 moderate: the role of commercial movements as a driver on the (re)emergence of a disease in belgium is moderate. disease can be prevented if biosecurity measures are tightened. high: the role of commercial movements as a driver on the (re)emergence of a disease in belgium is high. disease is hard to control via the current biosecurity measures. transport vehicles of live animals score 0 score 1 negligible: the role of transport vehicles of live animals as a driver for the (re)emergence of the disease in belgium is negligible score 2 low: the role of transport vehicles of live animals as a driver for the (re)emergence of the disease in belgium is low. moderate: the role of transport vehicles of live animals as a driver for (re)emergence of the disease in belgium is moderate. high: the role of transport vehicles of live animals as a driver for (re)emergence of the disease in belgium is high g5 bioterrorism potential score 0 score 1 negligible: the role of bioterrorism as a driver for a disease to (re)emerge is negligible: agent is available but difficult to handle or has a low potential of spread or generates few economic consequences score 2 low: the role of bioterrorism as a driver for a disease to (re)emerge is low: agent is available and easy to handle by professionals and labs but has a low spread score 3 moderate: the role of bioterrorism as a driver for a disease to (re)emerge is moderate: agent available and easy to handle by professionals and labs and rapidly spreads score 4 high: the role of bioterrorism as a driver for a disease to (re)emerge is high: agent is available and easy to handle by individuals and rapidly spreads g6 inadvertent release of an exotic infectious agent from a containment facility, for example laboratory score 0 score 1 negligible: the pathogen is not currently present in any laboratory score 2 low: the pathogen is present in a containment facility but its release is very unlikely as it is very easily contained score score 0 score 1 negligible: modification of the disease status due to a reduced national budget has a negligible effect on the (re) emergence of the disease in belgium score 2 low: modification of the disease status due to a reduced national budget has a low effect on the (re) emergence of the disease in belgium score 3 moderate: modification of the disease status due to a reduced national budget has a moderate effect on the (re) emergence of the disease in belgium score 4 high: modification of the disease status due to a reduced national budget has a high effect on the (re) emergence of the disease in belgium decrease of resources allocated to the implementation of biosecurity measures at border controls (e.g. harbours or airports). score 0 score 1 negligible: decreasing the resources allocated to the implementation of biosecurity measures has a negligible effect on the (re)emergence of the disease in belgium. disease has never been detected in the past in a harbour or airport score 2 low: decreasing the resources allocated to the implementation of biosecurity measures has a low effect on the (re)emergence of the disease in belgium. the disease has been suspected to have entered other countries because of deficient biosecurity at border controls. score 3 medium: decreasing the resources allocated to the implementation of biosecurity measures has a moderate effect on the (re) emergence of the disease in belgium. the disease has been introduced in other countries because of deficient biosecurity at border controls score 4 high: decreasing the resources allocated to the implementation of biosecurity measures highly increases the risk of (re)emergence of the disease in belgium. in the past, the disease has been introduced in other countries and in belgium because of deficient biosecurity at border controls most likely influence of (il)legal movements of live animals (livestock, pets, horses, etc.) from neighbouring/european union member states (ms) for the disease to (re)emerge in belgium. (continued) h5 influence of increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states for the disease/pathogen to (re)emerge in belgium score 0 score 1 negligible: increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states have a negligible influence on the pathogen/disease (re)emergence in belgium. score 2 low: increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states have a low influence on the pathogen/disease (re)emergence in belgium. moderate: increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states have a moderate influence on the pathogen/disease (re)emergence in belgium. high: increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states have a high influence on the pathogen/disease (re)emergence in belgium. most likely influence of increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states for the disease/pathogen to (re)emerge in belgium. score 0 score 1 negligible: increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states have a negligible influence on the pathogen/disease (re)emergence in belgium. score 2 low: increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states have a low influence on the pathogen/disease (re)emergence in belgium. moderate: increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states have a moderate influence on the pathogen/disease (re)emergence in belgium. high: increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states have a high influence on the pathogen/disease (re)emergence in belgium. most likely influence of (il)legal movements of live animals (livestock, pets, horses, etc.) from third countries for the disease to (re)emerge in belgium. most likely influence of increased imports of animal subproducts such as skin, meat and edible products from third countries, for the disease to (re)emerge in belgium. score 0 score 1 negligible: increased imports of animal subproducts such as skin, meat and edible products from third countries have a negligible influence on the pathogen/disease (re)emergence in belgium. score 2 low: increased imports of animal subproducts such as skin, meat and edible products from third countries have a low influence on the pathogen/disease (re)emergence in belgium. moderate: increased imports of animal subproducts such as skin, meat and edible products from third countries have a moderate influence on the pathogen/disease (re)emergence in belgium. high: increased imports of animal subproducts such as skin, meat and edible products from third countries have a high influence on the pathogen/disease (re)emergence in belgium. a p p e n d i x a (continued) h9 most likely influence of increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries, for the disease to (re)emerge in belgium. score 0 score 1 negligible: increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries have a negligible influence on the pathogen/disease (re)emergence in belgium. score 2 low: increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries have a low influence on the pathogen/disease (re)emergence in belgium. moderate: increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries have a moderate influence on the pathogen/disease (re)emergence in belgium. high: increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries have a high influence on the pathogen/disease (re)emergence in belgium. number of criteria = 9, hence 90 points to be distributed within this domain for the intra-domain weighing. a p p e n d i x b list of experts enrolled (n = 14) in the phase i (questionnaire assessment) with their gender, affiliation, country and field of expertise the schmallenberg virus epidemic in europe-2011-2013. preventive veterinary medicine molecular characterization of peste des petits ruminants viruses in the marmara region of turkey climate change and infectious diseases: from evidence to a predictive framework schmallenberg virus continues to spread across europe. the veterinary record antibodies to schmallenberg virus in domestic livestock in turkey surveillance, detection and response: managing emerging diseases at national and international levels building a picture: prioritisation of exotic diseases for the pig industry in australia using multi-criteria decision analysis evidence-based semiquantitative methodology for prioritization of foodborne zoonoses african horse sickness virus: history, transmission, and current status culicoides and the emergence of bluetongue virus in northern europe avian influenza. iowa state university evidence of schmallenberg virus circulation in ruminants in greece identification of wild boar-habitat epidemiologic cycle in african swine fever epizootic multi-criteria decision analysis tools for prioritising emerging or re-emerging infectious diseases associated with climate change in canada emerging infectious diseases of wildlife-threats to biodiversity and human health regional and international approaches on prevention and control of animal transboundary and emerging diseases the highly pathogenic avian influenza a (h7n7) virus epidemic in the netherlands in 2003-lessons learned from the first five outbreaks amending annexes i and ii to council directive 82/894/eec on the notification of animal diseases within the community scientific opinion on african swine fever scientific opinion on porcine epidemic diarrhoea and emerging porcine deltacoronavirus situation zoosanitaire et maladies à déclaration obligatoire en belgique notification obligatoire federal ministry of food and agriculture of germany animal production health phylogeographic analysis of african swine fever virus prioritizing emerging zoonoses in the netherlands multidisciplinary and evidence-based method for prioritizing diseases of food-producing animals and zoonoses emerging and re-emerging zoonotic diseases: challenges and opportunities. in 72nd general session world organisation for animal health outbreak of foot-and-mouth disease virus serotype o in the uk caused by a pandemic strain le service public de l'accès au droit le service public de l'accès au droit schmallenberg virus infection in small ruminants -first review of the situation and prospects in northern europe summer 2018: african swine fever virus hits north-western europe nederlandse voedsel-en warenautoriteit emergence of a highly pathogenic avian influenza virus from a low-pathogenic progenitor the 2010 foot-and-mouth disease epidemic in japan unexpected discovery of african swine fever in belgium west nile virus in europe: emergence, epidemiology, diagnosis, treatment, and prevention list of notifiable diseases great britain complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium bluetongue in europe: past, present and future animal-health-in-the-world/ oie-listed-disea ses-2018/ world organisation for animal health detection and partial sequencing of schmallenberg virus in cattle and sheep in turkey. vector-borne and zoonotic diseases contagious caprine pleuropneumonia 3,617.45 (1,099.65) 11 3,247.25 21 2 epizootic haemorrhagic disease 3 african swine fever 3 key: cord-354729-dpaz01np authors: huan, changchao; pan, haochun; fu, siyao; xu, weiyin; gao, qingqing; wang, xiaobo; gao, song; chen, changhai; liu, xiufan title: characterization and evolution of the coronavirus porcine epidemic diarrhoea virus hljby isolated in china date: 2019-08-22 journal: transbound emerg dis doi: 10.1111/tbed.13321 sha: doc_id: 354729 cord_uid: dpaz01np a strain of porcine epidemic diarrhoea virus (pedv), namely hljby, was isolated in heilongjiang province, china. to provide insight into the understanding of the phylogenetic and the current epidemiological status of pedv, pedv hljby was compared with cv777 and other pedv strains deposited in the genbank. the homology between the entire genomic nucleotide sequences of pedv hljby and cv777 was 97.7%. the homology of m gene was the highest (99.0%). however, the homology of orf3 gene was 97.7%, and protein of orf3 was 90.1%. in addition, hljby showed the highest nucleotide identity (99.9%) with pedv‐sx/china/2017 strain and lowest similarity (91.2%) to pedv/belgorod/dom/2008 strain. we analysed the changes in s gene and its protein of pedv hljby with 65 historic pedv strains. the highest nucleotide identity was 99.9% compared with pedv‐sx/china/2017 strain, and the lowest nucleotide identity was 60.0% compared with pedv/belgorod/dom/2008 strain. the length of deduced amino acid sequences of s proteins varied from 1,372 to 1,390 amino acids (aa). compared with most aa sequences of s proteins, hljby exhibited 5 aa deletions (position 55, 59–61, 144). analysis and comparison of open reading frame 3 (orf3) proteins between hljby strain and other pedv strains were also focused in this study. we revealed that the length of deduced amino acid sequences of orf3 proteins was 80–224 aa among tested strains and the identity of hljby orf3 amino acids with other pedv strains was 71.4%–98.9%. orf3 protein of both hljby strain and pedv‐sx/china/2017 strain consists of 91 aa, with 133 aa deletions at their c' end in relation to the other tested pedv strains. the phylogenetic tree based on different proteins or genes resulted in different phylogenetic groups. for pathogenicity evaluation of pedv hljby strain, colostrum deprivation piglets were challenged with pedv hljby, and pedv reference strain cv777 as a control, the results showed that animals challenged with either of these pedv strains developed diarrhoea, and histopathological examination of small intestines of challenged animals showed acute viral enteritis with villous atrophy in either pedv hljby‐p10 or pedv cv777‐p8 inoculated piglets. cluding a positive-sense single-strand rna genome, which can cause a devastating enteric disease characterized with dehydration and watery diarrhoea (pensaert & de bouck, 1978) . pedv is the causative agent of porcine epidemic diarrhoea (ped), which has high mortality in suckling piglets (debouck & pensaert, 1980; pijpers, nieuwstadt, terpstra, & verheijden, 1993) . this disease was initially documented in the united kingdom in 1971. since 1990s, ped was not serious. however, outbreak of ped suddenly occurred in the united states, canada and mexico causing huge economic losses (mole, 2013; stevenson et al., 2013; vlasova et al., 2014) . in addition, ped caused tremendous economic losses to the swine industry in europe and asia, including china, korea and japan (kocherhans, bridgen, ackermann, & tobler, 2001; sun et al., 2012) . pedv is a nonsegmented and infectious rna virus. the genome of pedv is 27-33 kb in length containing a 5'cap and a 3'polyadenylated tail (pensaert & de bouck, 1978) . in addition, the genome includes seven open reading frames (orfs) encoding three nonstructural proteins (replicase 1a,1b and orf3) and four structural proteins (the spike (s), envelope (e), membrane (m) and nucleoprotein (n)). these proteins arrange in the order of 5'-replicase(1a/1b)-s-orf3-e-m-n-3' (kocherhans et al., 2001) . to reveal the characteristic and the diversity between pedv strains currently circulating in china and other pedv strains outside, the complete genomic sequence of pedv hljby strain was determined and analysed, and the pathogenicity of pedv hljby strain in newborn piglets was also evaluated. pedv hljby strain was isolated from the intestinal contents of a piglet with diarrhoea from heilongjiang province, china at 2011. vero cells were grown in dulbecco's modified eagle's medium (dmem, hyclone) supplemented with 8% foetal bovine serum (fbs, gibco) and were maintained in maintenance medium (dmem supplemented with 2% fbs) at 37 °c in a 5% co 2 incubator. strain hljby was passaged ten times in vero cells. pedv n-specific antibody used in immunofluorescence assay (ifa) was gifted by professor xiang mao. pedv reference strain cv777 was purchased from china institute of veterinary drug control. when pedv-infected vero cells showed 70%-80% cytopathic effect (cpe), cell culture flasks were frozen and thawed three times, and cell debris was pelleted by centrifugation for 30 min at 12,000 rpm. culture supernatants were collected and used for preparation of viral rna. total rna was extracted using trizol reagent (vazyme biotech) according to the manufacturer's instructions. total rna was used for synthesis of cdna with hiscript reverse transcriptase (hiscript ii 1st strand cdna synthesis kit; vazyme biotech) according to the manufacturer's instructions. specific primers for pedv were designed based on pedv-cv777 genome (table 1) . pcr was performed to amplify for the six overlapping dna fragments using lamp dna polymerase (vazyme biotech). the expected bands in agarose electrophoresis of pcr products were excised, and a genclean column gel extraction kit (generay biotech) was used to purify the synthesized dna, and the dna products were cloned into pjet1.2 vector (thermo). the positive clone was sequenced by biotechnology co, ltd. the validated genome sequence of pedv hljby strain was submitted to genbank and acquired accession number: kp403802.1. sequence data was employed to assemble and analyse by dnastar software package (dnastar inc. genes of pedv strains were used for sequence alignments and phylogenetic analyses. nucleotide sequences of full-genomes, orf3 genes and s genes of pedv strains were aligned using the clustalx 2.0 program (thompson, gibson, plewniak, jeanmougin, & higgins, 1997 ). vero cells grown on coverslips were infected with 0. fifteen newborn piglets without colostrum were purchased from one pig farm and were free of pedv, transmissible gastroenteritis virus, porcine deltacoronavirus, and porcine rotavirus. the piglets were divided randomly into three groups: the sham-inoculated control group (n = 5), cv777-p8-inoculated group (n = 5), and hljby-p10-inoculated group (n = 5). the piglets were fed commercial milk replacer (8 times daily). the piglets in the challenge groups received an oral 1 ml dose of 10 7.0 tcid 50 /ml of pedv cv777 or hljby. the sham-inoculated pigs were given dmem medium (1 ml) orally. all animals were monitored for mortality and signs of vomiting and/or diarrhoea (observed and recorded for every 3 hr during whole experiment). all piglets were euthanized at 7 d post-challenge and checked for macroscopic and microscopic lesions. to reveal the characteristics of pedv hljby strain and determine more precisely the relationship among the pedv strains currently circulating in china and those from other nations, the full-length genome sequence of strain hljby was deduced by combining the sequences of several overlapping cdna fragments. the genome sequence of strain hljby was 27,953 nucleotides (nt) in length, excluding the 3' poly(a) tail. the genomic organization was typical of previously sequenced pedv strains and was summarized as 5'utr-orf1a/1b-s-orf3-e-m-n-3'utr ( figure 1 ). of note, compared with classical pedv cv777, the genome of hljby contains four deleted nucleotides or regions including 72 nt, 89-93 nt, 3403-3426 nt, 21092-21094 nt, respectively. the four deleted nucleotides or regions were located at 5'utr, orf1a/1b, s, orf3, respectively. to investigate the molecular characteristics of pedv hljby strain, utr (5' and 3') and the nucleotide and predicted amino acid sequences of the nonstructural and structural proteins (replicase orf1a/1b, s, orf3, e, m, n) of pedv hljby strain were compared with cv777. as shown in figure 2a and table 3 , the nucleotide of 5'utr of pedv hljby had 5 nt deletions and 1 nt insertion and the identity is 96.0% compared with cv777. 24 nt deletions were found in orf1a/1b of pedv hljby (figure 2b ), and the nucleotide sequence identity was 98.0%, and the amino acid sequence identity was 98.0% (table 3) . 3 nt deletions showed in s of pedv hljby ( figure 2c ), and the nucleotide sequence identity was 97.0%, and the amino acid sequence identity was 96.0% (table 3) . 