Identification and Epidemiology of a Rare HoBi‐Like Pestivirus Strain in Bangladesh

The genus pestivirus of the family flaviviridae consists of four recognized species: bovine viral diarrhoea virus 1 (BVDV‐1), bovine viral diarrhoea virus 2 (BVDV‐2), classical swine fever virus and border disease virus. A new putative pestivirus species tentatively named as either ‘HoBi‐like pestivirus’ or BVDV‐3 has recently been identified in Brazil, Italy and Thailand. Despite reports of serological evidence of BVDV in Bangladesh, the types of the virus circulating in cattle have not been identified. We conducted surveillance in cattle from May 2009 to August 2010 in three government veterinary hospitals to characterize BVDV in cattle of Bangladesh. We tested serum for BVDV using an antigen‐capture ELISA. Of 638 cattle samples, 3% (16/638) tested positive for BVDV antigen. The ELISA‐positive samples were selected for further molecular detection and characterization of BVDV. Molecular analysis of the partial 5′ untranslated region (UTR) nucleotide sequences of BVDV‐positive samples identified the rare HoBi‐like pestivirus or BVDV‐3 virus circulating in cattle of Bangladesh. The identification of this rare HoBi‐like pestivirus or BVDV‐3 strain in Bangladesh warrants further surveillance to evaluate its impact on livestock production.     

Serological relationship between a novel ovine pestivirus and classical swine fever virus

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV‐1) and type 2 (BVDV‐2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross‐neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.     

Evidence of porcine circovirus‐like virus P1 in piglets with an unusual congenital tremor

Outbreaks of trembling and shaking were reported among pigs at two pig farms in Jiangsu Province, China. Serum and tissue samples tested positive for porcine circovirus‐like virus P1 and negative for classical swine fever virus, porcine circovirus type 2, astrovirus and porcine pestivirus using PCR/RT‐PCR and immunohistochemical techniques. High P1 viral genome loads were identified in sera, brain and lymph node tissue samples by qPCR. In addition, one of the most notable pathological changes was dissolution of the nucleus in Purkinje cells. The results of this study provide molecular evidence of an association between congenital tremor in pigs and P1 virus.     

Identification of Natural Infections in Sheep/Goats with HoBi‐like Pestiviruses in China

The natural infections of HoBi‐like pestiviruses in cattle have been reported in South America, Europe and Asia. In China, although the detections of HoBi‐like pestivirus have been reported, the epidemiological investigation was limited. From January 2014 to October 2015, several flocks of sheep/goats in Henan province in central China suffered respiratory diseases which were recovered slowly after antibiotics treatment. To test whether it is the HoBi‐like pestivirus caused this symptom, 49 serum samples and 22 nasal swabs were then collected for analysis by serology and RT‐PCR. Serological result revealed that prevalence of pestivirus in small ruminants was 12.2% (6/49) in central China. Sequence analysis of partial 5′‐UTR nucleotides of pestivirus‐positive samples suggested that HoBi‐like pestivirus might have circulated in sheep/goats of China for a period and have evolved into new genotype clusters. It is apparent that the study provides the molecular evidence of natural infections in goat/sheep species with HoBi‐like pestiviruses in China.     

Characterization of a Novel Chimeric Swine Enteric Coronavirus from Diseased Pigs in Central Eastern Europe in 2016

