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