399 nt deletions exhibited in orf3 of pedv hljby (figure 2d ), and nucleotide sequence identity was 91.7%, and the amino acid sequence identity was 90.1% (table 3 ). in protein e, the nucleotide sequence identity was 97.0%, and the amino acid sequence identity was 97.0% (table 3) , resulting in amino acid changes in e (11val to ala and 76 val to ile) ( figure 2e ). in protein m, the nucleotide sequence identity was 99.0% and the amino acid sequence identity was 98.0% (table 3) , resulting in 4 amino acid changes in m ( figure 2f ). in protein n, the nucleotide sequence identity was 98.0% and the amino acid sequence identity was 98.0% (table 3) , resulting in 9 amino acid changes in n ( figure 2g ). the nucleotide sequence identity of 3'utr was 97.0% (table 3) . the complete genome sequences of pedv strains from different locations and years were compared, and the results revealed that hljby had a nucleotide identity of 99.9%-91.2% with other entire pedv genomes available in genbank ( the orf3 protein (an accessory protein) was located between s and to investigate the evolution of pedv, phylogenetic analysis based on the entire genomic nucleotide sequences of pedv hljby strain the group ⅲ was further divided into subgroup ⅲa and ⅲb. the the group ⅱ included the other pedv strains (figure 7) . to in recent years, pedv has re-emerged as one of the deadliest and most contagious pathogens in swine, causing large economic (li et al., 2012) . therefore, the relationship of (wang et al., 2012) . orf3 may contribute to the virulence of pedv. the orf3 was a key gene for pedv culture in vitro. orf3 gene was usually used to differentiate between field and vaccine-derived isolates and altered the virulence of pedv (park et al., 2008) . in addition, the pedv s protein is an important viral gene for studying genetic relatedness among isolates and epidemiology of pedv (chen et al., 2014; gerber et al., 2014; lee, 2015; lee et al., 2010; oh et al., 2014) . nucleotide sequencing of s gene analysis revealed that 3 nt deletions were found in s gene of pedv hljby compared with cv777 ( figure 2c ) and the nucleotide sequence identity is 97.0%. the amino acid sequence identity of s protein was 96.0% between pedv hljby and cv777. 3 nt deletions of s gene were located in the amino terminus of s gene, which was suitable to analyse the significant differences in epidemiology of pedv (li et al., 2012; temeeyasen et al., 2014; vlasova et al., 2014) . s gene functions as a receptor-binding domain (belouzard, millet, licitra, & whittaker, 2012) . and epidemiology of pedv. our study will provide more information about diversity, evolution, and in particular, the epidemic characin the phylogenetic tree of the whole genome, s aa and orf3 aa, the phylogenetic tree based on both the whole genome and s aa was similar, but the phylogenetic tree of orf3 aa was different from the phylogenetic trees for the s protein and genomic nucleotide sequences. all pedv strains were divided into three groups based on the phylogenetic tree of the whole genome and s protein. in the phylogenetic tree of the whole genome and s protein, pedv/belgorod/ dom/2008 strain located at group ⅰ, and pedv hljby belonged to the group ⅱ. in the phylogenetic tree of orf3 protein, the group ⅰ only harboured pedv/belgorod/dom/2008 and hljby. the phylogenetic tree based on different proteins implied the different evolution events. this will provide further information for virus evolution. the phylogenetic trees suggested that pedv hljby might originate from a genetic recombination event between pandemic strains and classical strains. we need to explore the mechanism and/or cause of the different phylogenetic trees based on different proteins or genes. this will lay a foundation for the recombination and evolution of pedv. biological characteristics of pedv hljby including the cytopathic effect, and pathogenicity, indicated that pedv hljby induced an apparent and classical cytopathic effect (cpe) in vero cells, and caused acute viral enteritis with villous atrophy in small intestine of challenged piglets. but all hljby-p10 or cv777-p8 inoculated piglets had no deaths during the challenge experiment. these results suggested that pedv hljby might have similar pathogenicity in experimentally infected piglets compared with that of pedv reference strain cv777. in summary, we found that the evolutionary trees are different based on different proteins or genes of pedv strains. this phenomenon needs further study. pathogenicity evaluation of pedv hljby indicated that pedv hljby potentially possessed equal pathogenicity compared with that of pedv reference strain cv777. this study might provide a reference for the evolutionary and variation of pedv. we thank james allen, phd, for editing the english text of a draft of this manuscript. this work was supported by the natural the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. all experimental protocols were approved by the animal care and mechanisms of coronavirus cell entry mediated by the viral spike protein prevalence of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus infection in korean pigs. the veterinary record isolation and characterization of 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virus hljby isolated in china key: cord-351564-nikcd44o authors: zhang, xiaozhan; deng, tongwei; lu, jianzhou; zhao, pandeng; chen, lulu; qian, mengwei; guo, yiwen; qiao, hongxing; xu, yaohui; wang, yan; li, xinzheng; zhang, guizhi; wang, zeng; bian, chuanzhou title: molecular characterization of variant infectious bronchitis virus in china, 2019: implications for control programmes date: 2020-01-24 journal: transbound emerg dis doi: 10.1111/tbed.13477 sha: doc_id: 351564 cord_uid: nikcd44o infectious bronchitis virus (ibv), an ongoing emergence enveloped virus with a single‐stranded positive‐sense rna genome, belongs to the gammacoronavirus genus in the coronaviridae family. ibv‐associated tracheitis, nephritis, salpingitis, proventriculitis and egg drop have caused devastating economic losses to poultry industry worldwide. since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with ibv in china. here, we described two ib outbreaks with severe respiratory system or kidney injury in ibv‐vaccinated commercial poultry farms in central china. other possible causative viral pathogens, including avian influenza virus (aiv), newcastle disease virus (ndv) and kedah fatal kidney syndrome virus (kfksv), were excluded by reverse transcription‐polymerase chain reaction (rt‐pcr), and three virulent ibv strains, hen‐1/china/2019, hen‐2/china/2019 and hen‐101/china/2019, were identified. although the gross pathologic appearance of these two ib outbreaks was different, the newly identified ibv strains were all closely related to the ck/china/i0529/17 strain and grouped into gi‐19 genotype clade based on the sequencing and phylogenetic analysis of the complete s1 genes. moreover, there are still some evolutionary distance between the newly identified ibv strains, hen‐101/china/2019 in particular, and other gi‐19 strains, suggesting that chinese ibv strains constantly emerge and evolve towards different directions. in conclusion, this study provided an insight of the recently emerging ibv outbreaks in ibv‐vaccinated commercial poultry farms and identified the genetic characteristics of three virulent gi‐19 ibv strains, which shows the need to carry out proper preventive measures and control strategies. avian infectious bronchitis (ib), a common avian disease with high morbidity and mortality, has drawn great attention for causing devastating economic losses to poultry industry worldwide (jackwood & wit, 2013) . infectious bronchitis virus (ibv), the causative agent, is an ongoing emergence enveloped, single-stranded, positive-sense rna virus, with genome length about 27.6 kb. it is the prototype of the gammacoronavirus genus in the coronaviridae family (bande et al., 2017; masters & perlman, 2013) . ibv mainly targets the respiratory tract of its natural host-chicken, causing severe respiratory system disease, and spreads via respiratory and feco-oral routes. additionally, ibv also infects the uro-genital tract, digestive system and reproductive system, resulting in nephritis, salpingitis, proventriculitis and egg drop (bande et al., 2017; jackwood & wit, 2013; sjaak de wit, cook, & van der heijden, 2011) . a great number of ibv serotypes, genotypes and pathotypes have been identified worldwide since its first description in 1931 in america (bande et al., 2017; lin & chen, 2017) . to prevent ibv infection, researchers have developed various commercial inactivated and live attenuated vaccines over the past decades, which successfully prevented ibv infections (jordan, 2017) . as a highly mutable coronavirus, however, the continuous emergence of novel ibv strains greatly emasculate vaccines efficacy, and the low cross-protection rates of ibv vaccines inevitably hamper the prevention and control of the disease (fan et al., 2018; jordan, 2017) . since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with ibv in china, especially in the intensive poultry raising regions, such as henan, hebei and shandong provinces. herein, we reported two ib outbreaks in ibv-vaccinated commercial broiler farms in central china, and the complete s1 genes of the newly identified ibv strains were sequenced, and its genotype, phylogeny and variations were analysed further. this study systematically described the genotype and evolutionary characteristics of the emerging ibv strains and highlighted the importance of continuous extensive surveillance to help choose suitable vaccines and develop control programmes reasonably. case 1: in april 2019, an acute outbreak of fatal respiratory disease occurred in a commercial broiler farm in henan province, central china. according to the breeder's description, approximately onetenth of 20,000 25-day-old broilers, vaccinated with commercial live attenuated ibv vaccine h120 at 7-day-old, showed severe respiratory symptoms, such as gasping, nasal discharge, tracheal rales, sneezing, and some affected flocks also presented greenishyellow diarrhoea, watery eyes and lethargy. the outbreak started from 18 april 2019, and antibiotic-antimycotic combination therapy did not work. a total of ten dead broilers (5/10) and illness broilers (5/10) were selected randomly to send to the laboratory for diagnosis. case 2: in september 2019, an outbreak of fatal nephritis disease occurred in another commercial layer farm in henan province. according to the breeder's description, the farm has approximate 9,000 layers with 21-day-old, which were vaccinated with commercial live attenuated ibv vaccine h120 at 7-day-old. the great majority of the flocks started to show moderate respiratory symptoms from 10 september 2019, and mitigated in a week with the antibiotic and traditional veterinary drugs combination therapies. since 20 september, about 200 chickens per day died acutely without obvious clinical symptom. six moribund layers were selected randomly to send to the laboratory for diagnosis. the sick chicken examined in this study was approved by the poultry farm owners. diagnosis and experimental protocols in this study were approved by the henan university of animal husbandry and economy animal care committee. the clinical samples used in this study were collected in strict accordance with the guidelines for animal ethics committees. all the chickens were euthanized by cervical dislocation and then examined the anatomical changes, samples of lungs, tracheas and kidneys were collected from sick chicken and stored at −80°c until used for diagnosis and virus isolation. the samples were homogenized and centrifuged to collect supernatants. total viral rna was extracted by using trizol (invitrogen, cat no: 15596026) according to the manufacturer's instructions, and further reversely transcribed to cdna as described in our previous report . ibv, aiv, ndv and kfksv (palya et al., 2019) , which can cause severe respiratory symptoms, nephritis and acute death, were analysed by rt-pcr using specific primers as previously described (nguyen et al., 2013) . moreover, the positive samples were homogenated and then isolated in 9-day-old specific pathogen-free (spf) eggs via the allantoic cavity, and the allantoic fluid was harvested sterilely and stored at −80°c. to identify the genotype of ibv strains identified in this outbreak, the complete s1 genes were amplified by using primers as follows: ibv-s1 forward: 5′-gaacaaaagaccgacttagt-3′ and ibv-s1 reverse: 5′-tatgtactcatctgtracagt-3′, which were designed based on the conserved regions flanked the s1 gene of ibv strains downloaded from the vipr database (http://www.viprb rc.org/brc/home.spg?decor ator=vipr). the correct size amplicons, with length about 2000 bp, were then cloned into pcloneez vector (clone smarter) and sequenced. the complete s1 genes of the newly identified strains were subjected to online blast program (https ://blast.ncbi.nlm.nih.gov/blast.cgi), and sequences sharing more than 95% nucleotide identify were downloaded for further genetic analysis. additionally, the s1 genes of representative ibv strains within different genotypes and lineages jiang et al., 2017; ma et al., 2019; valastro et al., 2016) were also downloaded for sequence alignment with clustal omega (https ://www.ebi.ac.uk/tools/ msa/clust alo/), and the phylogenetic tree was conducted by mega 7.0 with the neighbour-joining method using 1,000 bootstrap replicates, which were then visualized by itol v4 software (letunic & bork, 2019) . recombination events among ibv strains were further investigated using the rdp4 software, a widely used tool for analysing individual recombination events and overall recombination patterns, by seven different algorithms recombination detection program (rdp), bootscan, maxchi, geneconv, chimaera, siscan and 3seq (martin, murrell, khoosal, & muhire, 2017 ). previous studies have revealed that most of the variable amino acids in s1 region were distributed in the three hypervariable regions (hvrs), including hvr i (38 aa-67 aa), hvr ii (91 aa-141 aa) and hvr iii (274 aa-387 aa), which were associated with neutralizing epitopes and receptor-binding domain and usually employed to classify ibv genotypes (moore, jackwood, & hilt, 1997; valastro et al., 2016) . to further investigate the hvrs and mutations in the newly identified ibv strain, the 3d structure of hen-2/china/2019 s1 protein was constructed by homology modelling method. from the pdb database, the glycoprotein s1 structure of vaccine m41 strain (accession number: 6cv0) was downloaded and employed as the template for modelling s1 monomer (shang et al., 2018) . all the hvrs and mutations of hen-2/china/2019 and hen-101/china/2019 were located on the structure and visualized by pymol software (http://www.pymol.org/). among the organs collected from the morbid chicken among two ib outbreaks, the predominant histologic lesions were in the trachea and kidney, respectively. in case 1, a total of ten dead broilers (5/10) and illness broilers (5/10) were selected randomly to examine the anatomical changes, and trachea, especially between bronchus and bronchioles, showed the gross pathologic appearance of typical tracheitis, with serous, catarrhal or caseous exudate in trachea (figure 1a-c) . moreover, some of them presented systemic colibacillosis or airsacculitis, and no obvious nephritis and proventriculitis were observed. in case 2, all the six moribund layers exhibited severe lesion in kidneys, which were characterized by pale, swollen and mottled (figure 1d,e) . some of them showed distended ureters filled with uric acid, but none of them presented tracheitis. to identify the causative agent of this outbreak, rt-pcr assays were used to detect the common potential viral pathogens, including ibv, ndv, aiv and kfksv. as shown in figure s1a to further characterize the biology and ecology of the newly identified ibv strains, we sequenced the complete s1 genes of phylogenetic relationship among ibv strains has been established on the analysis of complete s1 gene. as shown in figure 2 , the phylogenetic tree further revealed that the hen-1/china/2019, hen-2/china/2019 and hen-101/china/2019 strains were f i g u r e 2 phylogenetic analysis of the complete s1 genes of newly identified ibv in 2019. phylogenetic analysis based on the deducted amino acid sequences of complete s1 gene of hen-1/china/2019, hen-2/china/2019, hen-101/china/2019 and other ibv isolates available from genbank database. phylogenetic tree was constructed by mega 7.0 software using the neighbour-joining method with 1,000 bootstrap replicates. the seven different ibv genotype, with 35 distinct lineage ibv strains were illustrated by the colour scale with itol software. meanwhile, the available ibv vaccine strains in china were indicated by blue star, and the three ibv isolates in this study were indicated by red star clustered together with ck/china/i0529/17, qx and lx4 strains and grouped into the gi-19 genotype, which were distantly from the available commercial vaccines h120, 4/91 and ldt3-a. as a highly variable coronavirus, numerous ibv variants have been identified and nucleotide substitutions or recombination between field strains and vaccines occurred frequently. previous study revealed that the recombinant events have already occurred in european strains within the s1 gene (moreno et al., 2017) . we further investigated the recombinant events between gi-19 and other ibv genotypes using the rdp4 software. the results showed that several recombinant events occurred between gi-19 and other ibv genotypes with high score (p < .01, recombinant score >0.6), including strains of the newly identified genotype gi-28, gi-25 and unassigned strain ldt3 ( figure s2) . interestingly, the gi-19 genotype plays a two-faced role in these recombinant events, not only providing fragments for other strains, but also receiving fragments during infection. to further explore the characteristics of the newly identified ibv strains, amino acid polymorphism of the s1 glycoprotein was compared with other ibv strains of all 35 genotypes jiang et al., 2017; ma et al., 2019; valastro et al., 2016) . the result revealed that most of the amino acid mutations in s1 region of the newly identified ibv strains were located in the three hvr regions. (table 1) . compared with the available capsid structure of m41 strain in the pdb database (shang et al., 2018) , the p87l and q89k mutations are adjacent to hvr i and hvr ii region structurally (figure 3c ) and located in the major receptor-binding domain (19 aa-253 aa; promkuntod, van eijndhoven, de vrieze, grone, & verheije, 2014) ; the q97e, s117a, t120r, a130e and r131s are in hvr ii region; the t300i and h329y are in hvr iii region (figure 3d ), indicating that these mutations might change virus antigenicity. ib is an economically important avian disease that affects the poultry industries worldwide, particularly in the large poultry-producing countries, such as usa, china and brazil (bande et al., 2017; jackwood & wit, 2013) . the first case of ib in china dates back to early 1980s, and since then, the outbreaks were constantly emerging and numerous ibv strains were identified, of which strains in genotype gi-19 (refers to the qx-like or lx4) became predominant among chickens currently, although the qx strain was initially identified in 1996. additionally, other genotypes of ibv, such as gi-1 (mass), gi-7 (tw-i or tw-like), gi-13 (4/91-like), gi-16 (q1-like) and gi-22 (yn-like and saibk-like), were also frequently detected in china (han et al., 2018; li et al., 2012; lin & chen, 2017; mo et al., 2013) . importantly, several novel ibv genotypes, including gi-28, gi-29 and gvii-1, were identified recently jiang et al., 2017; ma et al., 2019) . all these indicated that the ibv strains circulating in china are genetically diverse, providing a potential platform for recombination and greatly challenging the current biological control measures. (bande et al., 2017; han et al., 2011; li et al., 2012) . gi-1 genotype vaccine h120 has been the most widely used vaccine in china until now, which shared a very low homology of identity with the emerging gi-19 strain hen-1/china/2019 (32.2%) and hen-2/china/2019 (32.2%) in glycoprotein s1 gene region, and even the other gi-19 strains identified recently. these results indicated the h120 vaccine could not provide effective protection against gi-19 genotype strains infection, which might explain the clinical phenomenon that a number of ibv outbreaks emerged in vaccinated flocks (lin & chen, 2017 characterization and localization of specific nonsynonymous mutations in capsid protein of the newly identified ibv strains in comparison with other ibv isolates. the multi-alignment of s1 glycoprotein of all ibv genotypes was conducted by clustal-omega, and schematic diagram based on the identified protein functional domains of mature s1 protein (without 1aa-19aa) was illustrated (a); 3d 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china. infection vaccination against infectious bronchitis virus: a continuous challenge interactive tree of life (itol) v4: recent updates and new developments serotype and genotype diversity of infectious bronchitis viruses isolated during 1985-2008 in guangxi infectious bronchitis virus variants: molecular analysis and pathogenicity investigation novel genotype of infectious bronchitis virus isolated in china detecting and analyzing genetic recombination using rdp4 corornaviridae molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern china identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus a novel variant of the infectious