The inactivated vaccine of reassortant H3N2 canine influenza virus based on internal gene cassette from PR8 is safe and effective.

Canine influenza (CI) is a contagious respiratory disease in dogs, which poses a threat to canine health. A safe, high-yield vaccine seed virus is critical for CI vaccine development. We developed a PR8-based reassortant H3N2 canine influenza virus (RT CIV) using the reverse genetic method and evaluated its yield in canine kidney epithelial (MDCK) cells, Vero cells, and specific pathogen-free (SPF) chicken embryos. Mice and dogs were infected with RT CIV, and the pathogenicity was evaluated. The viral titers of RT CIV increased in MDCK cells, Vero cells, and SPF chicken embryos; the HA yield in SPF chicken embryos increased 4-fold. However, RT CIV was not lethal to mice, and it showed similar virulence as wild-type CIV. RT CIV also showed minimal pathogenicity in dogs, which manifested as mild fever and rhinorrhea for the first two days post-infection. Thus, RT CIV carrying the internal gene cassette from PR8 showed almost no pathogenicity in dogs. And the reassortant virus inactivated vaccine could provide complete protection against H3N2 CIV. To our knowledge, this is the first report on the pathogenicity of PR8-based reassortant H3N2 CIV in dogs. These studies are relevant for developing a high-yield and safe CI vaccine.

Phosphoproteomics to Characterize Host Response During H3N2 Canine Influenza Virus Infection of Dog Lung.

Avian-origin H3N2 canine influenza viruses (CIVs) cause severe contagious respiratory disease in dogs, and quickly adapt to new environments. To further understand the mechanism of virus infection and host-virus interactions, we characterized the complete phosphoproteome of dogs infected with H3N2 CIV. Nine-week-old Beagle dogs were inoculated intranasally with 106 EID50 of A/canine/Guangdong/04/2014 (H3N2) virus. Lung sections were harvested at 5 days post-inoculation (dpi) and processed for global and quantitative analysis of differentially expressed phosphoproteins. A total of 1,235 differentially expressed phosphorylated proteins were identified in the dog lung after H3N2 CIV infection, and 3,016 modification sites were identified among all differentially expressed proteins. We then performed an enrichment analysis of functional annotations using Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) database analyses to predict the functions of the identified differential phosphoproteins. Our data indicate that H3N2 CIV infection causes dramatic changes in the host protein phosphorylation of dog lungs. To our knowledge, this is the first study to assess the effect of H3N2 CIV infection on the phosphoproteome of beagles. These data provide novel insights into H3N2-CIV-triggered regulatory phosphorylation circuits and signaling networks and may improve our understanding of the mechanisms underlying CIV pathogenesis in dogs.