During a severe outbreak of diarrhoea and vomiting in a pig herd in Central Eastern Europe, faecal samples were tested positive for porcine epidemic diarrhoea virus (PEDV) and negative for transmissible gastroenteritis virus (TGEV) using a commercial RT‐qPCR assay that can detect both of these coronaviruses. However, further analyses, using other TGEV‐ and PEDV‐specific RT‐qPCR assays, provided results inconsistent with infection by either of these viruses. Sequencing of an amplicon (ca. 1.6 kb), generated by an RT‐PCR specific for the PEDV S‐gene, indicated a very close similarity (ca. 99% identity) to recently described chimeric viruses termed swine enteric coronaviruses (SeCoVs). These viruses (with an RNA genome of ca. 28 kb) were first identified in Italy in samples from 2009 but have not been detected there since 2012. A closely related virus was detected in archived samples in Germany from 2012, but has not been detected subsequently. Building on the initial sequence data, further amplicons were generated and over 9 kb of sequence corresponding to the 3′‐terminus of the new SeCoV genome was determined. Sequence comparisons showed that the three known SeCoVs are ≥98% identical across this region and contain the S‐gene and 3a sequences from PEDV within a backbone of TGEV, but the viruses are clearly distinct from each other. It is demonstrated, for the first time, that pigs from within the SeCoV‐infected herd seroconverted against PEDV but tested negative in a TGEV‐specific ELISA that detects antibodies against the S protein. These results indicate that SeCoV is continuing to circulate in Europe and suggest it can cause a disease that is very similar to PED. Specific detection of the chimeric SeCoVs either requires development of a new diagnostic RT‐qPCR assay or the combined use of assays targeting the PEDV S‐gene and another part of the TGEV genome.     

Evidence for Tunisian‐Like Pestiviruses Presence in Small Ruminants in Italy Since 2007

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV‐1, BVDV‐2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5′‐UTR and the whole N^pro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV‐1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian‐like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian‐like pestivirus from this species.     

Clinical Presentation Resembling Mucosal Disease Associated with ‘HoBi’‐like Pestivirus in a Field Outbreak

The genus Pestivirus of the family Flaviviridae consists of four recognized species: Bovine viral diarrhoea virus 1 (BVDV‐1), Bovine viral diarrhoea virus 2 (BVDV‐2), Classical swine fever virus (CSFV) and Border disease virus (BDV). Recently, atypical pestiviruses (‘HoBi’‐like pestiviruses) were identified in batches of contaminated foetal calf serum and in naturally infected cattle with and without clinical symptoms. Here, we describe the first report of a mucosal disease‐like clinical presentation (MD) associated with a ‘HoBi’‐like pestivirus occurring in a cattle herd. The outbreak was investigated using immunohistochemistry, antibody detection, viral isolation and RT‐PCR. The sequence and phylogenetic analysis of 5′NCR, N^pro and E2 regions of the RT‐PCR positive samples showed that four different ‘HoBi’‐like strains were circulating in the herd. The main clinical signs and lesions were observed in the respiratory and digestive systems, but skin lesions and corneal opacity were also observed. MD characteristic lesions and a pestivirus with cytopathic biotype were detected in one calf. The present study is the first report of a MD like presentation associated with natural infection with ‘HoBi’‐like pestivirus. This report describes the clinical signs and provides a pathologic framework of an outbreak associated with at least two different ‘HoBi’‐like strains. Based on these observations, it appears that these atypical pestiviruses are most likely underdiagnosed in Brazilian cattle.     

Pre‐Clinical Evaluation of a Real‐Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses

African swine fever (ASF) and classical swine fever (CSF) are two highly infectious transboundary animal diseases (TADs) that are serious threats to the pig industry worldwide, including in China, the world's largest pork producer. In this study, a duplex real‐time PCR assay was developed for the rapid detection and differentiation of African swine fever virus (ASFV) and classical swine fever virus (CSFV). The assay was performed on a portable, battery‐powered PCR thermocycler with a low sample throughput (termed as ‘T‐COR4 assay’). The feasibility and reliability of the T‐COR4 assay as a possible field method was investigated by testing clinical samples collected in China. When evaluated with reference materials or samples from experimental infections, the assay performed in a reliable manner, producing results comparable to those obtained from stationary PCR platforms. Of 59 clinical samples, 41 had results identical to a two‐step CSFV real‐time PCR assay. No ASFV was detected in these samples. The T‐COR4 assay was technically easy to perform and produced results within 3 h, including sample preparation. In combination with a simple sample preparation method, the T‐COR4 assay provides a new tool for the field diagnosis and differentiation of ASF and CSF, which could be of particular value in remote areas.     

