Abstract

H9N2 avian influenza virus (AIV) remains a global threat to poultry health and has zoonotic potential. Antigenic drift in the hemagglutinin (HA) protein complicates vaccine efficacy and diagnostic accuracy, highlighting the need for precise epitope characterization. In this study, the HA protein of H9N2 AIV was expressed in a eukaryotic system, and two monoclonal antibodies (mAbs), 9C12 and 9F4, were generated. Both mAbs specifically bound HA, as shown by ELISA, Western blot, and immunofluorescence, but lacked hemagglutination inhibition activity. Epitope mapping revealed two minimal linear epitopes: 123 FSSSRSYQ 130 within the vestigial esterase domain and 201 NLYTRTDTT 209 within the receptor-binding domain. Alanine scanning revealed key residues required for antibody binding, whereas structural modeling confirmed that both epitopes are surface exposed. Sequence analysis demonstrated strong conservation across H9N2 strains, with the 9F4 epitope showing near-complete invariance, whereas both epitopes exhibited low conservation among other influenza A virus subtypes. These findings define two novel, nonneutralizing epitopes on H9N2 HA that expand the antigenic map and represent promising targets for subtype-specific diagnostic assays.