Molecular Epidemiology Questions Transmission Pathways Identified During the Year 2000 Outbreak of Classical Swine Fever in the UK.

The last outbreak of classical swine fever (CSF) in the UK occurred in 2000. A total of 16 domestic pig holdings in the East Anglia region were confirmed as infected over a 3-month period. Obtaining viral genome sequences has since become easier and more cost-effective and has accordingly been applied to trace viral transmission events for a variety of viruses. The rate of genetic evolution varies for different viruses and is influenced by different transmission events, which will vary according to the epidemiology of an outbreak. To examine if genetic changes over the course of any future CSF outbreak would occur to supplement epidemiological investigations and help to track virus movements, the E2 gene and full genome of the virus present in archived tonsil samples from 14 of these infected premises were sequenced. Insufficient changes occurred in the full E2 gene to discriminate between the viruses from the different premises. In contrast, between 5 and 14 nucleotide changes were detected between the genome sequence of the virus from the presumed index case and the sequences from the other 13 infected premises. Phylogenetic analysis of these full CSFV genome sequences identified clusters of closely related viruses that allowed to corroborate some of the transmission pathways inferred by epidemiological investigations at the time. However, other sequences were more distinct and raised questions about the virus transmission routes previously implicated. We are thus confident that in future outbreaks, real-time monitoring of the outbreak via full genome sequencing will be beneficial.

High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a "hot-spot"-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the "hot spot" of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

Molecular characterization of equine infectious anaemia virus strains detected in England in 2010 and 2012.

Equine infectious anaemia virus (EIAV) is a retrovirus with worldwide distribution which is notifiable to the OIE. Despite its importance to the equine industry, most information regarding its biology have been obtained using only two strains (EIAVWYO and EIAVLIA ) from the USA and China, respectively. Recently full genome sequences from Ireland, Italy and Japan have been published; however, this is still not representative of the number of EIAV outbreaks experienced globally each year. The limited availability of published sequences makes design of a universal EIAV PCR difficult, hence diagnosis is solely reliant on serology. Accordingly, it is important to further investigate the re-emerging cases in other areas of the world. Here, we provide information regarding the outbreaks of EIA in England in 2010 and 2012 including the molecular characterization of strains. Full genome was obtained for two symptomatic cases but could not be resolved for the asymptomatic cases. The two British genomes from 2010 (EIAVDEV ) and 2012 (EIAVCOR ) each represent a new phylogenetic group, each differing genetically from the other available full genome sequences by 21.1%-25.5%. That the majority of new EIAV full genome sequences to be published adds another phylogenetic group indicates that the surface of EIAV global diversity is just being scratched. These data highlight that further work is needed to fully understand EIAV genetic diversity, namely the full genome sequencing of EIAV cases from a variety of locations and time points. This would aid both the use of phylogenetics in parallel with horse tracing as the epidemiological tool of disease tracking and the design of a universally applicable molecular diagnostic method.

Detection and molecular characterization of bovine leukemia virus in various regions of Iran.

Purpose. Bovine leukemia virus (BLV) infects cattle worldwide, imposing an economic impact on the dairy cattle industry. The purpose of this study was to evaluate the molecular epidemiology of BLV in Iran.Methodology. Blood samples taken from 280 cows aged over 2 years old from 13 provinces of Iran were used for leukocyte count and blocking ELISA. Genomic DNA was extracted from the peripheral blood leukocytes of BLV-infected samples and fetal lamb kidney cells to perform PCR of partial env, rex and tax genes and long-terminal-repeat region. The PCR products were sequenced, the phylogenetic tree of each gene was constructed, and nucleotide and amino acid sequence pair distances were calculated.Results. The frequency of BLV infection was 32.8 % among animals and was 80 % among provinces. In BLV seropositive animals, the rate of persistent lymphocytosis was 36.9 %. The constructed phylogenetic trees showed the presence of two BLV genotypes (1 and 4) in Iranian strains. As previous studies, our results showed that the env gene was more variable than previously thought, the Rex protein could withstand more amino acid changes compared to the Tax protein, and no significant differences were observed in average changes of the nucleotide of these genes between clinical stages.Conclusions. Our data indicates an increase in the frequency of this infection in Iran. This is the first study report of the presence of BLV genotype 4 in Iranian farms. These findings may have an important role in the control and prevention of BLV infection in Iran and other countries.