Virus Load in Pigs Affected with Different Clinical Forms of Classical Swine Fever

Summary Classical swine fever (CSF) is an endemic disease in India, but the real magnitude of the problem is not known as only outbreaks of acute CSF are reported and many cases of chronic and clinically inapparent forms of the disease, which manifest a confusing clinical picture, remain undiagnosed. The real status of classical swine fever virus (CSFV) infection can only be known by testing pigs with highly specific and sensitive diagnostic assays. To obtain the baseline prevalence of CSFV infection among pigs in an endemic region where no vaccination was being performed, a real‐time PCR assay was used to detect viral genetic material in tissue samples collected from a slaughterhouse in the northern state of Uttar Pradesh in India. In total, 1120 slaughtered pigs were examined for the presence of CSF suggestive pathological lesions and tissues from suspected cases were tested for the presence of CSFV antigen and nucleic acids by indirect immuno‐peroxidase test and real‐time PCR, respectively. Based on the detection of viral genetic material in the tonsils, the prevalence of CSFV infection among slaughtered pigs was found to be 7.67%. Pigs detected positive for viral genome by quantitative real‐time PCR assay when categorized into different forms of CSF, depending upon the pathological lesions observed, the viral load in the tonsils of some of the pigs with chronic or clinically inapparent form of the disease was similar to that detected in pigs with acute CSF. The results of the study suggested that the risk posed by pigs with chronic disease or those infected but showing no clinical disease may be relatively higher as they can transmit the virus to new susceptible hosts over a longer period of time.     

Infection Dynamics of Pandemic 2009 H1N1 Influenza Virus in a Two‐Site Swine Herd

Influenza A viruses are common causes of respiratory disease in pigs and can be transmitted among multiple host species, including humans. The current lack of published information on infection dynamics of influenza viruses within swine herds hinders the ability to make informed animal health, biosecurity and surveillance programme decisions. The objectives of this serial cross‐sectional study were to describe the infection dynamics of influenza virus in a two‐site swine system by estimating the prevalence of influenza virus in animal subpopulations at the swine breeding herd and describing the temporal pattern of infection in a selected cohort of growing pigs weaned from the breeding herd. Nasal swab and blood samples were collected at approximately 30‐day intervals from the swine breeding herd (Site 1) known to be infected with pandemic 2009 H1N1 influenza virus. Sows, gilts and neonatal pigs were sampled at each sampling event, and samples were tested for influenza virus genome using matrix gene RRT‐PCR. Influenza virus was detected in neonatal pigs, but was not detected in sow or gilt populations via RRT‐PCR. A virus genetically similar to that detected in the neonatal pig population at Site 1 was also detected at the wean‐to‐finish site (Site 2), presumably following transportation of infected weaned pigs. Longitudinal sampling of nasal swabs and oral fluids revealed that influenza virus persisted in the growing pigs at Site 2 for at least 69 days. The occurrence of influenza virus in neonatal pigs, but not breeding females, at Site 1 emphasizes the potential for virus maintenance in this dynamic subpopulation, the importance of including this subpopulation in surveillance programmes and the potential transport of influenza virus between sites via the movement of weaned pigs.     

Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine

Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti‐IAV antibodies using homologous and heterologous haemagglutination‐inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)‐blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut‐off of S/N ≤ 0.60, the sensitivity and specificity of the NP‐blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post‐inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost‐effective approach for the detection and surveillance of IAV infections in swine populations.     

