Background: The canine influenza virus (CIV) outbreak has garnered considerable attention as it poses a significant threat to dog health. During the H3N2 CIV evolution in beagles, the virus formed a new clade after 2019 and gradually became more adaptable to other mammals. Therefore, successfully elucidating the biological characteristics and constructing a canine influenza infection model is required for CIV characterization.

Methods: We performed genetic analyses to examine the biological characteristics and infection dynamics of CIV.

Results: The genotype of our H3N2 CIV strain (from 2019 in Shanghai) belonged to the 5.1 clade, which is now prevalent in China. Using MDCK cells, we investigated viral cytopathic effects. Virus size and morphology were observed using transmission electron microscopy. Beagles were also infected with 104, 105, and 106 50% egg-infectious doses (EID50). When compared with the other groups, the 106 EID50 group showed the most obvious clinical symptoms, the highest virus titers, and typical lung pathological changes. Our results suggested that the other two treatments caused mild clinical manifestations and pathological changes. Subsequently, CIV distribution in the 106 EID50 group was detected by hematoxylin and eosin (H&E) and immunofluorescence (IF) staining, which indicated that CIV primarily infected the lungs.