Biological characterization of two evolutionarily related pathogens, equine and canine H3N8 influenza A viruses

Equine influenza H3N8 virus (EIV) jumped into dogs around the year 2000 and consequently caused the emergence of canine influenza H3N8 virus (CIV). This dog-specific virus has since been circulating primarily in the United States in animal shelters and places with high density dog populations. Host specificity and adaptation of influenza A viruses (IAVs) are not well understood, and so we compared the biological properties of EIV and CIV in order to further elucidate these properties. We used a variety of assays to characterize virus growth, infections in different host cells, receptor specificity, hemagglutinin (HA) cleavage, and infections in tracheal cultures. Despite numerous mutations between the genomes, we found minimal biological differences comparing EIV and CIV. Both viruses grew similarly in dog cells (MDCK) while they could not infect horse (EQKD) and human (A549) cells. Both viruses' receptor binding HA protein preferred alpha2-3 over alpha2-6 linked sialic acids, and there was also no difference comparing HA cleavage efficiency. Interestingly, infections in tracheal cultures showed CIV could not establish a productive infection in horse trachea compared to EIV. We also characterized an ancillary protein, PA-X, from both viruses by using reporter assays and RNA sequencing (RNA-seq). Reporter assays showed EIV and CIV PA-X had ribonuclease activity and suppressed beta-galactosidase and GFP expression. Notably, EIV PA-X had significantly stronger activity compared to CIV PA-X. Using site directed mutagenesis we found this difference was due to a mutation at amino acid position 231 and the truncation at the C-terminus. RNA-seq of cells transfected with plasmids encoding EIV and CIV PA-X revealed that they up-regulated the expression of many host genes compared to the controls. These altered genes were involved in various functions such as modulating the immune response, protein ubiquitination, ER-Golgi sorting and trafficking, and transcription. The RNA-seq analysis did not reveal any differences in gene expression comparing EIV and CIV PA-X samples however. Taken together our results showed EIV and CIV's biology was very similar despite many genetic differences between the two viruses, and this implies IAV host-switching and adaptation may be mediated by more subtle factors.