Amino acid mutations I69L, L222R in HA of H9N2 AIV improve replication capacity in chicken embryos and immunogenicity

we mutated amino acids 69L and 222R 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 69L and 222R increased the EID50 and number of viral copies. Cross hemagglutination inhibition (HI) and cross-neutralization tests indicated that the 69L, 222R, 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.