The emergence of triple-reassortant H3N3 avian influenza viruses (AIVs) poses a threat to poultry and public health. This study characterized three novel H3N3 isolates (SQ2049, HN0120, and GT2125) from chickens in China, evaluating their evolution, pathogenicity, and vaccine efficacy. Phylogenetic analysis revealed these isolates originated from H10N3 and H3N8 lineages, carrying an H9N2-derived internal gene backbone. Although identified as low pathogenic AIVs (IVPI=0), they exhibited distinct replicative fitness. GT2125 showed reduced efficiency in embryos, and carried unique PB2 substitutions (V598I and K702). Pathogenicity trials in SPF chickens demonstrated that SQ2049 achieves efficient horizontal transmission in SPF chickens without clinical signs. Notably, cloacal viral shedding persisted for 11 days, whereas oral viral shedding only lasted 2 to 3 day, with mild histopathological lesions observed in the trachea and immune organs. Importantly, the inactivated SQ2049 vaccine elicited high cross-reactive antibody titers (HI≥8log2) and provided 100% protection in SPF chickens. Mouse infection studies identified two HA substitutions (G174E and N209D) after viral replication in mice. These two mutations have previously been associated with mammalian adaptation and enhanced binding to human-type α-2,6 receptors, though their functional effects in the H3N3 context were not evaluated in this study. These findings expand our understanding of H3N3 AIV evolutionary dynamics and highlight the importance of surveillance for reassortant influenza viruses. The demonstrated vaccine efficacy provides a foundation for future H3N3 prevention and control strategies.

To Cite This Article: Liu L, Liu J, Chang J, Cao X, Huang Z, Zhao K, Gao W, Li B, Zhou X, Liu Y, Zhou W, Pei X, Ren Y, Song Y and Li X, 2026. Pathogenesis and cross-species potential of emerging h3n3 influenza viruses with mammalian-adaptive mutations. Pak Vet J, 46(9): 2230-2241. http://dx.doi.org/10.29261/pakvetj/2026.208