Mutation of D201G near the receptor binding site significantly drives antigenic drift of circulating H9N2 subtype avian influenza virus

The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused significant losses in chicken flocks throughout China. Our previous research has shown that field isolates of H9N2 underwent antigenic drift to evolve into distinct groups with significant antigenic divergence from the commercially available vaccines. The present study sought to identify which single mutations that have naturally appeared in isolates from the past 5 years have driven antigenic drift. Six high-frequency mutation sites in/near the receptor binding site region were screened by comparing amino acid alignments of the H9N2 AIVs isolated from China between 2014 and 2019. Two substitutions (A168N and D201G) were demonstrated to have a significant impact on the antigenicity but did not change the growth kinetics of the virus. It is worth noting that the D201G substitution not only significantly changed the antigenicity but also caused immune escape against the parental virus. In conclusion, A168N and D201G substitution are newly discovered determinants that can significantly change the antigenicity of H9N2 AIV, which should be tracked during outbreaks.