Adaptive mutations in HA of avian H9N2 influenza viruses facilitate their transmission to swine.

The cross-species transmission of avian H9N2 influenza viruses to swine increases the risk of viral adaptation to mammalian hosts. However, the mechanisms by which these H9 viruses can overcome the barriers posed by swine hosts have not yet been fully elucidated. In previous studies, we identified avian H9 strains exhibiting either infective or noninfective phenotypes in swine. Here, we investigated the role of surface genes in cross-species transmission by replacing the surface genes of noninfective strains with those of their infective counterparts. We demonstrated that the surface genes of the infective strains, particularly the hemagglutinin (HA) gene, restored infectivity in pigs for two previously noninfective strains. Surface genes from infective strains significantly increased viral replication efficiency in both CEF and PK15 cells, and recombinant viruses carrying these genes presented superior thermal stability. Amino acid sequence analysis of HA identified six critical residues (30T, 39A, 327R, 373K, 465K, and 490R) associated with infectivity in pigs. Avian H9 viruses bearing these swine-adaptive molecular signatures began emerging in terrestrial poultry before the 2000s and subsequently achieved dominance through widespread dissemination. These findings suggest that molecular changes in HA accumulated during the adaptation of avian H9 viruses in terrestrial poultry may drive the emergence of swine-infective strains. This study elucidates key molecular determinants that enable avian H9 viruses to infect swine and highlights the public health implications of prolonged H9N2 circulation in terrestrial poultry, which facilitates mammalian adaptation. These insights underscore the need for intensified surveillance of avian-to-swine influenza transmission dynamics.