bronchitis virus resulting from recombination events in italy and spain multiplex nested rt-pcr for detecting avian influenza virus, infectious bronchitis virus and newcastle disease virus novel orthobunyavirus causing severe kidney disease in broiler chickens mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins infectious bronchitis virus variants: a review of the history, current situation and control measures s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification. infection identification and genomic characterization of the emerging senecavirus a in southeast china the authors declared no potential conflict of interests with respect to the research, authorship and publication of this article. the data that support the findings of this study are available in genbank at ncbi, reference number [mn055627, mn055628 and mn635798] . these data were derived from the following resources available in the public domain ncbi. https://orcid.org/0000-0002-0280-4964zeng wang https://orcid.org/0000-0002-3274-4296 key: cord-302819-oj33i2ma authors: pasick, j; berhane, y; ojkic, d; maxie, g; embury-hyatt, c; swekla, k; handel, k; fairles, j; alexandersen, s title: investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada date: 2014-08-07 journal: transbound emerg dis doi: 10.1111/tbed.12269 sha: doc_id: 302819 cord_uid: oj33i2ma summary: in january 2014, approximately 9 months following the initial detection of porcine epidemic diarrhea (ped) in the usa, the first case of ped was confirmed in a swine herd in south-western ontario. a follow-up epidemiological investigation carried out on the initial and 10 subsequent ontario ped cases pointed to feed as a common risk factor. as a result, several lots of feed and spray-dried porcine plasma (sdpp) used as a feed supplement were tested for the presence of pedv genome by real-time rt-pcr assay. several of these tested positive, supporting the notion that contaminated feed may have been responsible for the introduction of pedv into canada. these findings led us to conduct a bioassay experiment in which three pedv-positive sdpp samples (from a single lot) and two pedv-positive feed samples supplemented with this sdpp were used to orally inoculate 3-week-old piglets. although the feed-inoculated piglets did not show any significant excretion of pedv, the sdpp-inoculated piglets shed pedv at a relatively high level for ≥9 days. despite the fact that the tested pedv genome positive feed did not result in obvious piglet infection in our bioassay experiment, contaminated feed cannot be ruled out as a likely source of this introduction in the field where many other variables may play a contributing role. porcine epidemic diarrhea (ped) was first recognized in england in 1971 as an enteric disease affecting feeder and fattening pigs with less of an effect on suckling pigs (wood, 1977) . initially, it was this characteristic that distinguished it from transmissible gastroenteritis (tge). as the disease spread throughout europe, acute outbreaks of diarrhea began to be observed in pigs of all ages. in 1978, it was determined that this new type of epidemic viral diarrhea in pigs was caused by a coronavirus (debouck and pensaert, 1980) . this new coronavirus, porcine epidemic diarrhea virus (pedv), along with tge virus (tgev) have been classified into group 1 of the genus alphacoronavirus (song and park, 2012) . porcine epidemic diarrhea outbreaks were to 27 or more states (stevenson et al., 2013; chen et al., 2014; www.aasv.org/pedv/pedv_weekly_report_140319. pdf, accessed march 24, 2014). the initial usa pedv strain from april 2013 has been shown to phylogenetically cluster within subgroup iia along with a strain/lineage that was detected in china in 2012 (huang et al., 2013; stevenson et al., 2013; chen et al., 2014) . a second pedv strain, described by iowa state university (www.vetmed.iastate. edu/sites/default/files/vdpam/disease_ topics/swine-corona-viruses2-27-14_0.pdf, accessed march 7, 2014) and the ohio department of agriculture (wang et al., 2014) , was subsequently detected in late 2013/early 2014. based on the full-length spike (s) protein gene, this pedv strain clusters with another chinese lineage from 2010 to 2012 and is distantly related to the pedv strains that were initially detected in the usa, suggesting that multiple introductions, or a single introduction of multiple strains, of this virus have occurred. in late january 2014, the first case of ped was diagnosed in a swine herd in south-western ontario (manuscript accepted for publication). an epidemiological investigation of the ontario ped cases pointed to feed as a common risk factor. as a result, several lots of feed and spray-dried porcine plasma (sdpp) that was imported from the usa and used as a feed supplement were tested for the presence of pedv genetic material. several of these tested positive, supporting the possibility that contaminated feed may have been responsible for the introduction of pedv into canada (cfia news release, 2014; omaf, 2014; d. ojkic and g. maxie, personal communication) . this epidemiological link initiated bioassay trials to determine whether the sdpp supplement or the feed itself contained sufficient infectious pedv to infect weaned piglets under controlled laboratory conditions. nucleic acid extraction several nucleic acid extraction methods were employed depending on the sample matrix. all extractions included known positive and negative samples as well as a water negative control. for intestinal swab specimens taken at postmortem and placed in virus transport medium, 500-ll samples were extracted using the qiagen rneasy â mini kit (qiagen, mississauga, on, canada) following the manufacturer's instructions. this extraction method was used on the initial confirmatory submission received from the animal health laboratory, guelph. for rectal swab specimens taken from bioassay pigs and placed in phosphate-buffered saline (pbs), 50-ll samples were extracted using the mag-max tm -96 viral isolation kit (life technologies, burlington, on, canada) and a magmax tm express-96 instrument. this high-throughput extraction method was used to deal with the large number of samples that were generated during the bioassay experiment. for pelleted feed and sdpp samples, 10% (wt/vol) emulsions were first prepared in sterile pbs and then vortexed thoroughly until fully suspended/dissolved. three different methods were then evaluated for rna extraction: (i) magmax tm -96 viral isolation kit, (ii) qiagen rneasy â mini kit and (iii) tri-pure isolation reagent (roche diagnostic corporation, indianapolis, in, usa) . for the magmax tm -96 method, total rna was extracted from 50 ll of the unclarified 10% emulsion and eluted in 20 ll of nuclease-free elution buffer. for the qiagen rneasy â mini kit method, the 10% emulsion was clarified by brief centrifugation and total rna was extracted from 500 ll of the clarified supernatant and eluted in 50 ll of nuclease-free water. for the tripure method, 100 ll of unclarified emulsion was added to 900 ll of tripure isolation reagent and vortexed thoroughly. two-hundred microlitres of chloroform was then added, the mixture vortexed thoroughly for~30 s and incubated for 10 min. the aqueous and organic phases were separated by centrifugation and the aqueous phase added to an equal volume of isopropanol. the rna precipitate was then pelleted, and the pellet dried and dissolved in 20 ll of nuclease-free water. in an attempt to better determine the state of pedv in sdpp, a protocol using detergent treatment and differential centrifugation prior to magmax tm extraction was used. ten grams of sdpp was prepared as a 10% (wt/vol) suspension in pbs with or without 0.25% (vol/vol) nonidet p-40 (roche), a non-ionic, non-denaturing detergent. the suspension was first centrifuged at 10 000 g for 30 min at 4°c and the resulting supernatant was removed and centrifuged at 100 000 g for 1 h at 4°c. supernatants and pellets collected following both the 10 000 and 100 000 g centrifugation steps were then used for magmax extraction. total rna extracted by the various methods was screened using a real-time rt-pcr (rrt-pcr) assay that targets the nucleocapsid (n) gene of pedv (manuscript accepted for publication). the assay, consisting of forward primer pedn+2733-f: 5 0 -tatgctcagatcgccagt-3 0 , probe pedn+27354-pb: 5 0 -fam-gcaccaaatgttgcagcatt gct-bhq-1-3 0 , and reverse primer pedn-27395-r: 5 0 -cagccacattaccaccaaag-3 0 was run with two different chemistries -agpath-id tm one-step rt-pcr kit (life technologies) and the qiagen â one-step rt-pcr kit (qiagen) on two instrument platforms -smart cycler ii (cepheid, sunnyvale, ca, usa) and 7900ht sds (applied biosystems, foster city, ca, usa). each sample was amplified in a 25-ll rt-pcr reaction mix containing either 17 ll of agpath-id tm one-step rt-pcr mix or qiagen â one-step rt-pcr mix and 8 ll of rna. amplification was carried out for a total of 45 cycles. positive and negative controls as well as a notemplate control (water) were included in each run. conventional n and s gene rt-pcr followed by sequencing was used for confirmatory purposes on all diagnostic submissions and selected bioassay results. the primers used to amplify the partial n and s genes of pedv were based on those obtained from the national veterinary services laboratory (nvsl), ames, iowa with minor modifications. forward primer pedn253 -5 0 -ggcatttctactacc tcgga-3 0 and reverse primer pedn992 -5 0 -atagcct gacgcatcaacac-3 0 and forward primer peds218 -5 0 -gctagtggcgttcatggtat-3 0 and reverse primer peds442 -5 0 -taggcaattacgacctgttg were used to amplify 739 and 224 bp n and s1 gene products, respectively. these were then used directly for sequencing or gelpurified prior to sequencing or in some cases cloned prior to sequencing. for amplification of the full-length s gene of pedv, primer set s-f1 5 0 -tgctagtgcgtaataatgac-3 0 (pedv genome map position 20573-20592) and s-r1 5 0 -catctttgacaactgtgt-3 0 (pedv genome map position 24825-24842) described by huang et al. (2013) was used with a superscript ii rt/platinum taq one-step rt-pcr kit (invitrogen). the~4270-bp product was gelpurified and cloned using a clonejet tm pcr cloning kit (fermentas, burlington, on, canada). additional primer pairings that were also used to obtain s gene sequence information are as follows: s-f1 5 0 -tgctagtgcg taataatgac-3 0 (map position 20573-20592) and peds-2127-r 5 0 -acatatgcagcctgctctga-3 0 (map positions 22741-22760) that produced an~2187-bp amplicon and peds-mod-1581-f 5 0 -ccaaccttattgcatct-gac-3 0 (map positions 22214-22233) and s-r1 5 0 -cat ctt tgacaactgtgt-3 0 (map positions 24825-24842) that produced an~2628-bp amplicon. gel purification was carried out depending on the purity of the generated amplicons. sanger-sequencing reactions were performed using big dye terminator chemistry version 3.1 (foster city, ca, usa) and cycle-sequencing products were resolved on an applied biosystems 3130xl genetic analyser. virus isolation was carried out on vero cells as previously described (hofmann and wyler, 1988) with modifications. vero cells were cultured in dulbecco's minimal essential medium (dmem) high-glucose supplemented with 10% c-irradiated foetal bovine serum, 2 mm l-glutamine and 50 lg/ml gentamicin. confluent vero cell cultures in 25 cm 2 flasks were rinsed twice with pbs and then inoculated with 500 ll of the intestinal swab sample placed in virus transport medium or 10% (wt/vol) intestinal tissue emulsion. prior to inoculation, the intestinal swabs and intestinal tissue emulsions were treated with a 1009 antibiotic cocktail (109 final concentration) containing 10 000 international units/ml penicillin g, 10 mg/ml streptomycin, 10 mg/ml kanamycin, 5000 units/ml nystatin and 1500 units/ml polymyxin b sulphate at 1 : 10 for 30 min at room temperature. the 109 antibiotic cocktail treated swabs and emulsions were then clarified by centrifugation. after adsorbing the inoculum at 37°c with 5% co 2 for 1-2 h with continuous rocking, 4.5 ml of virus maintenance medium consisting of dmem supplemented with 2 mm l-glutamine, 0.33% (vol/vol) tryptose phosphate broth, 19 antibiotic cocktail and 1.25 lg/ml tpck-trypsin (sigma-aldrich, oakville, on, canada) was added to each flask. cultures were incubated at 37°c with 5% co 2 until 50-75% of the monolayer exhibited cytopathic effect (cpe) or for 4 days. isolation attempts involved a maximum of three passages. four percentage of paraformaldehyde was added to the tubes containing the plasma/feed, feces or the intestinal content samples taken from the bioassay pigs. the samples were then centrifuged for 20 min at 3800 g and negatively stained with 2% phosphotungstic acid. partial post-mortem examinations were performed and intestinal samples collected within 15 min (usually <5 min) following euthanasia. for each piglet, multiple sections of intestine (six sections of jejunum, six sections of ileum, one section of colon) were collected and fixed in 10% neutral phosphate-buffered formalin, routinely processed and stained with haematoxylin and eosin (he) for histopathological examination. for immunohistochemistry, 5-lm sections were cut, airdried overnight and placed into a 60°c oven for 1 h. the deparaffinized and rehydrated sections were quenched for 10 min in aqueous 3% hydrogen peroxide and rinsed in milliq water. epitopes were retrieved using dako target retrieval solution (dako, carpinteria, ca, usa) in a biocare medical decloaking chamber. once slides were cooled, they were placed into tris buffered saline plus tween (tbst; medimabs, montreal, qc, canada) for 5 min. the slides were incubated with mouse monoclonal antibody 66.31 (central veterinary institute wageningen ur, the netherlands) directed against the pedv s protein (van nieuwstadt and zetstra, 1991) at a dilution of 1 : 1500 for overnight incubation at 4°c. slides were rinsed with tbst and incubated for 30 min with an envision + anti-mouse kit (horseradish peroxidase labelled) (dako) and a tbst rinse. diaminobenzidine (dab; dako) was used as the substrate chromogen and the slides were counterstained with gill's haematoxylin. porcine epidemic diarrhea virus s protein antibodies were detected with a complex-trapping-blocking elisa that used monoclonal antibodies that were obtained from the central veterinary institute wageningen ur, the netherlands, and a protocol that was previously described by van nieuwstadt and zetstra (1991) with modifications. a british pedv isolate from 1987 (br1/87; have et al., 1992) , kindly provided by colleagues from the danish technical university-lindholm, denmark, was used to produce elisa antigen propagated in vero cells. samples with a blocking percentage >50% were considered positive, samples with <40% blocking were considered negative and samples between 40% and 50% blocking as dubious or suspicious. samples with initial results in the dubious/suspicious range were retested in the elisa and also tested by an immunofluorescence assay (ifa) using pedv br1/87 infected vero cells fixed with ice-cold methanol. forty 3-week old piglets were obtained from a single farm in manitoba (litters were weaned and mixed at the farm on the day of delivery but each animal was identified as to the sow of origin). piglets were randomly assigned (mixed on farm and selected at random at arrival) to four groups con-sisting of 12, 10, 10 and 8 piglets each. the groups were separately housed in containment level three animal cubicles at the national centre for foreign animal disease (ncfad) and cared for in accordance with canadian council on animal care guidelines and an animal use protocol approved by the institutional animal care committee. biosecurity features were used and standard operating procedures were followed to avoid cross-contamination among the four groups of pigs. each group of pigs was housed in physically separate animal cubicles with individually dedicated air systems. upon entry of each room, staff donned dedicated clothing and showered when exiting. additionally, staff worked in progressively 'infected' rooms, starting with the negative control room and finishing in the positive control room. following arrival but prior to inoculation, rectal swabs were taken from each piglet for pedv rrt-pcr testing and 5-ml blood taken to test for antibodies to pedv. a description of the different experimental groups is summarized in table 1 . feed and sdpp samples were processed as follows in preparation for inoculation. twenty-one grams of ground feed or sdpp was added to 210 ml pbs, mixed thoroughly and then divided into four aliquots of 50 ml for each piglet. the remaining 10 ml was stored at à70°c. animals received 25 ml of a feed or sdpp suspension via gastric tube while under light isoflurane anaesthesia and the remaining 25 ml orally after recovering from anaesthesia. three sdpp and two feed samples were tested with four piglets per sample as well as a positive and a negative control group. positive control animals received 50 ml (25 ml by gastric tube + 25 ml orally) of an intestinal tissue suspension positive for pedv. specifically, 2.0 ml of a 10% (wt/vol) tissue suspension of colonic tissue derived from the first-confirmed pedv case in ontario was diluted further in 500 ml pbs to give a final tissue dilution of 1 : 2500. negative control pigs received 50 ml pbs (25 ml by gastric tube + 25 ml orally). to prevent cross-contamination of the different inocula, the feed, sdpp and pedv positive intestinal tissue used as the positive control were all prepared in a biosafety cabinet that was thoroughly disinfected with a 1% virkon solution and allowed to clear for a minimum of 15 min between preparations. as an added precaution, the positive control inoculum from the pedv positive intestinal tissue was prepared last. at 7 days post-inoculation (dpi), the 10 negative control piglets were moved and allowed to co-mingle with the positive control (three contacts), sdpp (three contacts) and feed (four contacts) inoculated animals to assess transmission. piglets were observed daily for clinical signs, in particular vomiting or diarrhea, with the plan to euthanize any severely affected or moribund animals. rectal temperatures and swabs were taken daily from all animals. at 7 dpi, one piglet each from the positive control and the three sdpp inoculation groups were euthanized, postmortem examinations performed and various samples collected. at 12 dpi, one positive control piglet along with the three contacts (5 days post-contact) in that group were euthanized, post-mortem examinations performed and various samples collected. remaining animals were euthanized from 17 to 19 dpi and post-mortem examinations performed. a second bioassay experiment was run in parallel further evaluating the feed as a potential source of pedv (bioassay 2, table 2 ). these pigs were obtained from the same farm but housed separately from the first bioassay pigs and under similar conditions. forty-four 3-week-old piglets were randomly assigned to five groups, which included one group of 12 piglets that was mock inoculated and four groups of 8 piglets each that were inoculated with feed sample 1 as described above and with one of these four groups given the same feed (12.5 g per pig per day) as part of the normal daily feed for an additional 2 days. each of the four feed inoculated groups had an additional 4 piglets added in as contacts at day 2. in late january 2014, four suckling piglets that originated from a closed herd in south-western ontario were submitted to the animal health laboratory (ahl), university of guelph. this herd had a history of sudden onset of diarrhea in piglets less than a week of age and based on laboratory test results a diagnosis of ped was made (manuscript accepted for publication). briefly, intestinal swab specimens were tested for the presence of porcine respiratory coronavirus (prcv), transmissible gastroenteritis virus (tgev) and pedv by a real-time rt-pcr assay with all four swab samples producing strong positive results (c t s = 20.47, 21.85, 22.92 and 25.96) on the pedv n gene rrt-pcr assay. samples were forwarded to the national centre for foreign animal disease (ncfad) in winnipeg where the diagnosis of pedv was confirmed by pedv n and s gene conventional rt-pcr assays and sequencing. based on partial sequence information for the n and s genes, the ontario pedv isolate was found to be 99.8% identical to pedv isolated in the usa in 2013. an additional 10 cases that followed the ontario index case were submitted by ahl, guelph to ncfad, winnipeg for confirmation. all were confirmed by pedv n and s gene conventional rt-pcr assays with one of the submissions yielding a virus isolate. an epidemiologic investigation carried out on the index and 10 subsequent cases by the ontario ministry of agriculture and food (omaf) indicated feed as a common risk factor (cfia news release, 2014; omaf, 2014; d. ojkic and g. maxie, personal communication). this led ahl guelph to test lots of sdpp that were imported from the usa and the associated feed in which they were used as an additive for pedv nucleic acid by rrt-pcr assay. several of these gave positive results including the lot mentioned below (data not shown). five samples of sdpp from different pallets of a single lot and five feed samples that contained 6% (wt/wt) of the aforementioned plasma that were epidemiologically linked with the first cases of ped in south-western ontario, were tested by pedv n gene rrt-pcr and s gene conventional rt-pcr assays. at ncfad, all five sdpp samples gave weak positive reactions (c t s of 36. 