Characterisation and haemagglutinin gene epitope mapping of a variant strain of H5N1 subtype avian influenza virus

H5N1 avian influenza virus (AIV) undergoes rapid evolution, and its antigenicity needs to be constantly evaluated in order to update the vaccine strain. In this report, a clade 7.2 AIV isolate named A/Chicken/Huadong/4/2008 (DT) is identified. Antigenic analysis revealed that DT had a significant low cross-reactive HI titre with antiserum against a clade 7 representative AIV, A/Chicken/Shanxi/2/2006 (Re-4). Animal experiments demonstrated that the Re-4 + Re-5 vaccine provided 80% protection against DT challenge in chickens. Antisera cross-reactivity showed that a mutant with a change of residues 129, 139, 140 in site A in the HA protein had reduced reactivity with DT antiserum and increased reactivity with Re-4 antiserum. Furthermore, residue Leu129 in site A of the HA protein was confirmed to be critical for maintenance of the reactivity with the DT antiserum, and Asn140, possessing a new glycosylation site, was confirmed to be critical for reducing reactivity with the Re-4 antiserum. These results imply that there is an antigenic drift within clade 7 viruses, and insertion and glycosylation of amino acid residues in site A of the HA protein may contribute to the antigenic variation.