Objective To analyze the molecular mechanism of the improved replication of influenza A (H3N2) virus in chicken embryos during 2023-2024 in Changsha, Hunan province, and provide evidence for the screening of for vaccine strain.

Methods The positive samples of influenza A (H3N2) virus nucleic acid of 6 influenza like illness (ILI) cases during the influenza A (H3N2) epidemic in Changsha from 2023 to 2024 were selected and inoculated into dog kidney epithelial cells and specific pathogen-free chicken embryos for virus isolation. The virus titer was identified by red blood cell agglutination test and agglutination inhibition test. The full-length sequence of hemagglutinin (HA) gene was sequenced by the Illumina sequencing platform. The phylogenetic tree was constructed with neighbor-joining method by using MEGA 11.0 software. The amino acid mutations of antigenic determinants and receptor binding sites were analyzed by multiple sequence alignment, and the key sites related to chicken embryo adaptability were identified.

Results The HA titer of 6 H3N2 chicken embryo strains ranged from 1∶8 to 1∶64, and the positive rate of chicken embryo first-generation isolation was 83.33%. Phylogenetic analysis showed that the H3N2 epidemic strains in Changsha from 2023 to 2024 belonged to the 3C.2a1b.2a.2a.2a.3a.1 lineage and the sequence consistency with the vaccine strains recommended by World Health Organization from 2023 to 2024 was 99.10%−99.60%. Comparison with the HA protein sequence of the reference strain of chicken embryo dysplasia found H156S, Y159N, G186D, F193S and other amino acid mutation sites related to the development and adaptation of influenza virus in chicken embryos. In the passage in chicken embryos, H3N2 virus had two adaptive mutations of D186N and N246K in chicken embryos, which were not found in the antigenic determinant.

Conclusion From 2023 to 2024, the H3N2 strain spread in Changsha regained good growth and replication in chicken embryos through mutation of key amino acid sites in the HA protein receptor binding domain. The adaptive mutation D186N and N246K in chicken embryo has limited effect on viral antigenicity. This study provides molecular marker for vaccine strain screening, but the potential impact of these mutations on viral transmissibility and vaccine efficacy needs continuous monitor.