HA198 mutations in H9N2 avian influenza: molecular dynamics insights into receptor binding

The H9N2 avian influenza virus is widely disseminated in poultry and poses a zoonotic threat, despite vaccination efforts. Mutations at residue 198 of hemagglutinin (HA) are critical for antigenic variation and receptor-binding specificity, but the underlying molecular mechanisms remain unclear. This study explores the molecular mechanisms by which mutations at the HA 198 site affect the antigenicity, receptor specificity, and binding affinity of the H9N2 virus. Using the sequence of the A/Chicken/Jiangsu/WJ57/2012 strain, we constructed recombinant H9N2 viruses, including rWJ57, rWJ57/HA , and rWJ57/HA , using reverse genetics. These variants were analyzed through hemagglutination inhibition (HI) assays, receptor-destroying enzyme (RDE) assays, enzyme-linked immunosorbent assays (ELISA) and solid-phase receptor binding assays. Additionally, molecular dynamics (MD) simulations were performed to further dissect the atomic-level interactions between HA and sialic acids (SA). The results demonstrated that HA mutations significantly altered the receptor-binding properties of the virus. Specifically, rWJ57 (HA ) exhibited 4-fold and 16-fold higher overall receptor-binding avidity compared to rWJ57/HA and rWJ57/HA , respectively. Furthermore, HA mutations significantly enhanced viral binding to human-type α2,6 SA receptors (