Amino acid mutations 60L, 212R in HA of H9N2 AIV improve replication capacity in chicken embryos and immunogenicity

H9N2 avian influenza virus (AIV) is a low-pathogenicity virus that primarily affects the respiratory system of poultry. It is globally prevalent and causes substantial economic losses to the poultry industry. Hemagglutinin (HA) is a major surface glycoprotein of influenza viruses, and amino acid mutations in HA play crucial roles in viral replication and antigenicity. In this study, we mutated amino acids 60L and 212R of the HA protein of the wild-type strain DSS, Using the influenza PR8 reverse genetics system, we rescued recombinant viruses to examine the effects of these mutations on the replication efficiency in chicken embryos and the immunogenicity of H9N2 AIV. Replication assays in chicken embryos revealed that both single and combined mutations at positions 60L and 212R increased the EID50 and number of viral copies. Cross hemagglutination inhibition (HI) and cross-neutralization tests indicated that the 60L, 212R, and combined mutations increased the virus-specific antibody titers. In the cross-protection test, inactivated vaccines prepared from the mutated recombinant viruses elicited higher antibody levels than those derived from the recombinant rDSS or wild-type virus. Furthermore, the inactivated vaccine derived from the mutated recombinant virus provided a higher immunoprotection rate than those based on rDSS or the wild-type strain DSS. This study demonstrates that the 60L and 212R mutations enhance both the replication capacity in chicken embryos and the immunogenicity of H9N2 AIV, which is of significance for the production of H9N2 AIV inactivated vaccines.