Objective To sequence, identify H5N6 subtype avian influenza virus (AIV) from three environment samples from live poultry market and analyze the genetic characteristics of, so as to provide a basis for the prevention of human infection with H5N6 subtype AIV.

Methods The real-time fluorescence quantitative PCR amplification was used to determine the type of AIVs. The next generation sequencing method was used to sequence virus hemagglutinin (HA) gene and neuraminidase (NA) gene. The sequences were aligned by NCBI BLAST module, and the MEGA7.0 software was used to construct the phylogenetic tree and analyze the genetic characteristics.

Results The three AIV strains were identified as H5N6 subtype, belonging to clade 2.3.4.4 by sequencing. There were six consecutive basic amino acids (LRERRRKRGL, where R and K are basic amino acids) in the HA protein. The amino acids of in the receptor binding sites of 238-240 in the HA protein of the virus were QRG (amino acid sequence of H3-HA as reference). The receptor characteristics were avian origin. There was no stem deletion of amino acids at sites 69-73 of NA protein.

Conclusions A novel H5N6 subtype of AIV was identified in three environment samples that were positive of nucleic acid of H5 subtype AIV. The virus stains were high pathogenic AIV but without receptor binding characteristics feasible to infect human. The persistence of H5N6 virus in live poultry markets poses a threat to human health and public health. It is necessary to continue to strengthen the AIV surveillance.