Molecular and in vitro characterisation of hepatitis E virus from UK pigs.

Hepatitis E virus (HEV) infection is widespread in the global pig population. Although clinically inapparent in pigs, HEV infection is the cause of Hepatitis E in humans and transmission via the food chain has been established. Following a 2013 study that investigated prevalence of HEV infection in UK slaughter-age pigs samples indicating highest viral load were selected for further characterisation. High throughput sequencing was used to obtain the complete coding sequence from five samples. An in-frame insertion was observed within the HEV hypervariable region in two samples. To interrogate whether this mutation may be the cause of high-level viraemia and faecal shedding as observed in the sampled pigs virus isolation and culture was conducted. Based on viral growth kinetics there was no evidence that these insertions affected replication efficiency in vitro, suggesting as yet undetermined host factors may affect the course of infection and consequently the risk of foodborne transmission.

UK Pigs at the Time of Slaughter: Investigation into the Correlation of Infection with PRRSV and HEV.

Hepatitis E virus (HEV) and porcine reproductive and respiratory syndrome virus (PRRSV) and are both globally prevalent in the pig population. While HEV does not cause clinical disease in pigs, its zoonotic potential has raised concerns in the food safety sector. PRRS has become endemic in the United Kingdom (UK) since its introduction in 1991, and continues to cause considerable economic losses to the swine industry. A better understanding of the current prevalence and diversity of PRRSV and HEV in the UK, and their potential association, is needed to assess risks and target control measures appropriately. This study used plasma, tonsil, and cecal content samples previously collected from pigs in 14 abattoirs in England and Northern Ireland to study the prevalence of several pathogens including PRRSV and HEV. The diversity of PRRSV strains detected in these samples was analyzed by sequencing open reading frame 5 (ORF5), revealing no substantial difference in PRRSV strains from these clinically unaffected pigs relative to those from clinical cases of disease in the UK. Despite the potential immuno-modulatory effect of PRRSV infection, previously demonstrated to affect Salmonella and HEV shedding profiles, no significant association was found between positive PRRSV status and positive HEV status.

Molecular characterization of bovine leukemia virus from Moldovan dairy cattle.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a disease that has worldwide distribution. Whilst it has been eradicated in most of Western Europe and Scandinavia, it remains a problem in other regions, particularly Eastern Europe and South America. For this study, in 2013, 24 cattle from three farms in three regions of Moldova were screened by ELISA and nested PCR. Of these cattle, 14 which were PCR positive, and these were molecularly characterized based on the nucleotide sequence of the env gene and the deduced amino acid sequence of the encoded gp51 protein. Our results demonstrated a low level of genetic variability (0-2.9%) among BLV field strains from Moldova, in contrast to that observed for other retroviruses, including human immunodeficiency virus (HIV) (20-38%) Mason IL (Trudy vologod moloch Inst 146-164, 1970) and equine infectious anemia virus (EIAV) (~40%) Willems L et al (AIDS Res Hum Retroviruses 16(16):1787-1795, 2000), where the envelope gene exhibits high levels of variation Polat M et al (Retrovirology 13(1):4, 2016). Sequence comparisons and phylogenetic analysis revealed that BLV genotype 7 (G7) is predominant in Moldova and that the BLV population in Moldovan cattle is a mixture of at least three new sub-genotypes: G7D, G7E and G4C. Neutrality tests revealed that negative selection was the major force operating upon the 51-kDa BLV envelope surface glycoprotein subunit gp51, although one positively selected site within conformational epitope G was detected in the N-terminal part of gp51. Furthermore, two functional domains, linear epitope B and the zinc-binding domain, were found to have an elevated ratio of nonsynonymous to synonymous codon differences. Together, these data suggest that the evolutionary constraints on epitopes G and B and the zinc-binding domains of gp51 differ from those on the other domains, with a tendency towards formation of homogenous genetic groups, which is a common concept of global BLV diversification during virus transmission that may be associated with genetic drift.

From the field to the lab - An European view on the global spread of PEDV.