35, 36.2, 36.97, 36.65 and 36.69) on the n gene rrt-pcr assay. these were also positive by s gene rt-pcr with three of the samples considered as moderately strong positives producing clearly visible bands of the correct size. 41.29, 40.55, 40 .21 on the second run. when the feed sample that was initially positive on the n gene assay was retested, only one of the five aliquots produced a c t value on first (39.85) and second (37.22) runs indicating that the levels of pedv nucleic acid in this sample are at the limit of detection. the~224 s1 gene amplicons from the five sdpp samples were sequenced and found to be 100% identical to one another. although the sequence obtained for the amplicon from the positive feed sample was a match to pedv (70-90% match to pedv sequences in genbank by using blast), the quality of the sequence was too poor (below 80% quality values) for accurate comparisons. attempts made to clone this amplicon to obtain better sequence data were not successful. in an attempt to better understand the state in which pedv exists in sdpp, a protocol involving treatment with the non-ionic, non-denaturing detergent nonidet p-40 followed by differential centrifugation was employed. ten grams of sdpp was used to produce a 10% (wt/vol) suspension in pbs containing or not containing 0.25% (vol/vol) nonidet p-40. this was initially centrifuged at 10 000 g and the resulting supernatant centrifuged at 100 000 g to sediment intact virus or viral nucleocapsids. on the sdpp sample that was tested, the following n gene rrt-pcr results were observed: c t of 35.84 for pbs supernatant after 10 000 g, c t of 36.74 for the pbs pellet after 10 000 g, c t of 38.83 for the pbs + nonidet p-40 comparison of s protein gene sequences obtained from bioassay piglets versus those of field cases. one-hundred and eighty-eight nucleotides of the~224-bp-small s gene conventional rt-pcr amplicon (primers excluded) obtained from rectal swab samples of bioassay piglets and canadian and usa field cases were aligned. ncfad 2014-022 is the identical sequence of the 5 sdpp samples sequenced directly. plasma #21-22, #25-26, #29-30 and #32 are from sdpp-inoculated bioassay piglets. ontindex is the ontario index case. ncfad2014-18 #1-10 are the 10 ontario field cases that followed the index case. pellet # 35 is from a rectal swab specimen taken at 3 dpi from piglet # 35 of the feed group. pei is from a field case from prince edward island. ncfad 2014-35 is from a field case from quebec. mafri is from a field case from manitoba and ncfad 2014-41 is from an environmental sample from manitoba. colorado (kf272920) and indiana (kf452323) are usa pedv isolates. supernatant after 10 000 g, c t of 36.74 for the pbs pellet after 100 000 g, and c t of 35.94 for the pbs + nonidet p-40 pellet after 100 000 g. c t values of 35.94 and 36.74 for both nonidet p-40 treated and untreated 100 000 g pellets are an indication that the plasma sample contained intact virions or at least viral nucleocapsids (risco et al., 1996; krempl and herrler, 2001) . finally, no coronavirus-like particles could be identified by direct electron microscopical examination of sdpp or feed samples. no clinical signs were observed in the negative control group. in contrast, animals in the positive control group were depressed, off feed and had diarrhea/soft feces beginning at 1 dpi and a small amount of vomiting noted at 11 dpi (4 days post-contact for contact animals). rectal temperatures, however, remained within the normal range (38.7-39.8°c). the piglet group inoculated with the three sdpp samples had a few piglets with diarrhea or soft feces beginning at 2 dpi onwards, however, rectal temperatures were also within the normal range. the piglet group inoculated with the two feed samples were mildly depressed with one or two animals with diarrhea or soft feces beginning at 1 dpi onwards and decreased feed intake from 3 to 7 dpi. rectal temperatures as with the other experimental groups were within the normal range. rectal swab samples collected from all of the piglets prior to inoculation were pedv n gene rrt-pcr negative. all of the negative control piglets remained n gene rrt-pcr negative up to and including 7 dpi at which time they were moved in with the other experimental groups as non-inoculated contacts. the eight piglets inoculated with the feed samples along with the four contact piglets introduced into this group beginning at 7 dpi also remained negative up to and including 17 dpi (day 10 post-contact). in contrast, piglets in the positive control and sdpp-inoculated groups were pedv n gene rrt-pcr positive beginning at 1-2 dpi and remained positive until 7 dpi for all piglets and beyond for many of them (fig. 1a,b) . some of the contact piglets introduced at 7 dpi became positive beginning at 3 days post-contact (fig. 1c,d) . no significant difference was observed in the kinetics of n gene rrt-pcr positivity in animals that were inoculated with the three sdpp samples that were tested, suggesting that each contained infectious virus. however, as these piglets were housed in the same animal cubicle, we cannot unequivocally state that all samples were infective but that at least one of the three samples contained infectious virus. negative contrast staining electron microscopy of fecal samples collected at 4 dpi from the sdpp-inoculated piglets and the positive control group piglets showed the presence of virus-like particles consistent with coronavirus virions. similar virus-like particles were also found in the content of the small intestine of a sdpp-inoculated piglet at 7 dpi and a positive control group contact piglet at 5 days post-contact. no virus-like particles were observed in fecal samples from the feed-inoculated group. the partial s1 gene rt-pcr was also carried out on rectal swabs collected from the sdpp and feed-inoculated piglets at 3 and 4 dpi to confirm the n gene rrt-pcr results described above. rectal swabs from all 12 sdppinoculated piglets were strongly positive while one of the feed-inoculated piglets (piglet 35) produced a very weakly visible amplicon of the correct size, and a few others produced weakly visible amplicons of a different size indicating non-specific amplification. seven amplicons from the sdpp-inoculated piglets (piglets 21, 22, 25, 26, 29, 30 and 32) at 3 dpi, the weakly visible amplicon from feed-inoculated piglet 35, and spurious bands from the remaining seven feed-inoculated piglets were gel-purified and sequenced. all 7 amplicons from the sdpp-inoculated piglets were pedv with sequences that were similar to the strain found in the usa since april/may 2013, detected in the initial ontario field cases and in the sdpp samples (fig. 2) . the sequence from feed-inoculated piglet 35 was also pedv but of poor quality and difficult to analyse. the seven spurious amplicons selected for sequencing were unrelated to pedv (e.g. thermoplasmatales archeon and candidatus methonomethylophilus using blast). the weak amplicon from piglet 35 was cloned and of the 8 clones sequenced, one had a typical pedv sequence (188/ 188 to pedv strain usa/indiana/17846/2013 genbank accession no. kf452323) similar to those from the sdppinoculated piglets, indicating that the 3 dpi rectal swab from piglet 35 contained pedv rna/virus, albeit at trace levels. sequence results are summarized in fig. 2 . the sequences are identical with the exception of a singlenucleotide polymorphism (snp) at nucleotide 20 961 (the s gene maps from nucleotide 20 634 to 24 794 of the pedv genome). individual sequences were found to have a c, a t or both at this site, consistent with a mixed population as indicated by some of the plasma-inoculated piglets. this snp results in either a histidine (c at 20 961) or a tyrosine (t at 20 961) at amino acid 100 of the s protein. the full s gene sequence was obtained from a rectal swab collected at 3 dpi from one of the sdpp-inoculated piglets (piglet 30; genbank accession no. km196110). comparison of this sequence to that obtained from the ontario index case (genbank accession no. km189366), the ontario pedv isolate (genbank accession no. km189367), a single case from prince edward island (genbank accession no. km189368) which was also epidemiologically linked to the same feed and sdpp as the ontario cases, and to those reported initially in the usa (e.g. usa/colorado/2013 and usa/indiana/17846/2013; genbank kf272920 and kf452323) showed them to be 99.8-99.9% identical at both the nucleotide and amino acid levels. comparison of the ontario index case and sdpp piglet 30 s gene sequences revealed 5 synonymous and 2 non-synonymous (h110y and s1099a) changes. comparison of the ontario pedv isolate and sdpp piglet 30 s gene sequences showed that they differed by 1 synonymous and 1 non-synonymous (h110y) change, while the prince edward island and sdpp piglet 30 s gene sequences were 100% identical at the nucleotide and amino acid levels. overall, this is consistent with a somewhat mixed population of genomes, but all with a very high degree of similarity to the sequences reported from the initial outbreaks in the usa. of the 3 piglets from the sdpp-inoculated group on which post-mortem examinations were performed at 7 dpi, piglet 26 had a significant enteropathy characterized by a small intestine that was flaccid, thin walled in some areas, and filled with fluid mixed with brown flecks. all three piglets had some degree of watery intestinal content, which primarily affected the lower jejunum and the ileum. microscopically, small intestinal villi were shortened and often had a 1 : 1 crypt to villus ratio (fig. 3a) . there was a mild increase in inflammatory cells in the lamina propria, dilation of lymphatics and submucosal edema. in some areas, enterocytes had cuboidal morphology and were vacuolated (fig. 3b) . pedv antigen was detected by ihc within enterocytes of all 3 animals with moderate numbers of positive enterocytes observed in piglet 32 and occasional positive enterocytes observed in piglets 22 and 26 (fig. 3c,d) . no pathological changes or pedv antigen staining were observed in the remainder of the piglets in the sdpp group that were examined at 18 dpi, which included contact animals at 11 days post-contact. of the animals in the feed-inoculated group that were examined at 7 dpi, the intestinal walls appeared normal although some of them had watery intestinal content. none of the contact animals in this group that were examined on post-contact day 12 had any significant lesions. microscopically, some sections of intestine showed normal length of the small intestinal villi (1 : 3 crypt to villus ratio), but in other areas, villi were shortened and there was a mild increase of inflammatory cells in the lamina propria. the positive control piglet examined at 7 dpi (piglet 12) had thin-walled small intestines that were flaccid and fluid filled. microscopically, the intestinal villi were blunted with a 1 : 1 crypt to villus ratio in some areas. the surface enterocytes were low columnar to columnar with an easily discernible brush border. a mild increase in inflammatory cells within the lamina propria was also observed. two of the positive control group contact animals examined on day 5 post-contact (piglets 8 and 9) had a significant enteropathy characterized by flaccid, distended fluid-filled intestines. microscopic lesions were similar to those described above for the sdpp-inoculated group except for the presence of significant necrotic debris in the lumen, which was only observed in the positive control day 5 post-contact animals (fig. 3e) . pedv antigen was detected extensively within enterocytes, primarily within the brush border but also within the cytoplasm of the cell body (fig. 3f) . although no significant gross pathological changes were observed in the intestinal wall of the positive control piglet examined at 12 dpi (piglet 17), the intestinal contents were still watery. no significant macroscopic lesions, microscopic changes, or pedv antigen staining were observed in the three positive control group pigs examined at 19 dpi. because the negative control group from this bioassay was co-mingled with the experimental groups, negative control piglets were not available for microscopic examination. thus, negative control piglets from the second bioassay experiment euthanized at 12 dpi were used for the histopathology and immunohistochemistry observations. microscopically, there were some areas where the small intestinal villi appeared shortened and there was some evidence of mild inflammation in the lamina propria (fig. 3g) . ihc was performed on tissues from 5 of the negative control animals, and pedv antigen was not detected (fig. 3h) . in summary, the only significant finding in relation to the bioassay was the presence of significantly shortened villi and the presence of pedv antigen in both the sdpp group of piglets at 7 dpi and the positive control contact animals at 5 days post-contact. overall, there was some indication of mild enteritis in some piglets from all groups. this latter finding is considered non-specific and could be due to a number of factors. however, it is important to note that in negative controls, this inflammation was not associated with pedv infection as no antigen was detected and all other test results showed these piglets to be negative for pedv. serum samples from all 40 piglets collected before inoculation were negative for antibodies to pedv as determined by complex-trapping-blocking elisa. the ten piglets in the negative control group were serologically negative at 7 dpi and remained so throughout the remainder of the experiment. all eight piglets in the feed-inoculated group were negative for pedv antibodies at 7, 14 and 17 dpi. in the sdpp group, 4 of 12 piglets (piglets 22, 24, 31 and 32) were dubious/suspicious reactors on day 7 and, of the two samples tested on day 14, one was positive by elisa and ifa (piglet 29) and the other a dubious/suspicious reactor in the elisa and positive in ifa (piglet 28). at 18 dpi, of the 9 piglets that were tested, 7 (piglets 21, 23, 24, 27, 28, 29 and 30) were positive while the remaining two (piglets 25 and 31) gave dubious/suspicious results on the initial test and, on retest, piglet 25 was a weak positive reactor in the elisa and positive by ifa. in the positive control group, one piglet (piglet 16) was positive and another (piglet 17) a dubious/suspicious reactor at 7 dpi. at 14 dpi, three of the four animals tested (piglets 13, 18 and 19) were positive for antibodies to pedv. by 19 dpi, all 8 animals tested (piglets 11, 13, 14, 15, 16, 18, 19 and 20) were seropositive although piglet 11 had a high variability and scored negative/dubious upon retest. of the 4 contact piglets introduced into the feed group at 7 dpi and tested at 10 days post-contact, one (piglet 2) scored as weak positive (50.75% blocking) by elisa. upon repeat testing, it was negative by elisa and ifa. the three contact piglets introduced into the sdpp-inoculated group and the three contact piglets introduced into the positive control group tested negative for pedv antibodies at 11 and 5 days postcontact, respectively. the practice of weaning piglets at 21 days of age or less has led to the use of complex diets containing a variety of supplemental ingredients that include sdpp. the procedure used to produce this feed supplement involves collecting blood from healthy pigs at the abattoir into tanks containing an anticoagulant. the pooled blood is then shipped to a processing facility where the cells and plasma are separated by centrifugation. following centrifugation, the liquid plasma, which is approximately 7% crude protein, is concentrated by evaporation, nano-filtration or ultra-filtration which results in crude protein levels that are in the 20-25% range. this material is then spray-dried to achieve a final crude protein concentration of at least 80%. studies have shown that the addition of sdpp to weaner pig diets can result in positive benefits including increased feed intake and weight gain and decreased requirement for antibiotics (reviewed by ferreira et al., 2009) . although blood from apparently healthy animals can be assumed to be sterile, the risk for virus contamination exists if blood from subclinically affected and viremic animals is collected or if the product becomes contaminated during any step of the process. viremia is not a known feature of pedv infection. blood collected at 7 dpi from three sdpp and one positive control piglet tested negative on the n gene rrt-pcr assay, as did 1 of the positive control piglets at 12 dpi, and three of the positive control group contact piglets at 5 days postcontact. although transport of pigs in inadequately cleaned trailers has been an implicated source of transmission of pedv (lowe et al., 2014) , other modes of transmission may also exist. our study indicates that pedv-contaminated sdpp may be one such mode for introducing the virus into a na€ ıve pig herd. while a typical pedv sequence could be obtained directly from the sdpp samples used for inoculation, only one of the two selected feed samples produced a pcr amplicon in the conventional s gene rt-pcr assay that was of the correct size. the sequence of this product, although consistent with pedv, was not of sufficient quality to make a definitive conclusion. using a pedv n gene rrt-pcr along with a number of ancillary tests, we demonstrated that piglets inoculated with contaminated sdpp could replicate and excrete the virus as well as transmit it to contact piglets. virus isolation was not used as a measure of virus excretion due to the inherent difficulties associated with cell culture isolation of this virus from clinical material. to illustrate this point, chen et al. (2014) have recently reported that of 33 fecal samples and 17 intestinal homogenates, only 2 pedv isolates were obtained giving a success rate of only 4%. moreover, none of the virus isolation attempts that they carried out on feces were successful. consequently, virus isolation for determination of pedv infectivity from clinical material is not viewed as a sensitive method. the duration of pedv shedding in experimentally infected 14-day-old piglets as determined by quantitative rt-pcr was previously reported to be 7 to 9 days (song et al., 2006) . the quantitative rt-pcr used in that study targeted the s gene of pedv generating a 651-bp amplicon. a two-step rt-pcr was employed in which the reverse transcription step was carried out separately to produce cdna. each cdna sample was then competed with 10 000 copies of an internal control dna during the pcr reaction as a means of quan-titating the amount of pedv rna present in the sample. in the study reported here, several piglets in the positive control and sdpp groups shed virus well beyond 9 days post-inoculation, with two animals in the positive control group and one animal in the sdpp group still producing positive fecal swab samples at 18 dpi and still capable of transmitting to some of the contact piglets that were introduced at 7 dpi. the longer duration of pedv shedding in our study could potentially be attributed to a greater sensitivity of the n gene rrt-pcr assay or to a more prolonged infection, perhaps caused by a lower initial infectious dose. regardless, more work needs to be performed in this area as an accurate assessment of the duration of shedding will have practical applications with respect to control of this disease. rectal temperatures of the inoculated piglets remained within normal range, and although some clinical signs in the form of depression, diarrhea and reduced feed intake were observed in the positive control group, similar signs were also observed, albeit less pronounced and in only a few piglets, in the other groups with the exception of the negative control group. however, such observations should not alone be interpreted as signs of pedv infection as piglets may have mild clinical illness