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.