H5N6 avian influenza virus (AIV) has posed a potential threat to public health since its emergence in China in 2013. To understand the evolution and emergence of H5N6 in the avian population we performed molecular surveillance of live poultry markets (LPMs) in Wugang prefecture, Hunan province, in central China during 2014-2015. Wugang prefecture is located on the Eastern Asian-Australian migratory bird flyway and a human death due to an H5N6 virus was reported in the prefecture on 21th November 2016. In total, we sampled and sequenced the complete genomes of 175 H5N6 AIVs. Notably, our analysis revealed that H5N6 contains at least six genotypes arising from segment reassortment, including a rare variant that possesses an HA gene derived from H5N1 clade 2.3.2 and a novel NP gene that has its origins with H7N3 viruses. In addition, phylogenetic analysis revealed that genetically similar H5N6 AIVs tended to cluster according to their geographic region of origin. These results help reveal the evolutionary behavior of influenza viruses prior to their emergence in humans.

IMPORTANCE The newly emerged H5N6 influenza A virus has caused more than ten human deaths in China since 2013. In November 2016 a human death due to H5N6 was confirmed by the WHO in Wugang prefecture, Hunan province. To better understand the evolution and emergence of H5N6 we surveyed live poultry markets (LPMs) in Wugang prefecture before the reported human death, with a focus on revealing the diversity and genomic origins of H5N6 in birds during 2014-2015. In general, H5N6 viruses in this region were most closely related to H5N1 clade 2.3.4.4, with the exception of one virus with an HA gene derived from clade 2.3.2 such that it represents a novel reassortant. Clearly, the ongoing surveillance of LPMs is central to monitoring the emergence of pathogenic