Porcine Epidemic Diarrhea Virus (PEDV) is a member of the genus Alphacoronavirus, in the family Coronaviridae, of the Nidovirales order and outbreaks of porcine epidemic diarrhoea (PED) were first recorded in England in the 1970s. Intriguingly the virus has since successfully made its way around the globe, while seemingly becoming extinct in parts of Europe before its recent return from Northern America. In this review we are re-evaluating the spread of PEDV, its biology and are looking at lessons learnt from both failure and success. While a new analysis of PEDV genomes demonstrates a wider heterogeneity of PEDV than previously anticipated with at least five rather than two genotypes, biological features of the virus and its replication also point towards credible control strategies to limit the impact of this re-emerging virus.

Prevalence of Hepatitis E Virus Infection in Pigs at the Time of Slaughter, United Kingdom, 2013.

Since 2010, reports of infection with hepatitis E virus (HEV) have increased in England and Wales. Despite mounting evidence regarding the zoonotic potential of porcine HEV, there are limited data on its prevalence in pigs in the United Kingdom. We investigated antibody prevalence, active infection, and virus variation in serum and cecal content samples from 629 pigs at slaughter. Prevalence of antibodies to HEV was 92.8% (584/629), and HEV RNA was detected in 15% of cecal contents (93/629), 3% of plasma samples (22/629), and 2% of both (14/629). However, although HEV is prevalent in pigs in the United Kingdom and viremic pigs are entering the food chain, most (22/23) viral sequences clustered separately from the dominant type seen in humans. Thus, pigs raised in the United Kingdom are unlikely to be the main source of human HEV infections in the United Kingdom. Further research is needed to identify the source of these infections.

Correction: Evidence for the Circulation of Equine Encephalosis Virus in Israel since 2001.

[This corrects the article on p. e70532 in vol. 8.].

Evidence for the circulation of equine encephalosis virus in Israel since 2001.

Equine encephalosis virus (EEV) distribution was thought to be limited to southern Africa until 2008 when we reported EEV in Israel. It was then assumed that the clinical presentation resembled the initial incursion in Israel. To investigate further we conducted a retrospective analysis of equine sera, which had been collected for diagnosis of other suspected diseases, via serum neutralisation test. The data demonstrated that EEV was circulating as early as 2001 with incidence ranging from 20-100% for time period 2001-2008. As the symptoms of EEV can be similar to other equine notifiable diseases this is a significant finding which highlights the need for vigilance and education to accurately diagnose new and emerging diseases.

Development of an improved real time PCR for the detection of bovine leukaemia provirus nucleic acid and its use in the clarification of inconclusive serological test results.

With the aim to erradicate Enzootic Bovine Leukosis from Poland, a more sensitive real-time polymerase chain reaction was required and developed to detect proviral Bovine leukaemia virus (BLV) DNA, the causative agent of Enzootic Bovine Leukosis (EBL). This new method proved more sensitive for our needs, than the current protocols available in the public domain. DNA was extracted from peripheral blood leukocytes of 51 cattle, which had given rise to doubtful serological test results by ELISA, and from mesenteric lymph nodes of six cattle that were slaughtered as EBL suspect cases. Additionally, fourteen DNA samples were obtained from animals with a strong BLV antibody response by ELISA. All real-time data were compared to results obtained from three different nested PCR methods. All 14 strongly positive ELISA samples were positive in all PCR tests. The real-time assay in comparison to the conventional PCR methods detected 7.8% (4/51) more specimens positive for BLV nucleic acid and showed a detection limit down to one copy. These observations represent the first report in the value of using a real-time method to help elucidate the disease status of animals when inconclusive ELISA results are obtained in the diagnostic laboratory. Thus, this method should be recommended for use in countries which have implemented an EBL-eradication programme, where a low level of BLV infection is evident.

The molecular characterization of bovine leukaemia virus isolates from Eastern Europe and Siberia and its impact on phylogeny.

