The contribution of different mechanisms of viral sequence variation to the evolution of positive-sense single-stranded RNA viruses

The Flaviviridae family of positive-sense single-stranded RNA (+ssRNA) viruses includes viral taxa which greatly impact public health worldwide. To explore how the viruses within the Flaviviridae family evolve, we examined the extent to which these viral taxa use nucleotide covariance, spontaneous mutation, and/or homologous recombination to vary their genotype as well as the resulting phenotype. We developed and used CovarView to assist us in simultaneously viewing and inspecting the results from whole genome covariance analyses. This resulted in the identification of previously-characterized RNA functional structures in the genomes of hepatitis C virus (HCV), as well as a new RNA functional region in the gp120 coding region of human immunodeficiency virus type 1 (HIV-1). We observed two distinct clades within HCV subtype 1a genomes when we used phylogeny to examine the prevalence of mutations in this species. These clades were further characterized and many nucleotide positions that contributed significantly to the separation between the two clades were identified. Several of these positions were located at or near sites responsible for encoding antiviral resistance mutations. While assessing the homologous recombination results for these species we found that HCV and DENV use it most frequently, while the novel individual event that we found in West Nile virus (WNV) confirms its rarity. We compared and contrasted the results measuring the separate variation mechanisms to determine the extent to which the viral genera and species within the Flaviviridae family use one or more of these mechanisms more frequently than other(s) to obtain sequence variation. We observed that HCV frequently exhibits nucleotide covariance while DENV and WNV use it only at the 5’ and 3’ untranslated regions. Although mutations occur in all Flaviviridae species, including DENV and WNV, they seem to be tolerated better by HCV due to its replicating within a single host. Homologous recombination is used customarily within HCV and DENV genomes, but is extraordinarily rare in WNV. We conclude that although +ssRNA viruses use nucleotide covariance, mutation, and homologous recombination to acquire sequence variation, different viral species use these mechanisms in varying frequency as they continue to evolve within their own distinct environments. Keywords: covariance, mutation, recombination, bioinformatics, virology, +ssRNA viru