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Investigation into emerging, evolving or novel viruses of swine in respiratory, reproductive and enteric pathologies: horizon scanning for risks to swine health and food security

Periodic Reporting for period 1 - PIGRISK (Investigation into emerging, evolving or novel viruses of swine in respiratory, reproductive and enteric pathologies: horizon scanning for risks to swine health and food security)

Reporting period: 2016-04-04 to 2018-04-03

Emerging viruses pose a significant threat to animal health and welfare, food security and rural prosperity. When porcine circovirus 2 (PCV2) emerged in the late 1990s it had a serious socioeconomic cost.

According to the United Nations Population Division the human population in 2050 is estimated to be 9.15 billion. Pork accounts for about half of all the meat consumed in Europe. Pathogenic microorganisms cause huge economic loss in terms of production, poor growth rates, and veterinary costs. In order to ensure efficient and effective pig production and to maintain EU competitiveness in the global food market, it is vital that the emergence of novel pathogenic viruses is closely monitored and any potential disease problems are quickly discovered and controlled. This project aimed to improve the preparedness of the community to respond to the threat of emerging viral pathogens for our food security.

The objectives of the project were to improve our preparedness for future viral outbreaks by the following activities: (1) Develop, enhance and apply a range of classic and beyond the state-of-the-art diagnostic methods for the detection of emerging, evolving or novel viruses in swine; (2) Statistically establish disease association of detected viruses with these; (3) Develop next generation sequencing (NGS) methods to allow characterisation of the entire viral flora of pigs with particular disease states; (4) Parallel these research activities with a comprehensive, multi-disciplinary training programme to enhance employability of the researcher.
Training of the research fellow:
During the project the researcher received training in classical virology techniques such as immunofluorescence, light microscopy, virus storage and purification as well as tissue culture. In addition, training in molecular techniques including next-generation sequencing (NGS) and transferable and complementary skills including project management grant writing, reference management software and bioinformatics was provided.
Creation of a sample bank:
A sample bank was set up to include a range of sample types originating from pigs with various clinical presentations. More than 1000 samples were included.
Establishment of a diagnostic tool kit for detection of emerging viral threats:
A wide range of diagnostic, degenerate and sequencing assays were implemented or designed for emerging viruses of swine. The assays that were designed or implemented included porcine circovirus, porcine parvoviruses 2-6, stool-associated virus, porcine cyclovirus, calicivirus, kobuvirus, astrovirus, coronavirus, pestivirus, atypical pestivirus and teschovirus as well as a number of assays for established viruses. A Manual of Diagnostic tests has been produced outlining theses assays.
Investigation into the prevalence of novel/emerging viruses in swine:
Apart from pestivirus and porcine cyclovirus all of the other viruses were detected by molecular assay with prevalences ranging from 1 to almost 60%. Kobuvirus (59 out of 101 samples, 58.4%) and torque teno virus (TTV) (126 out of 223, 56.5%) were found at particularly high levels. Genetic sequencing was carried out on a number of positive samples. Noroviruses were particularly divergent showing 75-90% genetic similarity to known strains. A sequencing study was carried out examining the genotypes of PCV2 from samples taken from before and after the introduction of vaccination. All PCV2 sequences from this study were conventional PCV2a or PCV2b. Sequencing was carried out on the emerging porcine parvoviruses, stool associated virus and porcine circovirus 3. Porcine circovirus 3, as an emerging virus with potential role in disease, was of particular interest to us. The virus was 98% similar genetically to the virus described in 2016 from the US.
A study was carried out into determining the levels antibodies in pigs to porcine bocaviruses 3 and 4. Three hundred samples were tested from 15 farms and antibodies were detected in 70-100% of pigs on all farms demonstrating widespread circulation of the virus.
A database of clinical information from post-mortem and veterinary reports associated with the sample bank was created. Statistical techniques were applied to look for a correlation between the presence of emerging viruses and particular disease symptoms. A number of the novel parvoviruses showed a significant association with respiratory disease, wasting symptoms, lymphoid depletion, enlarged lymph nodes and intracellular inclusions. Porcine circovirus type 3 had a weak association with enlarged lymph nodes and a strong association with lymph oedema and intracellular inclusions, providing further novel evidence of a potential role in disease.
Viral metagenomics by next generation sequencing (NGS):
An evaluation of NGS sample preparation methods. This was accomplished using spiked material containing known viruses. Sample preparation methods such as centrifugation, filtration and enzyme treatment were evaluated. This was done on faeces samples spiked a range of viruses representing large, small, RNA, DNA, linear and circular species. Next generation sequencing and bioinformatics methodologies were applied to 4 porcine samples. In one sample 6 divergent picobirnaviruses, a stool associated virus, a bocavirus a parvovirus and picornaviruses were found along with a range of bacteriophage and sequences from bacteria, archae and host. These results demonstrate these methods are suitable for characterisation of the entire viral flora of pigs when a no-diagnosis sam
The project has progressed viral detection beyond the state of the art. A range of new molecular assays and diagnostic protocols are available for the detection of emerging viruses in pigs. The study generated information on how viruses were retained through the steps in the next generation sequencing sample preparation method and how each step reduces unwanted host material. Little information has been published regarding sample preparation methods for large-scale sequencing in virology and our studies will have progressed our knowledge in this field.Some important information has been generated by applying these assays for detection of viruses in clinical samples. The work on porcine circovirus 3 in this project is of particular importance – it was only the second reporting of the virus in Europe and the first on the islands of Ireland and Great Britain, demonstrating new geographic ranges. This study was the first to detect the virus in archival samples indicating historical circulation. The work on porcine circovirus 2 is also of particular significance. A number of newly emerging strains of virus have been detected in recent years (PCV2d) and some researchers have suggested that these may have a role in vaccine failure. None of these were detected in our samples.
The statistical correlation between clinical signs from post-mortem and veterinary reports and presence of virus is a novel approach to investigate disease association, particularly with emerging viruses that may be resistant to growth in cell culture.
Antibody detection studies on porcine bocaviruses 3 and 4 continue to enhance our knowledge of the distribution of these viruses - currently there are no other laboratories that have successfully grown emerging parvoviruses in cell culture or produced antibodies to the viruses.
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