Virus discovery and characterization in archived diagnostic samples using next-generation sequencing
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New viruses have emerged over the past two decades to cause important diseases in animals and humans. Previously controlled diseases have re-emerged following changes in viral genomes as they are transmitted across species and respond to changes in herd immunity within species. The advent of next-generation sequencing (NGS) now permits detection of viral genomes while simultaneously assessing their diversity. In this dissertation, NGS was used to detect viruses and evaluate their genomic characteristics across nucleic acid samples archived at the veterinary medical diagnostic laboratory (VMDL), university of Missouri. In the first study, sequence independent single primer amplification (SISPA) coupled with NGS was employed to identify 4 families of DNA viruses and 8 families of RNA viruses in 240 sera collected from wild-caught deer submitted to the VMDL from 2022 -- 2024. The presence of viruses belonging to the Togaviridae, Flaviviridae, Paramyxoviridae and Pneumoviridae families support the circulation of viruses belonging to known families of pathogenic RNA viruses in wild deer populations. In the second study, amplicon sequencing was used to retrieve genome segments of epizootic hemorrhagic disease of deer virus (EHDV) from nucleic acid samples obtained from deer and cattle in Missouri between 2013 and 2023. The study showed that EHDV serotype 2 predominated among the deer population of Missouri during the study period. Evidence of co-infection with more than one serotype (1/2, 2/6, 1/2/6) was detected in 6 0f 17 isolates sequenced. Analysis of quasispecies structure showed moderate diversity of EHDV genomes over time. In the third study, we classified 103 (46.19%), 72(32.29%, and 48 (21.52%) of 223 isolates of Canine Parvovirus 2 (CPV-2) found in dogs as CPV-2a, CPV-2b, and CPV-2c based on the amino acid mutations at position 426 of the viral structural protein VP2. The evolutionary pattern of CPV-2 VP2 gene from 2012 to 2022 was investigated by conducting deep sequencing of CPV-2-positive samples. Mutations were detected at 80 nucleotide positions in 71 VP2 sequences assembled. Of these, 18 (23%) positions were common to CPV-2a, CPV-2b, and CPV-2c genotypes. However, 15, 22, and 11 mutation sites were unique to 2a, 2b, and 2c, respectively. Two sites were shared between CPV-2a and CPV-2b as well as CPV-2a and CPV-2c, while10 were common to 2b and 2c. There were more nucleotide transitions (64/80) than transversions (16/80). Transition/transversion ratio did not follow a consistent trend over 10 years, suggesting an inconsistent pattern of evolution over a decade. Bayesian phylogeny classified consensus VP2 sequences of CPV-2 isolates into 8 clades, while maximum likelihood analysis divided CPV-2 isolates into 5 clades, with strong bootstrap support for only two clades. Classification into variants using mutations at VP2 position 426 did not consistently align with phylogenetic classifications. Nonetheless, 67 of 71 isolates from Missouri possessed the S297A mutation associated with "novel" variants in other studies. Analysis of quasispecies diversity using Shannon entropy showed that CPV-2 isolates did not diverge widely over time and geographic locations. These studies highlight the multidimensional utility of NGS in virus detection and tracking of changes in viral genomes over time. The use of NGS to explore archival samples in veterinary diagnostic laboratories is a potential surveillance tool to monitor the emergence of new viruses of livestock and viruses that inhabit the human-animal interface.
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Ph. D.