Molecular Evolution of Canine Influenza A Virus NS1 Gene: Phylogenetic Origin and Mutation Rate Analysis of Antigenic Sites

Influenza A is a virus composed of eight single-stranded, negative sense RNA molecules of approximately 14,000 nucleotides in total length and a member of the family Orthomyxoviridae. Influenza A viruses (IAV) are known to carry gene segments from humans and animals, including equine, avian, and swine. Studies have documented the mutations responsible for the introduction of equine IAV into dogs and known gene reassortments responsible for swine IAV introduction into the 2009 H1N1 human pandemic. Recent H3N8 U.S. canine influenza A (CIV) sequences are of H3N8 equine origin and H3N2 avian origin. In addition, there are known cytotoxic T-cell lymphocyte (CTL) epitopes in many of the eight influenza segments, including the NS1 gene. The hypotheses that recent U.S. canine NS1 sequences have an origin in Southeast Asia and are of avian origin and that there are host-specific differences in mutation rates associated with antigenic regions were tested. Influenza A NS1 nucleotide sequences were collected using the Influenza Research Database and GenBank at the National Center for Biotechnology Information. Sequences were aligned using CLUSTALW implemented in MEGA 6.0. Phylogenies were constructed using the neighbor-joining algorithm. Nonsynonymous mutation rates were compared to synonymous mutation rates in antigenic versus non-antigenic regions from different hosts. Synonymous mutation rates were higher, indicating purifying selection. Examination of the phylogenies confirmed that there was a Southeast Asian origin of U.S. canine influenzas, with roots in avian lineages. Understanding phylogenetic origin as well as host-specific mutation rates may help predict future host shifts and be used for preventative measures, such as the development of vaccines and other treatments