Recent studies have shown that bovine leukemia virus (BLV) sequences can be classified into seven distinct genotypes based on full gp51 sequence. This classification was based on available sequence data that mainly represented the BLV population that is circulating in cattle from the US and South America. In order to aid with a global perspective inclusion of data from Eastern Europe is required. In this study we examined 44 BLV isolates from different geographical regions of Poland, Belarus, Ukraine, and Russia. Phylogenetic analysis based on a 444bp fragment of env gene revealed that most of isolates belonged to genotypes 4 and 7. Furthermore, we confirmed the existence of a new genotype, genotype 8, which was highly supported by phylogenetic computations. A significant number of amino acid substitutions were found in the sequences of the studied Eastern European isolates, of which 71% have not been described previously. The substitutions encompassed mainly the C-part of the CD4+ epitope, zinc binding peptide region, CD8+ T cell epitope, and overlapping linear epitope E. These observations highlight the use of sequence data to both elucidate phylogenetic relationships and the potential effect on serological detection of geographically diverse isolates.

Main achievements of the World Organisation for Animal Health/United Nations Food and Agriculture Organization network on animal influenza.

The World Organisation for Animal Health (OIE)/United Nations Food and Agriculture Organization (FAO) joint network of expertise on animal influenza (OFFLU) includes all ten OIE/FAO reference laboratories and collaborating centers for avian influenza, other diagnostic laboratories, research and academic institutions, and experts in the fields of virology, epidemiology, vaccinology, and molecular biology. OFFLU has made significant progress in improving its infrastructure, in identifying and addressing technical gaps, and in establishing associations among leading veterinary institutions. Interaction with the World Health Organization (WHO) Global Influenza Program is also critical, and mechanisms for permanent interaction are being developed. OFFLU played a key role in the WHO/OIE/FAO Joint Technical Consultation held in Verona (October 7-9, 2008), which provided an opportunity to highlight and share knowledge and identify potential gaps regarding issues at the human-animal interface for avian influenza. OFFLU experts also contributed to the working group for the Unified Nomenclature System for H5N1 influenza viruses based on hemagglutinin gene phylogeny (WHO/OIE/FAO, H5N1 Evolution Working Group, Towards a unified nomenclature system for highly pathogenic avian influenza virus (H5N1) in Emerging Infectious Diseases 14:el, 2008). OFFLU technical activities, led by expert scientists from OIE/FAO reference institutions and coordinated by OIE and FAO focal points, have been prioritized to include commercial diagnostic kit evaluation, applied epidemiology, biosafety, vaccination, proficiency testing, development of standardized reference materials for sera and RNA, and issues at the human-animal interface. The progress to date and future plans for these groups will be presented. OFFLU is also involved in two national projects implemented by FAO in Indonesia and Egypt that seek to establish sustainable mechanisms for monitoring virus circulation, including viral characterization, and for streamlining the process to update poultry vaccines for avian influenza.

Replication, pathogenesis and transmission of pandemic (H1N1) 2009 virus in non-immune pigs.

The declaration of the human influenza A pandemic (H1N1) 2009 (H1N1/09) raised important questions, including origin and host range [1], [2]. Two of the three pandemics in the last century resulted in the spread of virus to pigs (H1N1, 1918; H3N2, 1968) with subsequent independent establishment and evolution within swine worldwide [3]. A key public and veterinary health consideration in the context of the evolving pandemic is whether the H1N1/09 virus could become established in pig populations [4]. We performed an infection and transmission study in pigs with A/California/07/09. In combination, clinical, pathological, modified influenza A matrix gene real time RT-PCR and viral genomic analyses have shown that infection results in the induction of clinical signs, viral pathogenesis restricted to the respiratory tract, infection dynamics consistent with endemic strains of influenza A in pigs, virus transmissibility between pigs and virus-host adaptation events. Our results demonstrate that extant H1N1/09 is fully capable of becoming established in global pig populations. We also show the roles of viral receptor specificity in both transmission and tissue tropism. Remarkably, following direct inoculation of pigs with virus quasispecies differing by amino acid substitutions in the haemagglutinin receptor-binding site, only virus with aspartic acid at position 225 (225D) was detected in nasal secretions of contact infected pigs. In contrast, in lower respiratory tract samples from directly inoculated pigs, with clearly demonstrable pulmonary pathology, there was apparent selection of a virus variant with glycine (225G). These findings provide potential clues to the existence and biological significance of viral receptor-binding variants with 225D and 225G during the 1918 pandemic [5].