due to many different causes including stress associated with moving and change in feed. the mild clinical signs observed in this study may be related to piglet age at the time of inoculation. using a field strain of pedv to inoculate specific pathogen-free pigs between the ages of 2 days and 12 weeks, shibata et al. (2000) reported that severe clinical disease and death only occurred in 2-7-day-old piglets. while rectal swabs from the negative controls and the feed-inoculated piglets remained negative throughout the study, the positive controls and the sdpp-inoculated piglets began shedding significant amounts of pedv beginning at 1 and 2 dpi. although negative by n gene rrt-pcr, a single piglet (piglet 35) from the feed-inoculated group had a weak band in the conventional s gene rt-pcr at day 3 and sequencing of this product was consistent with pedv although, similar to what was found for the feed sample, too weak for accurate determination. however, cloning of the pcr product and subsequent sequencing revealed that it was similar to the other pedv sequences obtained in this study as well as to those associated with canadian and initial usa field cases. electron microscopy of feces and intestinal content and immunohistochemistry on intestinal tissues of sdppinoculated and positive control piglets were consistent with coronavirus-like particles and the presence of pedv antigen in enterocytes, respectively. furthermore, at least one piglet from the sdpp-inoculated group examined at 7 dpi had an obvious enteropathy macroscopically and blunted small intestinal villi microscopically consistent with pedv infection. finally, seroconversion of animals in the positive control and sdpp-inoculated groups towards the end of the study provides further support of an active pedv infection. other studies have addressed the risk of transmitting viral contaminants, most notably porcine circovirus 2 (pcv-2), via spray-dried porcine plasma (patterson et al., 2010; pujols et al., 2008; shen et al., 2011) . one study (patterson et al., 2010) found that pcv-2 could be transmitted to na€ ıve pigs given pcv-2-contaminated sdpp by oral gavage. by contrast, the other two studies (pujols et al., 2008; shen et al., 2011) showed that weaned pigs that were fed pcv-2-contaminated sdpp neither developed clinical signs, became viremic or seroconverted. all three studies differed with respect to the health status of the animals used, the pooling of plasma from numerous animals, the pcv-2 dna load and the presence of anti-pcv-2 antibodies, all of which could contribute to the differences in the reported results. in conclusion, we have shown that the tested sdpp contains infectious pedv as demonstrated by a relatively high level of pedv excretion detected for ≥9 days by pedv n gene rrt-pcr. this was supported by s gene rt-pcr results and sequences and by seroconversion. moreover, the infection spread to 2 of 3 contact piglets introduced at day 7. the 12 piglets in the sdpp group were inoculated with three samples from different pallets of the same lot number in groups of 4 piglets. the kinetics of pcr positivity did not appear to differ among these piglets indicating that all three samples may likely have contained infectivity. however, as the piglets were kept together in the same room, we cannot unequivocally determine that all three samples were infective but can state that at least one of the three samples contained infectivity and that the results indicate that all 3 may likely have done so. inoculation with the tested feed samples did not produce any significant excretion of pedv although genetic material could be detected in the feed at trace levels and a single inoculated piglet at day 3 had traces of pedv genetic material in its rectal swab that was shown to be similar in sequence to the other samples tested. thus, as the tested feed did contain the sdpp shown to be infectious and did contain pedv genetic material and, moreover, could be detected in a single inoculated piglet at 3 dpi, we consider the tested feed as inconclusive or not possible to determine whether it is infectious or not by bioassay. the one feed that did produce a borderline result in a single piglet (feed sample 1) was subjected to a separate, second bioassay. although this bioassay included a larger number of piglets (n = 32), infectivity of the feed was not demonstrated. nevertheless, contaminated feed cannot and should not be ruled out as a potential source of infection as it is very possible that the limited bioassay studies described here are likely much less sensitive than what might occur under field conditions. many more piglets than used in this study coupled with larger amounts of feed, more stressful field conditions and ongoing infections may influence susceptibility of animals to pedv-contaminated feed. lastly, the feeds in question only contain 6% sdpp and, furthermore, had been in use for several weeks in the field prior to their evaluation in our bioassay experiments which, although stored under warehouse conditions, may have resulted in a further loss of any potential minimal infectivity present initially. cfia statement on porcine epidemic diarrhea virus in feed isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states experimental infection of pigs with a new porcine enteric coronavirus, cv 777 spray dried plasma for pigs weaned at different ages coronavirus infection in mink (mustela vison). serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus propagation of the virus of porcine epidemic diarrhea in cell culture 2013: origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins new variants of porcine epidemic virus use of two enzyme-linked immunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhea virus porcine epidemic diarrhea investigation efficacy of experimentally produced spray-dried plasma on infectivity of porcine circovirus type 2 lack of transmission of porcine circovirus type 2 to weanling pigs by feeding them spray-dried porcine plasma the transmissible gastroenteritis coronavirus contains a spherical core shell consisting of m and n proteins commercially produced spraydried porcine plasma contains increased concentrations of porcine circovirus type 2 dna but does not transmit porcine circovirus type 2 to na€ ıve pigs isolation of procine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages porcine epidemic diarrhea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines use of an internal control in a quantitative rt-pcr assay for quantitation of porcine epidemic diarrhea virus shedding in pigs 2013: emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences usda technical note -porcine epidemic diarrhea (ped) new variant of porcine epidemic diarrhea virus an apparently new syndrome of porcine epidemic diarrhoea we gratefully acknowledge the hard and dedicated work of the animal care, laboratory, sample receiving and administrative staff involved in this project. we also acknowledge the contributions of dr. john copps and our collaborators at nvsl-ames, isu-ames, lindholm-denmark and lelystad-netherlands. key: cord-294468-0v4grqa7 authors: kasilingam, dharun; prabhakaran, s.p sathiya; dinesh kumar, r; rajagopal, varthini; santhosh kumar, t; soundararaj, ajitha title: exploring the growth of covid‐19 cases using exponential modelling across 42 countries and predicting signs of early containment using machine learning date: 2020-08-04 journal: transbound emerg dis doi: 10.1111/tbed.13764 sha: doc_id: 294468 cord_uid: 0v4grqa7 covid‐19 pandemic disease spread by the sars‐cov‐2 single‐strand structure rna virus, belongs to the 7(th) generation of the coronavirus family. following an unusual replication mechanism, it’s extreme ease of transmissivity has put many counties under lockdown. with uncertainty of developing a cure/vaccine for the infection in the near future, the onus currently lies on healthcare infrastructure, policies, government activities, and behaviour of the people to contain the virus. this research uses exponential growth modelling studies to understand the spreading patterns of the covid‐19 virus and identifies countries that have shown early signs of containment until 26(th) march 2020. predictive supervised machine learning models are built using infrastructure, environment, policies, and infection‐related independent variables to predict early containment. covid‐19 infection data across 42 countries are used. logistic regression results show a positive significant relationship between healthcare infrastructure and lockdown policies, and signs of early containment. machine learning models based on logistic regression, decision tree, random forest, and support vector machines are developed and show accuracies between 76.2% to 92.9% to predict early signs of infection containment. other policies and the decisions taken by countries to contain the infection are also discussed. coronaviruses, though uncommon, are serious pathogens responsible for infections that posit flu-like symptoms in infected individuals. these symptoms sometimes resemble the cold and cough symptoms caused by the rhinovirus. recently, the family has added its seventh generation coronavirus -sars-cov-2 (chengxin et al., 2020) . the virus shares 79% identity to severe acute respiratory syndrome (sars) and 50% identity to middle east respiratory syndrome (mers) epidemic outbreak in 2003 and 2012 (salute, 2020) . sarc-cov-2 that causes covid-19 mutated to transmit from animal to human. this virus is believed to have transferred to humans through bats from a meat market in wuhan, china (rajendran et al., 2020; shereen et al., 2020) . in march 2020, who declared the covid-19 to be a pandemic; a pandemic being described as an infection that has spread across countries and international borders rather than within a local region or neighbouring countries. the sarc-cov-2 is a deadly corona virus that is transmitted readily between humans and already infected more than 530,000 people all over the world in 198 countries as on 26 th march 2020 which led global shutdowns (who, 2020) . the fatality rate has varied among countries and age groups. until june 2020, the fatality rate averaged 5.5% with italy recording the highest of 14.49%. the fatality rate of us, germany, and india were 5.56%, 4.72%, and 2.86% respectively until june 2020 (our world in data, 2020) . of the total deaths, less than 5% belonged to the age group of less than 45 years thereby indicating that the younger population is much more resilient to the covid-19 (worldmeters, 2020) . while these fatality rates are significantly less than those of mers (34.4%) and sars (9.5%) (petrosillo et al., 2020) , covid-19 has severe transmissivity because of the possibility of asymptomatic people being carriers and spreaders of the virus (daw et al., 2020) . the reproduction number r 0 for sarc-cov-2 has been found to be between 2.06 to 2.52 (sheng et al., 2020) . a value of r 0 greater than 1 indicates that the disease can invade the human population and higher the value, the easier is it's spread. sarc-cov-2 is the largest single-strand rna virus known to the humankind; while other viruses have a single protein spike for attachment to the human cell, this coronavirus family has 10 to 12 spike proteins, which makes it easier for the virus to attach itself to the ace-2 protein in humans (paraskevis et al., 2020) . the virus follows an unusual double step replication mechanism, which leads to high rates of proliferation (luan et al., 2020) . the this article is protected by copyright. all rights reserved incubation period is typically 2 to 14 days, and the infected person often does not have serious symptoms, rather showing common symptoms associated with flu and pneumonia (rodeny, 2020) . general symptoms of pneumonia include fever, cough, chest pain, shortness of pain, fatigue, headache, myalgia, and arthralgia (sattar sba, 2020) . in addition to symptoms of pneumonia, covid-19 infected individuals may experience a loss of taste or smell, nausea, congestion, and diarrhoea (cdc, 2020) . there are a few drugs that are being recommended and used to manage the symptoms of covid-19, but there has, as yet, been no vaccines that are proven to be effective against the coronavirus family, including covid-19 (sexton et al., 2016; gautret et al., 2020) . in the absence of vaccines, it is imperative to check transmission of the virus by alternative ways (dey et al., 2020) . policy changes in pandemic and epidemic situations involve social distancing, lockdowns, travel restrictions, awareness campaigns etc. it has been speculated in past research that environmental conditions of countries like temperature and humidity also sometimes play a significant role in controlling pandemics (lin et al., 2020) . quantitative covid-19 impact analyses are scarce in literature, given the recency of the pandemic and more studies in this area are necessary, given the seriousness of the infection. epidemics are assumed to have an exponential growth at an early stage and the number of infections reduces over time, due to interventions like lockdowns, travel restrictions, awareness programs, etc. mathematical modelling studies using exponential growth analysis coupled with machine learning could provide a better prediction model for covid-19 transmission (keeling and danon, 2009; siettos and russo, 2013; victor, 2020) . such models must incorporate the various precautionary measures taken during the viral outbreak. the objective of the research is to develop a mathematical model using exponential growth analysis coupled with machine learning, to predict worldwide covid-19 early containment signs. models have been developed based on data collected from 42 countries. the objectives of this work are twofold. first, it seeks to identify countries that were successful in early containment of the covid-19 virus. secondly, the research aims at building supervised machine learning models with high accuracies for predicting signs of early containment with infrastructure availability, environmental factors, infection severity factors, and government policies of countries as independent variables. in the process of modelling, the significance of the above variables in containing the infection at early stages is this article is protected by copyright. all rights reserved also studied. this report also includes a discussion on other activities undertaken by the governments of various nations to flatten the infections curve and their corresponding effectiveness. covid-19 is believed to have originated in an animal meat market in wuhan, china and it is thought to have been transmitted from bats (shereen et al., 2020) . within few months, the virus has rapidly spread across the world, through transmissions of fluids and aerosol particles between humans. initially, all diagnosed cases outside china had a travel history to the wuhan market. soon, community transfer caused exponential increases in infections in countries like italy, us, uk, korea, japan, etc. the ability of the sars-cov-2 virus to double replicate with the spike protein, has posed significant challenges to the development of vaccines (shereen et al., 2020) . while hydroxychloroquine and azithromycin have been recommended by some researchers, to treat covid-19-infected people, there haven't been too many clinical trials to validate the claim (gautret et al., 2020) . thus, until a scientifically validate cure or vaccine is developed, countries have to rely on preventive measures to contain the spread. this, in turn, depends on epidemiological studies that can predict spreading patterns so that policymakers can take appropriate protective measures. several viruses including sars have been reported to be vulnerable to hot temperatures, which results in differences in spreading patterns across geographic locations (zhang et al., 2020) . however, such geographic variations have not yet been analysed for covid-19. other factors like government policies and interventions, infrastructure availability, and the severity of the infection itself can affect the ability of a country to contain epidemics and pandemics. this research seeks to explore all the above factors. the climatic conditions such as temperature and humidity play an important role in both airborne and aerosol virus transmissions. the 30-year human relationship with the influenza virus has proven that the mortality rate is directly related to temperature and humidity (lowen and steel, 2014) . hence, in order to minimize transmission of diseases, isolation wards in hospitals generally tend to have optimized pressure, temperature, and humidity (who, 2014) . research on the virus in the diamond cruise ship off the coast of japan showed that a one-degree rise in temperature and a one percent increase in pressure this article is protected by copyright. all rights reserved could reduce the reproduction number r 0 down by 0.0224 -0.0383. it must be mentioned that the generalizability of the study is questionable because the ship was a contained environment and the results may not apply to the real world (sheng et al., 2020) . certain studies in china and indonesia have investigated the relationship between the temperature and the spread of infection and resultant deaths and have reported a low to medium level of correlation (tosepu et al., 2020; yueling ma et al., 2020) . relative humidity was found to have low to no correlation with infection spread or deaths. global warming has also been a reason for recent temperature increases and certain studies indicate that this can reduce flu based viral infections (national research council, 2001; actuaries, 2010; dincer et al., 2010) . however, these statements need to be further validated. while the spread of virus may be affected by climatic conditions, once the virus enters the human body, it is independent of the outside environment. however, since the virus lives outside the human body for a period of at least 12 hours under normal conditions (richard, 2020) , it is necessary to study the effects of the environmental on the spreading patterns itself. social distancing, although a new terminology for the 21 st century, is not a new approach to epidemic control. it was used by the united kingdom in 1918 to control the influenza virus outbreak that caused about 100 million deaths. social distancing involves the avoidance of mass gatherings and distancing of at least six feet between people. such measures are combined with enhanced personal hygiene through regular hand wash, and wearing a protective mask for flu-like outbreaks (yu et al., 2017; leung et al., 2018) . this is done primarily because flu causing viruses are spread through aerosols generated from saliva and nasal fluid, which can be transmitted across distances as much as three feet. the average lifetime of covid-19 viruses in the outer environment is believed to be about 12 hours, which increases transmissivity because aerosols from infected people can settle on doorknobs, lifts, transports, hotels, malls etc. and stay active for a long time, thus increasing the window of transmission. direct physical contacts, such as hand-shaking, are also avenues of transmission of the virus. the reduction of social contact has been proven to significantly reduce flu-like diseases (maharaj and kleczkowski, 2012) . the closure of schools and malls flattened the this article is protected by copyright. all rights reserved spread curve during the influenza pandemic in 2009 (rashid et al., n.d.; moh, 2014) . thus, governments worldwide have stressed on social distancing and quarantining measures for at least 14 daysthe typical incubation period of covid-19 virus -to contain its spread (prem et al., 2020) . lockdown is a preventive strategy taken by local, central or global administration during the spread of epidemic or pandemic diseases and involves stopping transportation between cities, provinces or counties. the world has so far seen four major pandemics, viz., plague in the 14 th century, influenza in 1918, sars in 2009, and the current covid-19 in 2019 as reported by who (porta, 2008; east et al., 2020; pi, 2020) . in all these cases, lockdowns were implemented by various countries to control the outbreaks. china announced lockdown as early as january 2020, to flatten the curve of the covid-19 infections over time. in march, most countries around the globe announced lockdowns of local transport, office, industries, city and national borders to contain the virus (callaway et al., 2020) . although quarantine centres for the infected are available in hospitals, large-scale infections necessitate self-quarantines and lockdown measures, in addition to the hospital-based quarantines (wuhan, 2020) . during epidemic and