Molecular Evolution and Emergence of H5N6 Avian Influenza Virus in Central China

Abstract

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 AIV in the avian population, we performed molecular surveillance of live poultry markets (LPMs) in Wugang Prefecture, Hunan Province, in central China, during 2014 and 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 21 November 2016. In total, we sampled and sequenced the complete genomes of 175 H5N6 AIVs. Notably, our analysis revealed that H5N6 AIVs contain 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 tend to cluster according to their geographic regions of origin. These results help to 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 10 human deaths in China since 2013. In November 2016, a human death due to an H5N6 virus, in Wugang Prefecture, Hunan Province, was confirmed by the WHO. To better understand the evolution and emergence of H5N6 viruses, 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 and 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 influenza viruses.

Keywords: H5N6; avian influenza virus; evolution; molecular epidemiology; reassortment.

FIG 1

Time-scaled evolutionary history of H5N6 AIVs. (A) An MCC tree of the HA sequences of viruses sampled in China (n = 281) is shown, with the viruses collected in Wugang Prefecture (WG) highlighted in blue. A virus from a human case of H5N6 (A/Guangdong/ZQ874/2015) infection distributed within WG4 is marked with an asterisk. Shaded bars represent the 95% highest probability distribution for the age of each node. (B) Analysis of root-to-tip divergence against sampling date for the HA and NA gene segments. A group of clear outliers in the HA graph is marked (and was removed from the BEAST analysis).

FIG 2

Phylogenetic history of the H5N6 NA gene segment. This time-scaled phylogeny reveals two major groups: those with and without an 11-amino-acid deletion. The Wugang strains determined here are marked “WG.” Shaded bars represent the 95% highest probability distribution for the age of each node.

FIG 3

Genesis of H5N6 AIV. Virus particles are shown as colored ovals containing horizontal bars that represent the eight gene segments (from top to bottom: PB2, PB1, PA, HA, NP, NA, M, and NS). To illustrate the history of reassortant events, segments in descendant viruses are colored according to their corresponding source viruses.

FIG 4

Global phylogenetic history and geographic distribution of H5N6 AIVs. Viruses sampled from different locations (n = 487 in total), including Wugang (i.e., Hunan), are distinguished by their colors, and all reassortant viruses detected in this study are also indicated. All branch lengths are scaled according to the number of substitutions per site (subs/site). The tree is rooted using A/duck/Vietnam/LBM360c1-4-1/2013 (H5N6), which was collected on 6 February 2013.