Bioaerosol and surface sampling for the surveillance of influenza A virus in swine

Influenza A virus in swine is of significant importance to human and veterinary public health. Environmental sampling techniques that prove practical would enhance surveillance for influenza viruses in swine. The primary objective of this study was to demonstrate the feasibility of bioaerosol and surface sampling for the detection of influenza virus in swine barns with a secondary objective of piloting a mobile application for data collection. Sampling was conducted at a large swine operation between July 2016 and August 2017. Swine oral fluids and surface swabs were collected from multiple rooms. Room‐level air samples were collected using four bioaerosol samplers: a low volume polytetrafluoroethylene (PTFE) filter sampler, the National Institute for Occupational Safety and Health's low volume cyclone sampler, a 2‐stage Andersen impactor and/or one high volume cyclonic sampler. Samples were analysed using quantitative RT‐PCR. Data and results were reported using a mobile data application. Eighty‐nine composite oral fluid samples, 70 surface swabs and 122 bioaerosol samples were analysed. Detection rates for influenza virus RNA in swine barn samples were 71.1% for oral fluids, 70.8% for surface swabs and 71.1% for the PTFE sampler. Analysis revealed a statistically significant relationship between the results of the PTFE sampler and the surface swabs with oral fluid results (p < 0.001 and p < 0.01 respectively). In addition, both the PTFE sampler (p < 0.01) and surface swabs (p = 0.03) significantly correlated with, and predicted oral fluid results. Bioaerosol sampling using PTFE samplers is an effective hands‐off approach for detecting influenza virus activity among swine. Further study is required for the implementation of this approach for surveillance and risk assessment of circulating influenza viruses of swine origin. In addition, mobile data collection stands to be an invaluable tool in the field by allowing secure, real‐time reporting of sample collection and results.     

HoBi‐Like Virus RNA Detected in Foetuses Following Challenge of Pregnant Cows that had Previously Given Birth to Calves Persistently Infected with Bovine Viral Diarrhoea Virus

The ability of ruminant pestivirus including bovine viral diarrhoea virus (BVDV) and the related emerging pestivirus, HoBi‐like virus, to establish persistent infection (PI) following foetal infection is central to keeping these viruses in circulation. Non‐PI dams carrying BVDV PI calves develop high levels of immunity due constantly viral exposure. A study to determine whether the immunity developed following the generation of a BVDV PI is enough to prevent HoBi‐like virus infection of a subsequent foetus was performed. This study consisted of nine pregnant cows, four had birthed BVDV‐1 PI calves in a previous pregnancy, three cows had birthed BVDV‐2 PIs and two had birthed pestivirus negative calves. From this, six pregnant cows were challenged with HoBi‐like virus about day 85 of gestation (four BVDV‐1 and two BVDV‐2 cows) and three non‐challenged cows (negative control). At the day of challenge, the serum neutralizing titres against the homologous BVDV strains of the first inoculation ranged from 1148 to 5793. At day 6 post‐challenge, HoBi‐like RNA was detected in the serum of all four BVDV‐1 cows but not in the two BVDV‐2 cows. The foetuses harvested from five of the exposed dams (three BVDV‐1 and two BVDV‐2 cows) at day 30 post‐challenge were positive for HoBi‐like virus RNA. The sixth cow, BVDV‐1 cow #541, while pregnant at the time of exposure, had no foetus 30 days after exposure. Foetuses from HoBi‐like virus exposed dams were significantly smaller and lighter than control foetuses. HoBi‐like RNA was detected in samples of all challenged foetuses. The identification of viral RNA in the serum of 4 cows at day 6 post‐challenge, as well viral RNA detection in all foetuses 30 days post‐inoculation, indicates that the foetuses of dams with high antibodies titres against BVDV‐1 or BVDV‐2 would not be protected from challenge with a HoBi‐like virus.     