pandemic viral outbreaks, the availability of and access to health care infrastructure such as hospitals, beds, healthcare workers, clinical equipment, first aid kits, ventilators, and protective equipment are vital to pandemic management (bambas and drayton, 2000; persoff et al., 2018) . during the massive influenza outbreak of 1918, even developed countries had inadequate health care infrastructure, which further expanded the outbreak (george, 2008). the ebola outbreak in west africa became uncontrollable due to lack of infrastructure facilities (paweska et al., 2017) . after the outbreak, who in south africa had asked the hospitals to report their available facilities to plan for future infections optimally (murrin, 2018) . innovative measures have been recommended, to create necessary healthcare infrastructure during pandemic and epidemic situations by converting schools, colleges, theatres, and stadiums into hospitals and quarantine centres (wimberly, 2018; nuzzo et al., 2019) . health care workers supported by ngos, youth, and volunteers also play a significant role in containing outbreaks (itzwerth, 2013) . hence studying health care this article is protected by copyright. all rights reserved infrastructure availability across countries can predict covid-19 containment at an early stage. predictive modelling using machine learning and growth models can provide actionable insights to policy makers and governments to contain epidemic and pandemic infections (thompson et al., 2019) . during the onset of an epidemic, it is crucial to use exponential growth models to understand the infection rates and with proper policy implementations and behavioural changes among the susceptible group of the population, the slope reduces and the curve flattens over time (keeling and danon, 2009 ). for other outbreaks like smallpox, ebola, sars, and influenza, various studies have used mathematical and statistical modelling to understand the growth of infections (dietz, 2002; nishiura, 2011; kerkhove and ferguson, 2020) . in fact, the centres for disease control and prevention has an exclusive book with established procedures for analysing disease outbreaks, stressing on the importance of the such modelling studies. (dicker, 2006) in outbreaks, epidemiologists generally use the exponential growth model at the onset of an outbreak and proceed with prediction and classification techniques like regression, decision trees, neural networks deep learning, etc. to forecast outbreaks. (sameni, 2020; victor, 2020) . there are few studies on modelling and predicting containment of covid-19 so far (lin et al., 2020; prem et al., 2020) . the research work reported in this paper, sought to integrate crucial variables concerning infrastructure, environment, policies, and severity of the disease to predict initial signs of containment. the study used a machine learning and exponential growth model. the variables used as part of the predictive mode were, doctors per 1000 population, beds per 1000 population, average temperature, average humidity, days since official lockdown, percentage of lockdown days, total cases per million population, deaths per million population, days since the first contact, and percentage of serious cases of infected. data associated with the variables were collected from different official sources for a total of 42 counties with respect to covid-19 infections as on 26 th march 2020. this accounts for 448,989 covid-19 cases comprising of 84.78% of the total infections this article is protected by copyright. all rights reserved worldwide. the daily number of infections, recovery, and deaths were collected from the website of the who. the data for infrastructure-centred variables like the number of hospitals and the number of doctors were taken form (world bank, 2020) . environmentbased variables like average temperature and humidity since the onset of covid-19 was taken from (weather underground, 2020). day-wise covid-19 case distributions extracted from who were used to identify countries that showed sign of containment of the virus based on a novel exponential growth modelling approach. raw data from the sources were also consolidated and the variables physicians per thousand individuals, hospitals per thousand individuals, percentage of lockdown days since the first contact, cases per million population, deaths per million population, days since the first case, serious cases per thousand infections, average temperature since the first infection, and average humidity since the first infection were calculated to train the machine learning models. most epidemic and pandemic diseases grow exponentially in the initial stages of the outset in a country (ma, 2020) . a popular modelling technique that demonstrates this is the susceptible-infectious-recovered (sir) model (kermack et al., 1927) . if s denotes the fraction of susceptible individuals to a pandemic, i indicates the fraction of infectious people, r is the fraction of recovered patients, β indicates the transmission rate per infectious individual, and the recovery rate is denoted by γ, the infectious period is exponentially distributed with a mean of 1/ γ as shown below. linearizing this about the disease-free equilibrium, we get the following. hence from the above expression, if − > 0, then the infection function i(t) grows exponentially about the disease-free equilibrium point. in addition to this, at the onset of the infection, ≈ 1 and hence the incidence rate = also grows exponentially. hence, modelling the initial stages on a pandemic like covid-19 is both relevant and crucial in understanding the growth of the infection. although sub-exponential and polynomial modelling have worked in cases of outbreaks like ebola, hiv, and foot and mouth diseases (chowell et al., 2016) , they generally work well with proceeding generations. for pandemics like covid-19, the exponential growth model is relevant and the use of least-squares at the initial stages can afford precise insights. figure 1 shows the analysis plan to achieve the objectives of the research. this article is protected by copyright. all rights reserved the exponential growth model assumes that the onset of any outbreak follows an exponential distribution. however, due to government interventions, medical innovations, behavioural changes etc, at a later stage, the growth curve flattens and rate of infections gradually reduces (kermack et al., 1927) . to identify such signs, we looked at the infections in the last seven-day period and the deviation of the data points from the modelled exponential curve was captured using the mean absolute percentage error metric. based on the errors and the direction in which the actual data points were to the modelled growth curve, the countries were classified according to whether they showed initial sign of containment or not. in line with the objectives of the study, classifiers were built based on a set of independent variables to predict if a country that has covid-19 infections showed early signs of infection containment as a reflection of policy implementations and behaviour changes. logistic regression was used to understand the list of independent variables significantly affecting the infection containment and their corresponding importance in the model. then, to predict signs of early containment, machine learning algorithms like logistic regression, decision trees, random forest and support vector machines were used and their corresponding accuracies are compared. for all models, cross-validation was done in 5 folds to address overfitting. logistic regression by le cessie and van houwelingen, (1992) is a generalized linear model (glm) and is one of the most widely used classifiers. according to (kondofersky and theis, 2018) , when there is binary response, as in this research, by using logistic regression one typically aims at estimating the conditional probability . as with simple linear regression, bearing equation = + , estimating the dependent variable y directly, the logistic regression estimates p( = 1) using the following equation. this article is protected by copyright. all rights reserved as with multiple linear regression, logistic regression can also handle multiple independent variables and its probability estimate can be represented as follows. = 1 1 + −( 1 1 + 2 2 + 3 3 ……+ the conditional probability ( = 1| = ) can be calculated using the odds ratio /(1 − ). the significance of the beta coefficient values ( 1 , 2 , 3 , … , ) in the above equation can be tested to see if their corresponding independent variables ( 1 , 2 , 3 , … , ) are influencers of the dependent variable. a wald test is generally conducted to evaluate the statistical significance of the coefficients in the model. since logistic regression falls under the category of glm, the significance of each independent variable in predicting the outcome of the dependent variable, sign of early containment, can be studied. a decision tree is a decision support model that illustrates the consequences, chance, and event outcomes of certain decisions. decision trees are used as a predictive model to make statistical conclusions about an item's target value, based on observations. in this tree structure, leaves represent class labels and branches represent conjunctions of features that lead to those class labels. there are both classification trees where the response variable takes on a set of categorical values and regression trees where the response variable takes on a set of continuous values. the collective name for such trees is classification and regression trees (cart), first introduced and developed by (breiman et al., 1984) in classification and regression trees. decision trees use two metrics namely entropy and information gain to arrive at the final tree. entropy is the measure of the total amount of uncertainity in the dataset and is given as follows: s -the data set for which entropy is to be calculated cset of classes in the data set s p(c)ratio of number of elements in class c to the number of elements in set s this article is protected by copyright. all rights reserved when the entropy value is equal to zero, the dataset s is perfectly classified. the information gain metric is defined as the measure of the difference in the entropy from before to after the dataset s is split based on an atribute a and is given as follows. step 1: compute entropy for the dataset step 2: for every feature in the dataset, compute the following i. calculate the entropy for all the categorical values ii. find the average information entropy for current attributes iii. calculate the gain for curret attributes step 3: select the attribute with the highest gain step 4: repeat from step 1 till the desired tree is achieved introduced by (breiman, 2001) , random forest is a statistical supervised machine learning technique that we used for both regression and classification. this is an ensemble learning technique that uses an averaged combination of many decision trees for the final prediction. the technique of averaging a statistical machine learning model is called bagging and it improves stability and avoids overfitting (hastie et al., 2009) . normally, decision trees are not competitive to the best-supervised learning approaches in terms of prediction accuracy since they tend to have high variance and low bias. this is because building two different decision trees can yield in two different trees. bagging is therefore well suited for this article is protected by copyright. all rights reserved decision tress since it reduces the variance. the idea behind random forests is to draw bootstrap samples from the training data set and then build several different decision trees on the different training samples. this method is called random forest because it chooses random input variables before every split when building each tree. by doing this, each tree would have reduced covariance, which, in turn, would lower the overall variance even further (hastie et al., 2009) . the random forest algorithm has two stagesrandom forest creation followed by random forest prediction. the steps involved in the stages are as follows. step 1: randomly select 'k' features from the total 'm' features available in the dataset where k << m step 2: using the best split point, calculate the node 'd', among the selected 'k' step 3: split the node into daughter nodes using the best split step 4: repeat steps 1 to 3 until 'l' nodes are reached step 5: repeat steps 1 to 4 for 'n' number times to create a forest of 'n' number of trees stage ii: random forest prediction step 1: using the features and applying the rules of randomly selected decision tree, predict the outcome and store it as a predicted target step 2: calculate the votes for each predicted target step 3: the highest voted predicted target will be the prediction of the random forest algorithm the objective of support vector machine (svm) is to find a line that best separates the data into multiple groups. this is achieved by an optimization process supported by the data in the training set. these instances are called support vectors and they form a crucial role in the classification process (flake and lawrence, 2002) . finally, few datasets can be separated with just a straight line. sometimes a line with curves or even polygonal regions must be marked. this is achieved with svm by projecting the data into a higher-dimensional space to draw the lines and make predictions. svms calculate a maximum margin around the boundary that ultimately results in a homogenous partition. the ultimate goal is to establish a this article is protected by copyright. all rights reserved margin as wide as possible. in order to so, a lagrange multiplier has to be constructed as follows and maximized. ( , ) = + table 3 shows the result for logistic regression with early containment as the dependent variable. of all the independent variables, the availability of beds in hospitals and the percentage of lockdown days significantly and positively affected the signs of early this article is protected by copyright. all rights reserved containment. other variables did not significantly influence the dependent variables. the model had an accuracy of 78.57% in the classification. the true positive and false negative rates were found to be 78.6% and 21.6% respectively. precision and recall values were 0.788 and 0.786. the f1 score and roc values were found to be 0.786 and 0.755 respectively. a j48 decision tree was constructed for predicting early infection containment with the independent variables listed in figure 1 . the batch size was set to 10 and a confidence factor was selected as 0.25. the minimum number of objects on the tree was set as 2. the accuracy of the tree was found to be 80.95%. the variables in the decision tree were percentage lockdown days, days since official lockdown, and death rate per million population. the decision tree is shown in figure 4 . the true positive and false negative rates were found to be 81% and 25.4% respectively. precision and recall values were 0.857 and 0.81. the f1 score and roc values were found to be 0.796 and 0.852 respectively. a random forest ensemble algorithm was created with 100 combined trees. the batch size was selected as 10 and the depth of the trees was set to unlimited. other metrics for the random forest algorithm are given in table 4 . this model reported a high accuracy figure of 92.9% in correctly classifying countries that showed signs of early containment. the true positive and false negative rates were found to be 92.9% and 8.1% respectively. precision and recall values were 0.929 and 0.929. the f1 score and roc values were found to be 0.928 and 0.993 respectively. in order to make predictions for signs of early containment, an svm was modelled this article is protected by copyright. all rights reserved on 5-fold cross-validation with the data for all the algorithms and models, it can be inferred that the random forest design produces the minimum error and maximum accuracy as reported in table 4 . it outshines all the other machine learning algorithms constructed in the study. j48 decision tree, logistic regression and svm produce almost similar levels of accuracy in predicting the sign of containment of covid-19. this research is one of the first of its kind to integrate exponential growth modelling with machine learning techniques for predicting the spread of covid-19. the research presents machine learning models based on variables such as infrastructure, environment, policies, and the infection itself, to predict early signs of containment in the country. for the purpose, disease data from 42 leading countries in covid-19 infections were taken and exponential growth modelling was used to see if the countries showed signs of containment. then with the sign of the early containment of the infection as a dependent variable, supervised machine learning predictive models including logistic regression, decision tree, random forest, and support vector machine were developed. this research can directly be of use to countries and policymakers to understand if their proposed interventions are effective in containing infections even during early stages. (tosepu et al., 2020; yueling ma et al., 2020) . however, the long-term effect of environmental factors on the infection rates may prove to be significant. decision tree analysis also shows that early signs of containment are possible if the number of lockdown days is at least 33.7% of the days since the first contact to contain the infection. if that is not the case, countries show recovery signs if the lockdown is at least 10 days or more. for countries with a lockdown period less than 10 days, variable depicting the number of deaths per million population plays a significant role in containing the infection. this variable is indirectly related to the health care infrastructure of countries like beds, physician, ventilators, icus etc. hence in any pandemic situation, governments must be proactive and frame policies even at the onset, thereby reducing the risk of spread, which would ultimately lead to early containment. this also emphasises on the need for resilient health care infrastructure to contain infections at an early stage. the machine learning models random forest and support vector machines were able to classify the countries with respect to their signs of early containment with an accuracy of 92.9 and 76.2 percentages, respectively, proving random forest to be the best machine learning algorithm for the problem studied. although this research applies data from only 42 countries, the proposed models with their corresponding hyper parameters can be extended to predict early containment for the other countries as well. similarly, although these models were built only for the covid-19 pandemic, they can serve as a base for other future pandemics that have similar characteristics and reproduction numbers thereby giving governments the necessary information to take timely actions to protect both people and the economy. this article is protected by copyright. all rights reserved act called covid-19 act which has proven to be effective to contain the infection (library of congress, 2020). the number of hospital beds per 1000 population of austria was also high, which facilitated early recovery. chile has implemented sanitary barriers and intense screening mechanisms to track and quarantine the infected (us embassy, 2020). in addition to tough quarantine measures, denmark closed down schools and also announced lockdown in march. employers were also instructed to not cut salaries of the employees on quarantine thereby encouraging social distancing and hence containing the infection (carstensen, 2020a) . japan, south korea, and singapore did not announce any lockdowns. south korea used processes that led to early detection of the covid-19 and quarantining the infected, thus stopping spread. they also predicted the movement of viruses and tactical interventions were taken to minimize spread (npr, 2020). singapore had a ready infrastructure with isolation wards in place during the sars outbreak and was readily equipped, which led to early containment of covid-19. strong community engagement messages and communications from the government also led to better pandemic management in singapore (fisher, 2020a) . most other countries that showed early signs of recovery rigorously followed lockdowns, social distancing, travel restrictions, and testing to contain infections. another reason for the countries like japan, korea and austria to contain the infection was the presence of availability of strong health care infrastructures in these countries to address the infections. the various actions taken by the government to control the transmission of covid-19 are shown in table 5 . countries like italy, brazil, india, malaysia, pakistan, united kingdom etc. do not have the necessary health care infrastructure to support mass admission of covid-19 patients and hence need to rely on intense lockdowns to contain the infections. the increase in the number of covid-19 cases in the us and the inability to contain it is also due to late lockdown decision of the government post-outbreak. the percentage of lockdown days since the first infection continues to be low for these countries to be on a recovery path against the infection. with time, there is a high probability that the infection will be contained. however, in the long run, these countries must invest in improving health care facilities to reduce causalities during pandemics. countries must be prepared for epidemics and pandemics and proactive policies and infrastructure as in the case of singapore can save more lives than reactive measures. it is evident that covid-19, unlike sars, will not be controlled by environmental factors and any future outbreaks will still rely on healthcare infrastructures, timely lockdowns, and social distancing for containment. this article is protected by copyright. all rights reserved there is no conflict of interest with the authors. no funding received. the data is openly available in world health organisation report. the research confined to the highest level of ethics. