Abstract
The H3 subtype of avian influenza virus (AIV) stands out as one of the most prevalent subtypes, posing a significant threat to public health. In this study, a novel triple-reassortant H3N3 AIV designated A/chicken/China/16/2023 (H3N3), was isolated from a sick chicken in northern China. The complete genome of the isolate was determined using next-generation sequencing, and the AIV-like particles were confirmed via transmission electron microscopy. Subsequent phylogenetic analyses revealed that HA and NA genes of the H3N3 isolate clustered within the Eurasian lineage of AIVs, exhibiting the closest genetic relationship with other H3N3 AIVs identified in China during 2023. Interestingly, the HA and NA genes of the nove H3N3 isolate were originated from H3N8 and H10N3 AIVs, respectively, and the six internal genes originated from prevalent H9N2 AIVs. These findings indicated the novel H3N3 isolate possesses a complex genetic constellation, likely arising from multiple reassortment events involving H3N8, H9N2, and H10N3 subtype influenza viruses. Additionally, the presence of Q226 and T228 in the HA protein suggests the H3N3 virus preferentially binds to α-2,3-linked sialic acid receptors. The HA cleavage site motif (PEKQTR/GIF) and the absence of E627K and D701N mutations in PB2 protein classify the virus as a characteristic low pathogenicity AIV. However, several mutations in internal genes raise concerns about potential increases in viral resistance, virulence, and transmission in mammalian hosts. Overall, this study provides valuable insights into the molecular and genetic characterization of the emerging triple-reassortant H3N3 AIVs, and continued surveillance of domestic poultry is essential for monitoring the H3N3 subtype evolution and potential spread.