Background: Since the first human infection with H9N2 virus was reported in 1998, the number of cases of H9N2 infection has exceeded one hundred by 2021. However, there is no systematic description of the biological characteristics of H9N2 viruses isolated from humans.

Methods: Therefore, this study analyzed the pathogenicity in mice of all available H9N2 viruses isolated from human cases in China from 2013 to 2021.

Results: Although most of the H9N2 viruses analyzed showed low or no pathogenicity in mice, the leucine to glutamine substitution at residue 226 (L226Q) in the hemagglutinin (HA) protein rapidly emerged during the adaptation of H9N2 viruses, and was responsible for severe infections and even fatalities. HA amino acid 226Q conferred a remarkable competitive advantage on H9N2 viruses in mice relative to viruses containing 226L, increasing their virulence, infectivity, and replication.

Conclusion: Thus, our study demonstrates that the adaptive substitution HA L226Q rapidly acquired by H9N2 viruses during the course of infection in mice contributed to their high pathogenicity.