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved gesley, 2020) brazil employees at the airport were asked to wear a mask. borders were closed for flights from affected countries (cdcp, 2020) canada all travellers were forced to self-isolate for 14 days upon entry to control the outbreak (gc, 2020) chile screening in the airport was enhanced and people with symptoms were iran followed strict social distancing and lockdown (duddu, 2020) ireland invested in massive testing facilities. treated all patients equally irrespective of their income strata. all hospitals operated on a not for profit basis (bbc, this article is protected by copyright. all rights reserved 2020) used technology to track the movement of infected individuals with their mobiles and quarantined the people who came in contact with the individual (lomas, 2020) italy though italy closed borders during the onset, lack of proper testing facilities caused a massive outbreak. this was followed by a strict lockdown (gary, 2020) japan managed the outbreak with rules and excellent medical infrastructure. (japan, 2020) luxembourg quarantined people over 60 years to reduce casualties (piscitelli, 2020) malaysia the banned entry of people from infected countries followed with additional screening measures in the airport. promoted personal hygiene and eventually ended with a lockdown. (world, 2020a) netherlands travellers returning from affected countries were advised to visit doctors and medical facilities if symptoms were felt. post outbreak, the country went under lockdown. (world, 2020b) norway travel bans and closure of schools, public services like gums, malls, theatres etc. (norway panorama, 2020) formed a team to monitor situations and take necessary actions on a daily basis. (pakistan, 2020) portugal employed strict lockdown (ivo oliveira, 2020) qatar proper tracking, and strict screening and testing of travellers (health, 2020) republic of korea proactively built a centralized testing and quarantine facility before an outbreak in the country. china's reports triggered this action (beaubien, 2020) romania lockdown and border closing (gherasim, 2020) singapore with previous experience from sars pandemic, the country had a proper infrastructure facility with negative pressure room for pandemic control. the testing was done rigorously and the infected were not let back into society. migrants from other countries were not allowed to work until the pandemic is controlled. (fisher, 2020b) slovenia used innovative ways to spread covid-19 control messages before going into lockdown. (slovenija, 2020) south africa immediately implemented entry and exit to affected countries. declared as a this article is protected by copyright. all rights reserved national disaster and went for the lockdown to prevent a major outbreak (fihlani, 2020) spain local movement controlled by social distancing. travel to an affected country completely banned. enhanced medical attention at arrival to control the spread. (kate mayberry, 2020) after closing school, colleges and non-essential business, the country used their military and civilian support to enhance infrastructure and healthcare needs to contain the infection (keystone, 2020) the united kingdom people with symptoms were asked to self-quarantine. cancelled overseas travels and only tested people who were admitted. followed social distancing, lookdown, isolation and house quarantined. the country did not force people for testing. (yong, 2020) united states of america enforce travel restriction and implemented mandatory quarantine in new york. a level of screening and lockdown was implemented. 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coronavirus outbreak may have the opposite effect to what ' s needed 2020: tthe atlantic, the u.k.'s coronavirus 'herd immunity' debacle effects of reactive social distancing on the 1918 influenza pandemic effects of temperature variation and humidity on the death of covid-19 in sars-cov-2 turned positive in a discharged patient with covid-19 arouses concern regarding the present standard for discharge 2020: estimating the effective reproduction number of the 2019-ncov in china this article is protected by copyright. all rights reserved key: cord-331932-oujdl459 authors: lung, o.; ohene‐adjei, s.; buchanan, c.; joseph, t.; king, r.; erickson, a.; detmer, s.; ambagala, a. title: multiplex pcr and microarray for detection of swine respiratory pathogens date: 2015-12-12 journal: transbound emerg dis doi: 10.1111/tbed.12449 sha: doc_id: 331932 cord_uid: oujdl459 porcine respiratory disease complex (prdc) is one of the most important health concerns for pig producers and can involve multiple viral and bacterial pathogens. no simple, single‐reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with prdc. furthermore, the detection of most of the bacterial pathogens implicated in prdc currently requires time‐consuming culture‐based methods that can take several days to obtain results. in this study, a novel prototype automated microarray that integrates and automates all steps of post‐pcr microarray processing for the simultaneous detection and typing of eight bacteria and viruses commonly associated with prdc is described along with associated multiplex reverse transcriptase pcr. the user‐friendly assay detected and differentiated between four viruses [porcine reproductive and respiratory syndrome virus (prrsv), influenza a virus, porcine circovirus type 2, porcine respiratory corona virus], four bacteria (mycoplasma hyopneumoniae, pasteurella multocida, salmonella enterica serovar choleraesuis, streptococcus suis), and further differentiated between type 1 and type 2 prrsv as well as toxigenic and non‐toxigenic p. multocida. the assay accurately identified and typed a panel of 34 strains representing the eight targeted pathogens and was negative when tested with 34 relevant and/or closely related non‐target bacterial and viral species. all targets were also identified singly or in combination in a panel of clinical lung samples and/or experimentally inoculated biological material. the global pig industry produced approximately 963 million pigs and 109 million metric tons of pork in 2011 (http://faostat.fao.org). respiratory diseases are considered to be one of the main contributors to economic losses in the swine industry (opriessnig et al., 2011) . the 2006 united states department of agriculture (u.s.d.a.) national animal health monitoring system (nahms) study of 435 swine production sites with 100 or more pigs from 17 major pork-producing states showed that respiratory problems are the main cause of nursery deaths (53.7%) and grower/finisher pig mortality (60.1%) (united states department of agriculture, 2008) . in canada, 37-78% of pigs going for slaughter have cranioventral bronchopneumonia (hansen et al., 2010a) . porcine respiratory disease complex (prdc) is multifactorial, with both infectious and non-infectious factors contributing to respiratory disease and predominantly seen in pigs between the ages of 3 and 6 months (opriessnig et al., 2011) . the interaction of viral and bacterial pathogens, environmental factors, pig-specific factors and management conditions all contribute to the development and impact the severity of prdc (opriessnig et al., 2011) . the type of pathogens involved in prdc is specific to the regions and countries where production occurs (opriessnig et al., 2011) . however, viruses most commonly associated with prdc include porcine reproductive and respiratory syndrome virus (prrsv) (rammohan et al., 2012) , porcine circovirus type 2 (pcv2) (ellis et al., 2004; genzow et al., 2009) , influenza a virus (iav) and porcine respiratory corona virus (prcv) (pensaert et al., 1986; jung et al., 2009; renukaradhya et al., 2010) . bacteria such as mycoplasma hyopneumoniae (m. hyopneumoniae) (hansen et al., 2010b) , pasteurella multocida (p. multocida) (davies, 2004) , salmonella enterica serovar choleraesuis (s.e choleraesuis) (reed et al., 1986; asai et al., 2010) and streptococcus suis (s. suis) (done and paton, 1995; silva et al., 2006; baums et al., 2007) are also commonly associated with prdc. porcine reproductive and respiratory syndrome virus is a major cause of swine production losses worldwide, and in the united states, reproductive and growing pig losses are an estimated $560 million per year (neumann et al., 2005) . prrsv (genus arterivirus, family arteriviridae) is an enveloped virus with a single-stranded, positive-sense rna genome of approximately 15 kb. prrsv is classified into two types with type 1 predominating in europe and type 2 predominating in north america and asia. pcv2 (genus circovirus, family ciroviridae) is a non-enveloped virus with a circular, covalently closed single-stranded dna genome of 1767-1768 nucleotides (meehan et al., 1997; hamel et al., 1998) . iav (genus influenzavirus a, family orthomyxoviridae) are enveloped viruses with a genome composed of eight single-stranded negative-sense rna segments. prcv (genus alphacoronavirus, family coronaviridae) are enveloped viruses with a single-stranded positivesense rna genome of approximately 28.5 kb. it was first isolated in belgium in 1984, and it is a natural variant of transmissible gastroenteritis virus (tgev) (pensaert et al., 1986 ) that contains a 5 0 deletion (621-681 nt in size) in the s gene which is used to differentiate prcv from tgev in pcr-based assays (kim et al., 2000; costantini et al., 2004) . prcv often causes subclinical infections. m. hyopneumoniae (hansen et al., 2010b) causes mycoplasmal pneumonia of swine and is known to be one of the most common and economically important diseases found in pig farms worldwide, having low mortality, but high morbidity (otagiri et al., 2005) . p. multocida is a commensal found in the upper respiratory tract of pigs, but can also act as a primary or secondary pathogen responsible for pneumonia (davies, 2004) and atrophic rhinitis in pigs . s. e. choleraesuis is a host adapted salmonella serovar responsible for almost all types of salmonellosis in pigs in north america and europe (kingsley and baumler, 2000) . s. suis is a gram-positive, zoonotic bacterial pathogen important in polyserositis, septicaemia, arthritis, pneumonia and endocarditis in pigs (done and paton, 1995; silva et al., 2006; baums et al., 2007) . some of these bacteria are of low pathogenicity and exist as commensals in the upper respiratory tract of healthy pigs. they can cause severe respiratory disease by invading tissues already damaged due to a primary pathogen(s) (virus or bacteria), general immunosuppression and other factors such as poor environmental conditions and poor management practices. routine diagnostic methods for detection of viruses implicated in prdc include virus isolation in cell culture, antigen detection by direct fluorescent antibody staining, and enzyme immunoassay and culture-based methods for bacteria. such methods (grau-roma and segales, 2007) are time-consuming and require independent tests for each pathogen. furthermore, the detection of bacterial pathogens typically depends on time-consuming culture-based methods that can take several days to obtain results. due to their high sensitivity and ease of use, pcr and real-time pcr tests have been developed for several agents implicated in the prdc; however, these tests typically target single pathogens (lierz et al., 2008; lomonaco et al., 2009; tang et al., 2009) . a diagnostic test capable of simultaneous detection of multiple pathogens involved in prdc (atashpaz et al., 2009; wernike et al., 2012; xu et al., 2012) would save time, labour and cost by providing more information with each test performed. a multiplex pcr assay capable of detecting five porcine viruses including two porcine respiratory viruses was developed (giammarioli et al., 2008) . duplex and triplex real-time pcr for porcine respiratory viruses have also been recently described (chang et al., 2014; wu et al., 2014) . however, to date, there are no diagnostic tests capable of simultaneous detection of multiple major viral and bacterial porcine respiratory pathogens in a single reaction. microarray technology, with its capacity to incorporate a large number of capture probes, is a potentially useful tool for multiplexed detection and typing of pathogens. here, a microarray assay with associated multiplex rt-pcrs for detection and differentiation of four viruses and four bacteria involved in prdc using a novel user-friendly electronic microarray in which capture probe printing, hybridization, washing and reporting are fully integrated and automated is described. the electronic microarray contains 400 test sites which can be activated independently via electrodes and utilizes electrophoretically driven hybridization that can be completed instantaneously. databases containing all available full and partial sequences of the genomic regions encoding the matrix proteins of iav (n = 4373) and prrsv (n = 1829), prcv spike protein (n = 24), and pcv2 capsid protein (n = 2048) were compiled from the national centre for biotechnology information (ncbi). similarly, databases were compiled for the kmt1 (n = 16) and toxa (n = 50) genes of p. multocida, sly (n = 106) and orfb (n = 48) genes of s. suis, a 640 bp portion of intergenic region ig-070/071 (gardner and minion, 2010) of m. hyopneumoniae (n = 6) and a 708 bp region between open reading frames sc4343 and sc4353 previously identified as a metabolic island for s. e. choleraesuis (n = 1). sequences were retrieved by searching ncbi's 'nucleotide' database using the gene names as keywords, as well as performing blast homology searches (altschul et al., 1990 ) with a representative sequence for each target, and downloading the aligned portion of all blast hits. redundant sequences were removed based on accession numbers. multiple sequence alignments for each genetic target were generated with clustalx v. 2.0 ( thompson et al., 1997; larkin et al., 2007) or mafft v. 7.0 (katoh and standley, 2013) using default settings. databases were maintained with either mega4 (tamura et al., 2007) or bioedit sequence alignment editor v. 7.1.9 (hall, 1999) to ensure that all sequences were correctly oriented and aligned. similarly, representative whole-genome sequences, as well as full and partial sequences of homologous genes from related and unrelated non-targets such as tgev, porcine circovirus type 1 (pcv1), as well as other salmonella enterica serovars, and mycoplasma species were downloaded for in silico analysis of probe specificity. several published pcr primers suitable for the assay were adopted from the literature (table 1) . additional pcr primers (table 1 ) and all target-specific capture probes (table 2) were designed using either alleleid (premier biosoft international, palo alto, ca, usa) or bioedit sequence alignment editor v. 7.1.9 (hall, 1999) based on the databases described above. primers and probes were designed to be 18-25 bp in length, with the melting temperatures between 54 and 65°c and minimal secondary structures (dg ≥ à8.0 kcal/mol). primers and probes identified by each software were compiled and examined in silico for specificity by blast (altschul et al., 1990) analysis using custom inhouse databases containing representative whole-genome sequences, as well as full or partial sequences of homologous genes from related and relevant non-targets. primers or probes that showed significant homology to closely related or unrelated non-target species were excluded from further investigation. a list of the viruses, bacteria and clinical samples used in this study is presented in table 3 . the yield of the rneasy mini kit (qiagen, mississauga, on, canada), qiaamp viral rna mini kit (qiagen) and magmax total nucleic acid kit (ambion, austin, tx, usa) was evaluated with pcv2 (a non-enveloped dna virus) and prrsv (an enveloped rna virus) as per the manufacturers' recommendation. for these viruses, 100 ll of a quantified stock was serially diluted (10 à2 to 10 à6 ) in swab material from 2-dayold piglets (prairie swine centre, saskatoon, sk, canada), previously tested to be negative for the targets. similarly, the ultraclean tissue and cells dna isolation kit (mobio laboratories, carlsbad, ca, usa), dneasy blood and tissue kit (qiagen) and magmax total nucleic acid kit were tested to evaluate the nucleic acid extraction efficiency of a gram-positive bacteria (s. suis) and a gram-negative bacteria (p. multocida). for these bacteria, 100 ll of culture of known cfu/ml was serially diluted in the same swab material as above and extracted using each kit in parallel according to the manufacturers' instructions. the efficiency of the extraction kits was evaluated based on the limits of detection observed after rt-pcr amplification of the extracted material. the dneasy blood and tissue kit and the viral rna mini extraction kit were the most efficient for the tested bacteria and virus targets, respectively (data not shown). therefore, all subsequent nucleic acid extractions of laboratory amplified strains were performed using these kits. following preparation of nucleic acid extractions, the samples were subjected to pcr and microarray analysis. for the determination of the analytical sensitivity of the assay for viral targets, selected genes were amplified using the superscript tm iii one-step rt-pcr system with platinum â taq dna polymerase (life technologies, carlsbad, ca, usa) and cloned into the pjet1.2 cloning vector using the clonejet pcr cloning kit (fisher scientific, ottawa, on, canada) according to the manufacturers' specifications. plasmids were extracted from successfully transformed bacteria using the qiaprep miniprep kit (qiagen) according to the manufacturer's specifications and were confirmed by sequencing (eurofins genomics, huntsville, al, usa). vectors containing the target genes for each virus, except for pcv2, were linearized with the hind iii restriction enzyme (fisher scientific) and subjected to in vitro transcription using the megascript t7 transcription kit (life technologies) according to the manufacturer's specifications. template dna was eliminated using successive treatments with turbo dnase (life technologies,) before quantifying the rna using the rna br assay kit and the qubit 2.0 fluorometer (life technologies) according to the manufacturers' specifications. the rna was then serially diluted 1 : 10 in ultrapure distilled water (life technologies). the copy number was inferred using an online tool (http://endmemo.com/bio/dnacopynum. php) taking into account the nucleic acid concentration and nucleotide composition of the amplified region of each target. the copy number for the plasmid containing pcv2 capsid protein coding region was inferred based on the nucleic acid concentration and nucleotide composition over the entire plasmid. for the determination of the analytical sensitivity of the assay for bacterial targets (excluding m. hyopneumoniae), frozen cultures were streaked for single colonies onto 5% sheep blood agar plates (bbl tm blood agar base infusion agar; bd diagnostics, sparks, md, usa) and incubated at 37°c overnight. a single colony was inoculated into 5 ml of miller's lb broth (life technologies) and grown overnight at 37°c on a shaking incubator (150 rpm). the