Phylogenetic Analysis
We performed a phylogenetic analysis of the H5N6 viruses sequenced in this study, along with 144 complete H5N6 genomes between 2016 and 2019 (available from GISAID [The GISAID Initiative, 2016]) (Table S1). The HA phylogeny revealed that nearly all of the H5N6 sequences fell within Clade 2.3.4.4 [Figure 1(A)]. The HA gene of the H5N6 strains can be further divided into two groups (major and minor). The time of differentiation of the two groups of the HA gene was calculated by BEAST during May 2014 (Figure S2). Strains from 2018 to 2019 were mostly distributed in the minor group from the collection time analysis of the sequence. Except for one strain A/duck/guangdong/17460/2017 in Clade H6N6, the neuraminidase (NA) phylogeny showed that all of the N6 genes from the Chinese poultry belong to the Eurasian lineage [Figure 1(B)]. The N6 genes could be further classified into two groups, whereas the H5N6 NA genes belong to two groups: H5N6-N6/Major and H5N6-N6/Minor.
Interestingly, 69 of 153 H5N6 viruses (45.1%) had HA, and NA genes from the H5N6-H5/Minor and H5N6-N6/Major lineages (H5/Minor:: N6/Major), 49 of 153 (32%) have the H5/Major:: N6/Major combination and 35 of 153 (22.9%) have the H5/Major:: N6/Minor combination[Figure 1(A)][Table S1]. All of H5/Major: N6/Minor viruses were isolated during 2016, the H5/Major::N6/Major virus were mainly isolated during 2016-2017, and only two of the H5/Major::N6/Major viruses were isolated in January 2018. The H5/Minor::N6/Major viruses were isolated during 2016-2019[Table S1]. Our results showed that there was a distinct time scale distribution of the HA-NA gene combinations and that the H5/Minor:: N6/Major H5N6 viruses were prevalent in China [Table S1][Figure 2]. Both HA and NA genes showed three gene differentiation events between 2016 and 2019 by ML and BL phylogenetic tree analysis results. The evolution of the H5 influenza virus is similar to that of seasonal influenza, which typically only divides into a few small branches and evolves in one direction. These data illustrate the rapid evolution of influenza viruses in the current vaccine immune environment.