Objective
To characterize the genetic evolution and molecular characteristics of 17 H5N6 avian influenza virus (AIV) strains isolated from live poultry markets (LPMs) in Changsha.
Methods
From 2016 to 2023, environmental samples were collected from LPMs, nucleic acid were tested for type A influenza virus and H5/N6 subtypes using RT-PCR. Subsequently, 17 H5N6 AIV-positive specimens were subjected to next-generation sequencing (NGS). The sequencing samples were subjected to sequence similarity comparison using GISAID database, followed by comprehensive characterization of key amino acid residues, phylogenetic relationships, and molecular characteristics using MEGA-X software.
Results
The nucleotide similarity of HA genes of 17 H5N6 subtype avian influenza viruses ranged from 99.88% to 96.63%. The HA protein contained a polybasic cleavage site of RERRRKR↓GLF, which is characteristic of highly pathogenic avian influenza (HPAI) viruses. While the receptor-binding sites Q226 and G228 remained conserved and retained their affinity for avian-type preference for α2,3-linked sialic acid receptors; mutations at S123P, I155T, and T160A were identified, which may potentially enhance binding affinity to human-type α2,6-linked receptors and increased the transmission ability. The nucleotide similarity of NA genes of 17 H5N6 subtype avian influenza viruses ranged from 99.93% to 98.66%, with stalk region deletion residues 59-69, that has been associated with increased virulence in murine models. No mutations E119V, H274Y, R292K, or N294S conferring resistance to neuraminidase inhibitors were detected, suggesting retained susceptibility to oseltamivir and zanamivir. Phylogenetic analysis classified the isolates into multiple clades: 2.3.4.4b (n=3), 2.3.4.4e (n=1), 2.3.4.4h (n=5), 2.3.4.4g (n=3), and unclassified 2.3.4.4 (n=5), demonstrating significant genetic diversity in the HA gene among H5N6 viruses in Changsha LPMs.
Conclusion
From 2016 to 2023, there were highly pathogenic H5N6 AIVs in the LPMs in Changsha City. Some receptor binding sites have changed, and the virus has differentiated into multiple sub-lineages, and continuous monitoring is therefore necessary.