overnight lb broths were serially diluted 1 : 10 in pbs, and 100 ll of material was spread onto blood agar plates in triplicate and grown overnight at 37°c for enumeration using the viable plate count method. the cultures were standardized to 3.33 9 10 8 cfu/ml (s. e choleraesuis and p. multocida) and 3.33 9 10 6 cfu/ml (s. suis), so a 30 ll aliquot from each serial dilution in the series yielded cfus to the nearest power of base 10 (i.e. 1 9 10 6 , 1 9 10 5 , etc.). for m. hyopneumoniae, an aliquot of genomic dna was quantified on the qubit 2.0 fluorometer, and a genomic copy number was inferred based on the nucleic acid continued) concentration and nucleotide composition over the entire genome. the analytical specificity of the viral and bacterial multiplex pcr assays was assessed by amplifying panels of 14 non-target viruses and 21 bacteria, respectively (table 3) . the forward primers were modified with 5 0 -phosphorylation (idt, coralville, ia, usa). all reverse primers were modified with either 5 0 -tye665 â fluorophore using spc3 â attachment chemistry for the slide microarray or were synthesized with the reverse complementary sequence of the reporter probe at the 5 0 end for the electronic microarray as described in lung et al. (2012) . rt-pcrs were performed using the superscript tm iii one-step rt-pcr system with platinum â taq dna polymerase. a multiplex rt-pcr with 10 primers targeting the genomic regions encoding the iav and prrsv matrix proteins, prcv spike protein, pcv2 capsid protein, as well as an internal control, was developed (table 1) . a multiplex pcr with 14 primers targeting the kmt1 and toxa genes of p. multocida, sly and orfb genes of s. suis, intergenic space of m. hyopneumoniae, metabolic island of s. e. choleraesuis and an internal control was developed (table 1) . a plasmid containing a fragment of the dengue virus genome was used as an internal pcr control for both the bacterial and viral rt-pcrs. both assays were optimized for buffer and magnesium concentration, annealing temperature, cycle number and internal control concentration. the finalized rt-pcr mixtures consisted of 1 ll of nucleic acid, 0.01 pg internal control, 1 ll of enzyme mix, 1 lm of each primer in 19 reaction buffer in a final volume of 25 ll. reverse transcription was carried out for 15 min at 60°c, followed by 94°c for 2 min. pcr was carried out for 35 cycles of 94°c for 30 s, 50°c for laboratory samples and 58°c for clinical samples for 15 s, 68°c for 45 s, with a final extension step of 68°c for 7 min. following pcr, unpurified material was assayed on the qiaxcel capillary gel electrophoresis system (qiagen) for visualization of amplicons. analytical sensitivity of each multiplex assay was determined using serial dilutions of quantified dna or reverse-transcribed rna as appropriate for each pathogen. the serial dilutions were amplified using the multiplex pcr assays as well as the singleplex pcr for each target. all pcr amplifications were carried out on the veriti thermocycler (life technologies) and visualized on the qiaxcel using the biocalculator v. 3.0 software (qiagen). the limit of detection was considered to be the last dilution where amplification was greater than the default threshold on the electropherogram and described in terms of the approximate total number rna or dna copies in the sample. a total of 30 probes for the detection of four target viruses, 25 probes for the detection of four bacterial targets and three control probes were initially screened by passive hybridization on low-cost conventional epoxy glass slide microarrays (corning, corning, ny, usa) that were printed and processed in-house according to protocols described previously (lung et al., 2011) . the probes were screened against a panel of five isolates representing the four target viruses, and three non-target viruses, and 11 strains representing the four target bacteria, and 11 non-target bacteria (table 3) . microarray data were represented using the mean pixel intensity for each probe reaction. probe reactivity was calculated using the mean pixel fluorescent intensity (mfi) of all probes as a ratio of the non-template control. probe reactions above 29 the ratio of the non-template control were considered positive. probes that showed good reactivity and specificity were selected for testing and validation on a novel automated electronic microarray in which capture probes are printed on streptavidin-containing acrylamide hydrogels and hybridization, washing and reporting are automated and computer controlled. capture probes used on the electronic microarray were modified with 5 0 -biotin group to allow attachment to streptavidin-containing test sites (idt, coralville, ia, usa). a selected set of 12 probes targeting the viruses, 14 probes targeting the bacteria and three control probes (a negative probe and two probes targeting the internal control), which exhibited high reactivity and specificity on glass slide microarrays, were selected for validation on the electronic microarray. the viral probes were tested against an expanded panel of 14 strains or isolates of the four target viruses and 14 non-target viruses (table 3) . similarly, the bacterial probes were tested against a panel of 20 strains or isolates representing the four target bacteria and 20 related or unrelated non-target bacteria ( table 3 ). the electronic microarray assays were run using a protocol previously described (lung et al., 2012) with modifications. the modifications included the replacement of the 'touch down' washing protocol with a 'touch up' protocol in which washing steps were carried out using low salt buffer (nexogen, inc., san diego, ca, usa) with incremental increases rather than decreases in temperature. images were captured at each temperature increment. the red universal reporter probe was replaced with a 5 0 -alexa fluor 647 modified locked nucleic acid (lna) variant (5 0 -/5alex647n/tgt+ca+agc-g+at+at+act+gc-3 0 ) (idt, coralville, ia, usa) to increase its thermal stability over a more robust range of wash temperatures. all electronic microarray hybridizations were performed in duplicate, and a non-template pcr control (ntc) was included in all experiments. raw fluorescent intensity (fi) data from all utilized electrodes at each temperature increment were obtained and analysed using microsoft excel. for each probe, positive-to-non-template control (p : n) ratios were calculated by dividing the fi value from each analyte by the fi value produced by the ntc. for each assay, samples that produced p : n ratios of 2.0 or greater were considered positive. average p : n data derived from the microarrays were visualized with a heat map generated using treeview v. 1.60 (eisen et al., 1998) . oral and nasal swabs were obtained from specific pathogen free pigs at the cfia ottawa laboratory fallowfield, ontario, canada. oral and nasal swabs were screened for the target viral and bacterial pathogens, and pools of oral and nasal material that were negative for the target bacteria and viruses were used for spiking with target pathogens. bacterial strains were grown and quantified as described above, and supernatants containing virus from cell culture were used. for m. hyopneumoniae, culture was not performed and a freeze-dried cell pellet purchased from atcc was used after re-suspension in pbs and 60% glycerol. for inoculations with single agents, 120 ll aliquots of oral and nasal samples were experimentally inoculated with 20 ll of each live virus or bacteria. samples inoculated with multiple agents were prepared by pooling 20 ll of each pathogen together, and adding 20 ll of the pooled pathogens into 120 ll of oral and nasal material (table 4 ). nucleic acid from the full 140 ll volume of the samples was extracted, pcr amplified and assayed on the electronic microarray as described in previous sections. approximately 30-40 mg of a panel of lung tissue submitted in 2015 to manitoba agriculture, food and rural initiatives (mafri) for diagnosis of porcine respiratory pathogens was ground in 1.5 ml of rlt buffer (qiagen), transferred to 400 ll of magmax lysis buffer and processed for nucleic acid extraction in the kingfisher 96 instrument (ambion). five to 8 ll of extracted rna from 90 ll of elution buffer was tested by real-time rt-pcr at mafri, and 5 ll was tested at cfia by electronic microarray. multiplex pcr/rt-pcr two separate multiplex pcrs were developed for amplification of selected genes of four viruses and four bacteria involved in prdc, respectively. the multiplex pcr for bacteria consisted of a total of 12 primers for the detection of the four target bacteria, typing of p. multocida and a pair of primers for an internal control (table 1) . two genes from s. suis and p. multocida were each targeted for amplification by the multiplex pcr. amplification and detection of the toxa gene of p. multocida allowed for differentiation of virulent or pathogenic strains from non-pathogenic strains. primers for the orfb gene were added to a previously designed multiplex pcr with primers for the sly gene to allow detection of s. suis strains that lack the sly gene. the multiplex pcr generated products of the expected sizes ranging from approximately 205-708 bp (table 1) when a panel of 23 isolates representing the target species, including different serotypes of s. suis were tested (fig. 1) . similarly, a multiplex rt-pcr with 10 primers was developed and used to detect the four viruses and an internal control. the multiplex rt-pcr successfully amplified a panel of 14 targeted viruses and generated amplicons of the expected size of approximately 229-534 bp ( table 1) . the samples represented both genotypes of prrsv, as well as different subtypes of iav (fig. 1) . the internal control variably amplified in both the bacterial and viral multiplex pcrs as a result of competitive pcr. in instances where targets were strongly amplified, amplification of the internal control was either weak or absent. conventional glass slide microarrays were processed manually as an initial low-cost screening tool to assess the specificity of the probe set (n = 30) designed to detect the four target viruses, distinguish between genotypes 1 and 2 of prrsv, as well as differentiate prcv and tgev, and the probe set (n = 25) designed to detect the four target bacteria and differentiate toxigenic and non-toxigenic strains of p. multocida. a selected set of 12 probes targeting the viruses, 14 probes targeting the bacteria and three control probes (a negative probe and two probes targeting the internal control), which exhibited the highest reactivity and specificity on slide microarrays, were selected for validation on the user-friendly automated electronic microarray. all target viruses and bacteria were accurately detected, and prrsv and p. multocida were accurately typed using the viral and bacterial probe set on the electronic microarray platform (fig. 2) . the assay also successfully detected targeted pathogens in clinical lung specimens, as well as porcine oral and nasal swab material experimentally inoculated with single or multiple targets (table 4 ). the results obtained were consistent with those obtained by singleplex assays with the exception of lt-7 which was positive for p. multocida based on the electronic microarray assay, but negative by bacterial culture. for both the rt-pcr and microarray assays, the analytical sensitivity varied with the different targets. for some targets, the multiplex assay had comparable sensitivity with the respective singleplex assay, while for other targets, the singleplex assays were more sensitive ( table 5 ). the multiplex assay was most sensitive for detection of iav and s. suis for viral and bacterial targets, respectively (table 5) . non-target bacteria samples did not react with the probes on either the conventional slide microarray or electronic microarray. other than tgev, a natural variant and highly related virus to prcv, no other non-target viruses showed amplification in the virus multiplex rt-pcr (data not shown). due to the strong amplification of tgev by the multiplex rt-pcr, the internal control failed to amplify (fig. 2b, sample 21) . however, prcv and tgev were differentiated based on amplicon size, and in subsequent microarray characterization (fig. 2 ). a user-friendly microarray for the simultaneous detection and differentiation of four viruses and four bacteria associated with prdc was developed as a test case for a novel automated 'amplicon-to-answer' electronic microarray technology. the electronic microarray integrates and automates all post-pcr steps required for microarray analysis (capture probe printing, hybridization, washing, reporting) and allows for simultaneous identification of eight pathogens, differentiation of the two prrsv genotypes and pathogenic versus non-pathogenic p. multocida strains. although the amplification of bacterial dna did not require a reverse transcriptase phase, an rt step was included in the pcr protocol as the amplicon yield was better than without the rt step. using the same protocol would also allow potential combination of the bacterial and viral multiplex pcr into a single multiplex pcr targeting all eight pathogens. a likely explanation for the increased amplification yield observed with rt-pcrs for bacterial targets is rt-pcr could utilize not just genomic dna, but also rna transcripts of target genes as template. in addition, the proprietary quantity of taq polymerase in the rt-pcr kit that was used in the rt-pcrs may be higher than that used in the pcr. initial screening of capture probes was performed using a conventional slide microarray platform due to the lower cost of screening large number of probes that were printed in conjunction with other projects. subsequently, the assay was adapted to a novel, rapid and user-friendly microarray platform that automates and integrates capture probe printing with all post-pcr steps of the assay, including electrophoretically driven hybridization, washing and reporting. the automated electronic microarray assay reduces the labour, time and number of instruments needed to acquire microarray results when compared with conventional microarrays which typically require substantial manual processing, slower passive hybridization of amplicons with capture probes and multiple pieces of equipment. the electronic microarray has an open platform format with 400 test sites that can be individually activated and utilizes a single integrated instrument that automates capture probe printing and microarray processing using parameters set by the user. thus, the novel technology also reduces the skill level required to perform microarray assays and allows immediate, on-site modification of assays depending on the needs of the enduser. these unique features eliminate the need for anticipation of future needs, and the procurement and storage of manufactured arrays that are designed for a specific set of predetermined pathogens. for example, the system will allow immediate switching between assays that detects/ types all eight pathogens simultaneously to one that detects/types a subset of the pathogens, or to assays for detection and typing of other pathogens. as the user is able to control hybridization, wash and reporting temperature the assay can have excellent specificity and can be used for differentiating variants that are genetically very similar. however, the automated electronic microarray assay requires specialized arrays and investment in instruprobes p. multocida (toxigenic) fig. 2 . summary of microarray results from the electronic microarray representing the four bacterial targets (a), the four viral targets (b) on the electronic microarray. the reactivity of specific reactions between targets and each pathogen-specific probes is outlined in yellow. nsbp = non-specific binding probe negative control. p : n ratios ≥ 2.0 are shown in red, and p : n ratios < 2.0 are in black. ntc = no template control. ic = internal control. amplification and detection of the internal control are not always observed when a target is present in high amounts. mentation. the cost of consumables for testing a sample with an electronic microarray is expected to be between one and a few real-time pcr assays and will depend on the assay design (i.e. reducing the number of probes will decrease the cost by allowing more samples to be tested on each array with 400 test sites). in addition to glass and hydrogel (acrylamide)-based microarray matrices used in this study, the final probe sets have been tested successfully on reverse dot blots performed on nylon membranes (unpublished results). thus, the probe set described should have broad applications in hybridization-based assays that use a variety of different matrices. assays for simultaneous detection of multiple bacteria and virus implicated in prdc have not been described previously. the limit of detection of a lamp assay for pcv2 has been shown to be approximately 1 copy of dna plasmid, much more sensitive than conventional pcr whose detection limit was 1 9 10 4 copies (zhou et al., 2011) . however, lamp assays typically detect a single pathogen and are difficult to multiplex. in multiplex real-time pcr assays, a duplex pcr assay had a detection limit of 1 tcid 50 /ml for pcv2 and 6.3 tcid 50 /ml for prrsv (chang et al., 2014) , while a triplex real-time pcr assay had a detection sensitivity of 10 copies/ll for pcv2 and prsv and 100 copies/ll for ppv (wu et al., 2014) . the multiplex pcr described here did not reduce the limits of detection for five targets although the detection limit of the pcr for three pathogens was an order of magnitude lower in the multiplex format. thus, further improvements in sensitivity are desirable and may be partly achieved by increasing the number of pcr cycles, reducing amplicon lengths or reducing the amount of internal control used in the assay. the limit of detection for pcv2, prrsv, and m. hyoneumoniae was lower with the electronic microarray in comparison with the multiplex rt-pcr indicating the use of the electronic microarray platform reduces sensitivity, and unpublished results show that only amplicons that were visible after agarose gel electrophoresis can be detected by the electronic microarray. despite the reduced analytical sensitivity of the electronic microarray assay for some of the targets, the bacterial and viral targets were detected singly or in combination in clinical samples submitted for laboratory diagnosis. furthermore, the assay detected p. multocida in a clinical lung sample that was not detected by traditional culture methods. this discrepancy may be due to the higher sensitivity of the microarray assay, or the lack of active infection (e.g. the use of antibiotics). while primers and probes were evaluated using all sequences available on ncbi at the time of assay design and samples representative of both target and non-target bacteria and viruses were tested in this study, regular reevaluation of the coverage of the primers and probes and additional validation with clinical samples is desired. the four swine respiratory viruses targeted in this study have also been successfully detected with the virochip panviral array which consists of probes for detection of all known viruses at the time of design (nicholson et al., 2011) . the high-density virochip is a useful tool for virus discovery, but needs approximately 24 h to obtain results and requires high viral titre for positive detection. in contrast, the electronic microarray assay described here can be completed in less than 4 hours with little user handling plus approximately 1.5 h for the rt-pcr described. new instrumentation that further simplifies the workflow by integrating the pcr and array processes is now commercially available. to our knowledge, the automated microarray assay described here is the first one that simultaneously lipman, 1990: basic local alignment search tool molecular typing and antimicrobial resistance of salmonella enterica subspecies enterica serovar choleraesuis isolates from diseased pigs in japan rapid 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real-time pcr panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders 2012: development of multiplex pcr for simultaneous detection of six swine dna and rna viruses loop-mediated isothermal amplification for detection of porcine circovirus type 2 1.4 9 10 3 1.4 9 10 3 1.4 9 10 3 1.4 9 10 4 prcv 62 620 620 620 prrsv 3.8 9 10 3 3.8 9 10 3 3.8 9 10 4 3.8 9 10 4 iav 160 160 160 160 m. hyopneumoniae 480 480 480 4.8 9 10 3 p. multocida 1.0 9 10 3 1.0 9 10 3 1.0 9 10 3 1.0 9 10 3 s. e. choleraesuis 1.0 9 10 3 1.0 9 10 4 1.0 9 10 3 1.0 9 10 4 s. suis none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. 