Abstract
Influenza H3N8 viruses have been frequently isolated from chicken farms. However, comprehensive characterization of their virological properties, molecular evolution, virulence, and risk of spillover into mammals remains limited. In particular, little attention has been given to the transmission efficiency of H3N8 avian influenza viruses among chickens and their spillover risk. Here, we systematically characterized H3N8 isolates obtained from asymptomatic chickens through multidisciplinary approaches, including genomic surveillance, receptor binding profiling, and in vivo pathogenicity and transmission assays. All strains showed >98% nucleotide homology with human-infecting strains. Phylogenetic analysis revealed that their internal genes were derived from H9N2, while HA and PB2 genes shared high homology (bootstrap support >98%) with the novel H3N3 virus. All isolates maintained avian-type receptor-binding motifs (HA-Q226/G228) while exhibiting dual α2,3/α2,6-sialic acid binding and robust replication in mammalian cells (peak MDCK titer: 10(7)·(5) TCID(5)(0)/mL). ZJ07 demonstrated exceptional thermostability (HA activity persisting >3 hr at 56°C), while JS13 showed 1.8-fold elevated neuraminidase activity versus controls (p < 0.05). In vivo, all strains caused subclinical infections with broad tissue tropism in chickens and mice without adaptation, transmitting efficiently among direct-contact poultry. Strikingly, AH12 achieved 100% airborne transmission in chickens. These findings confirm H3N8's capacity for silent poultry circulation and identify key features conducive to cross-species infection, including dual receptor binding, infection in a mammalian model, and high genetic homology with human strains. The airborne transmissibility of AH12 underscores a heightened spillover risk, necessitating enhanced surveillance and vaccines targeting avian-human interface strains.