Biological characterization of African swine fever virus genotype II strains from north‐eastern Estonia in European wild boar

Due to its impact on animal health and pig industry, African swine fever (ASF) is regarded as one of the most important viral diseases of pigs. Following the ongoing epidemic in the Transcaucasian countries and the Russian Federation, African swine fever virus was introduced into the Estonian wild boar population in 2014. Epidemiological investigations suggested two different introductions into the southern and the north‐eastern part of Estonia. Interestingly, outbreak characteristics varied considerably between the affected regions. While high mortality and mainly virus‐positive animals were observed in the southern region, mortality was low in the north‐eastern area. In the latter, clinically healthy, antibody‐positive animals were found in the hunting bag and detection of virus was rare. Two hypotheses could explain the different behaviour in the north‐east: (i) the frequency of antibody detections combined with the low mortality is the tail of an older, so far undetected epidemic wave coming from the east, or (ii) the virus in this region is attenuated and leads to a less severe clinical outcome. To explore the possibility of virus attenuation, a re‐isolated ASFV strain from the north‐eastern Ida‐Viru region was biologically characterized in European wild boar. Oronasal inoculation led to an acute and severe disease course in all animals with typical pathomorphological lesions. However, one animal recovered completely and was subsequently commingled with three sentinels of the same age class to assess disease transmission. By the end of the trial at 96 days post‐initial inoculation, all animals were completely healthy and neither virus nor viral genomes were detected in the sentinels or the survivor. The survivor, however, showed high antibody levels. In conclusion, the ASFV strain from north‐eastern Estonia was still highly virulent but nevertheless, one animal recovered completely. Under the experimental conditions, no transmission occurred from the survivor to susceptible sentinel pigs.     

Genetic Diversity of Brazilian Bovine Pestiviruses Detected Between 1995 and 2014

Pestivirus infections in ruminants result in significant economic losses worldwide. The aetiological agents are three species from the genus Pestivirus, family Flaviviridae, including bovine viral diarrhoea virus type 1 (BVDV‐1), BVDV‐2, border disease virus (BDV), and an atypical pestivirus named HoBi‐like pestivirus. In this study, eighty‐nine pestivirus isolates that were collected in Brazil between 1995 and 2014 and that originated from either cattle, fetal bovine serum (FBS) or as cell culture contaminants were genotyped based on a comparison of gene sequences from their 5′ untranslated regions (5′UTR), N‐terminal autoprotease (N^pro) and envelope glycoprotein 2 (E2). Of these isolates, 53.9% of the sequences were genotyped as BVDV‐1, 33.7% as BVDV‐2 and 12.4% as HoBi‐like pestivirus. The prevalence of subgenotypes within the species was as follows: BVDV‐1a (35.9%), BVDV‐2b (31.4%), BVDV‐1b (10.1%), BVDV‐1d (6.7%), BVDV‐2c (2.2%) and BVDV‐1e (1.1%). BVDV‐2c and BVDV‐1e were detected for the first time in Brazil. This study revealed extensive genetic diversity among Brazilian pestivirus isolates, and the combination of pestiviruses that was detected is unique to Brazil. This information may serve as a foundation for designing and evaluating diagnostic tools and in the development of more effective vaccines; therefore, it may potentially contribute to pestivirus control and eradication.     

Detection of Influenza A Virus Nucleoprotein Antibodies in Oral Fluid Specimens From Pigs Infected Under Experimental Conditions Using a Blocking ELISA

In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek^™ Avian Influenza Virus MultiS‐Screen^® Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP‐blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen‐based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample‐to‐negative (S/N) ratio of influenza‐inoculated pigs was significantly different (P < 0.0001) from uninoculated controls (unvaccinated or vaccinated‐uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P < 0.0001). The results showed that oral fluid samples from influenza virus‐infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.     

Molecular Characteristics of Bovine Virus Diarrhoea Virus 1 Isolates from Turkey: Approaches for an Eradication Programme

Forty pestivirus isolates sampled from cattle in Turkey between 2002 and 2007 were characterized according to 5′ untranslated region (5′UTR) sequences and autoprotease (N^pro) gene sequences. The sampling of Bovine virus diarrhoea viruses (BVDVs) from 15 farms in five different regions indicated that BVDV 1‐l (18/40, 45%) was the predominant genotype in Turkey; the samples also contained the genotypes 1‐f (10/40, 25%), 1‐b (7/40, 17.5%), 1‐d (3/40, 7.5%), and 1‐a (2/40, 5%), respectively.