Avian influenza A(H5N6) virus detected during live-poultry market surveillance linked to a human infection in Changsha, China, from 2020 to 2023

In November 2022, we reported a fatal case of human infection caused by a highly pathogenic avian influenza A(H5N6) virus bearing a clade 2.3.4.4b HA gene in Changsha City. We investigated the transmission route and distribution of the H5N6 virus in the largest live-poultry market (LPM), which is linked to the human infection. A total of 1357 samples from the LPM were collected for avian influenza A virus detection from 2020 to 2023. The proportion of LPM samples positive for H5 subtype avian influenza virus was 14.30% (194/1357). Sequences of H5N6 (n = 10) and H5N1 (n = 4) avian influenza viruses were obtained from the LPM samples using next-generation sequencing. The complete genome sequence of the H5N6 virus from the human infection case, A/Changsha/1/2022(EPI_ISL_16466440), was determined and analyzed. The PB1 and PB2 segments shared 99.65% and 99.23% sequence identity with A/duck/Hunan/S40199/2021(H5N6) and A/Whooper swan/Sanmenxia/H615/2020(H5N8), respectively. The other segments showed the highest sequence similarity to strain A/Guangdong/1/2021(H5N6), which was isolated in Guangzhou. L89V and I292V substitutions in the PB2 protein were predicted from the A/Changsha/1/2022 genome sequence. Phylogenetic analysis based on the HA gene showed that A/Changsha/1/2022 and other H5 subtype isolates obtained from the LPM grouped together in the 2.3.4.4b branch. Bayesian evolutionary analysis of the HA gene showed that clade 2.3.4.4b of the H5N6 virus is likely to have been prevalent in Hunan Province around October 2021. In conclusion, we confirmed that the clade 2.3.4.4b HA gene of A/Changsha/1/2022 virus recombined with those of local strains. These results demonstrate the importance of continuous surveillance of H5N6 influenza viruses.