Abstract
Since their introduction into humans, H3N2 influenza A viruses have evolved continuously to escape immunity through antigenic drift, driven by mutations in and around the receptor-binding site. Recently, these changes resulted in viruses that recognize elongated glycans, which are less abundant in the human respiratory tract, complicating vaccine strain propagation. This study employed ELISA, glycan arrays, tissue staining, flow cytometry, and hemagglutinin (HA) assays to demonstrate the molecular determinants of recent H3N2 viruses that regained recognition of shorter glycans. Mutations Y159N/T160I in contemporary strains replace Y159/T160, weakening receptor binding. However, this is compensated by Y195F in the 190-helix. These findings highlight epistasis across critical residues in the HA receptor-binding site, including the 130-loop, 150-loop, and 190-helix. Interestingly, a positive correlation exists between binding to an asymmetrical N-glycan and binding to human and ferret respiratory tract tissues. These results elucidate the epistatic nature of receptor-binding specificity during influenza A virus H3N2 evolution.