Emergence of a novel reassortant H3N3 avian influenza virus with enhanced pathogenicity and transmissibility in chickens in China

The H3N3 avian influenza viruses (AIVs) are not as prevalent as H3N8 viruses in poultry. Yet, reassortant H3N3 viruses are occasionally detected in domestic poultry and wild birds. In this study, we isolated an H3N3 virus in chickens from a live poultry market in China. Full genomic analysis revealed that the virus had a single basic amino acid in the cleavage site of the hemagglutinin (HA) gene. Phylogenetic analysis revealed that eight genes of the H3N3 virus belong to the Eurasian lineage and the HA and NA genes were clustered with H3N2 and H11N3, respectively, while the internal genes were closely related to H3N8 and H9N2 viruses. The H3N3 virus demonstrated high and moderate stability in thermal and acid conditions, respectively, along with efficient replication capabilities in mammalian cells. Moreover, the H3N3 virus could infect and replicate in the upper and lower respiratory tract of BALB/c mice without prior adaptation, triggering hemagglutination inhibition (HI) antibody titers ranging from 80 to 160. The H3N3 virus replicated vigorously within the chicken respiratory and digestive tracts, transmitted efficiently and swiftly among chickens via direct contact, and resulted in elevated HI antibody titers in both the inoculated and contact birds. These findings indicate that the H3N3 virus might be a novel reassortant originating from viruses circulating in domestic poultry, demonstrating increased pathogenicity and transmissibility in chickens. Our findings demonstrate that H3N3 AIV may pose a potential threat to poultry industry and public health, thereby highlighting the importance of active surveillance of AIVs.