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\begin{document}

\title{Gene differentiation, reassortment, and evolution of H5N6 Avian
influenza virus in China during 2016-2019}



\author[1]{xiao Li}%
\author[1]{jianglin Chen}%
\author[1]{jing Liu}%
\author[1]{lezi Yin}%
\author[1]{shumin Xie}%
\author[1]{mengmeng Zhang}%
\author[1]{meifeng Liang}%
\author[1]{siru Lin}%
\author[1]{yiqi Liao}%
\author[1]{xuanjiang Jin}%
\author[1]{jingkai Hu}%
\author[1]{jinfeng Wang}%
\author[1]{yifan Wu}%
\author[1]{wenbao qi}%
\author[2]{Ming Liao}%
\author[2]{weixin Jia}%
\affil[1]{SCAU}%
\affil[2]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 28, 2024}


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\selectlanguage{english}
\begin{abstract}
Continuous monitoring and analysis of the evolution, recombination and
epidemic of avian influenza virus will help to prevent and control the
emergence of new avian influenza virus. The genes of 153 H5N6 avian
influenza viruses selected from our long-term surveillance data and the
GISAID for the 2016-2019 period were dissected in detail. Our analysis
shows that the genes of H5N6 AIVs have been divided into more lineages,
and that mutations leading to amino acid replacement of hemagglutinin
and neuraminidase occurred mainly between 2017 and 2018. All H5N6
viruses are differentiated into at least 20 distinct genotypes derived
from different evolutionary pathways. G1 genotype has replaced other
genotypes to become dominant genotype of AIV. The results of animal
experiments show that H5N6 of different genotypes has high virulence to
mice. which indicates that H5N6 AIVs, especially the reassortment
strains, are a potential threat to public health.%
\end{abstract}%



\sloppy




\textbf{Background}

Influenza A viruses are RNA viruses.To date, 18 major antigenic variants
of HA and 11 of NA have been detected(Fouchier et al., 2005; Tong et
al., 2012). In addition, more than 20 other mammalian host species as
well as various poultry species can be infected(Wille \& Holmes, 2019).
The first outbreak of the highly pathogenic H5N1 avian influenza was
reported in Guangdong Province in 1996. The following year human
infection of the H5N1 virus was reported in Hong Kong(Xu,Subbarao,Cox,
\& Guo, 1999). After that, the viral genes divided rapidly and produced
multiple lineages (Clades 0-9)(Duan et al., 2008). Reassortments with
other low pathogenic avian influenza produced numerous genotypes. Clade
2.3.2 H5N1, which is associated with the outbreak of avian flu of Lake
Qinghai, China, 2005, astonished the world, in that migratory birds were
observed with the infection, and over 6,000 birds died. Then, Clade
2.3.2 viruses evolved into Clade 2.3.2.1, which diversified into
2.3.2.1a, b, and c in 2009(Bi,Chen, \& Zhang et al., 2016; Bi,Chen, \&
Zhang et al., 2016). The first isolation of Clade 2.3.3.4 was reported
in Eastern China in 2010, and then spread to all parts of the
world(Lee,Bertran,Kwon, \& Swayne, 2017). Bi et al. reported that H5 has
reassorted with H6N6 and H9N2/H7N9 from 2013 to 2016, resulting in a
large number of genotypes. In China, 34 genotypes have been
identified(Bi,Chen, \& Wang et al., 2016). In recent years, clade2.3.4.4
and clade2.3.2.1 still can be detected in China(Bi et al., 2016; Sun et
al., 2016). According to the ministry of agriculture of China, six
outbreaks of highly pathogenic avian influenza have occurred in Sichuan,
Xinjiang, and Hunan, China in January and February of 2020, five of
which were caused by H5N6 (including four outbreaks in wild birds) and
one by H5N1. A total of 6,340 poultry died, and 20,089 were culled. Two
poultry outbreaks resulted in the deaths of 6, 340 poultry, and a total
of 20,089 poultry were disposed of in a bio-safety way. Four outbreaks
killed 48 wild birds(http://www.moa.gov.cn/gk/yjgl\_1/yqfb/).

Bi et al. studied the epidemiology of H5N6 in China from 2013 to 2016,
and analyzed in detail the geographic distribution and reassortment
evolution of the virus in live poultry markets(Bi et al., 2016). They
and many other studies have shown that live poultry trade markets play
an essential role in the proliferation of viruses, and people who work,
live, or have long-term contacts with the live poultry trade market
(LPM) are more likely to be infected by the virus than the general
population(Gao, 2014; Kim et al., 2018; Shi et al., 2018). The LPMs have
played an essential role in the evolution and reassortment of avian
influenza(Qi et al., 2018; Shi et al., 2018). The constant surveillance
of the LPM is an important procedure in the epidemiological study of
avian influenza and public health safety.With this in mind, we collected
oropharyngeal and cloacal swabs from an LPM every two weeks in the
Guangdong province and samples from poultry farmers in other provinces
of China during 2016-2019. We used these data to analyze in detail the
genetic evolution reassortment ,prevalence and pathogenicity in mice of
H5N6 avian influenza virus.

\textbf{Research methods}

\textbf{Sample collection and virus isolation}

We collected oropharyngeal and cloacal swabs of poultry (including
chickens, ducks, and geese) from live poultry markets in the Guangdong
Province during 2016-2019, which were kept in PBS with penicillin and
streptomycin. We also collected swabs or tissue samples from sick or
dead poultry in farms. The collected swabs were centrifuged, and 0.2 ml
of the supernatant was inoculated into nine to 10-day-old SPF chicken
embryos. After 48-72 hours of culture at 37\selectlanguage{ngerman}°C, the allantoic fluid was
collected, and Hemagglutinin-positive samples were further confirmed.

\textbf{Sequence and phylogenetic analysis}

A Simply P total RNA extraction kit was used to extract RNA according to
manufacturer instructions. The reverse transcription was performed in a
total volume of 60 \selectlanguage{greek}μ\selectlanguage{english}L containing 39 \selectlanguage{greek}μ\selectlanguage{english}L of RNA, 3 \selectlanguage{greek}μ\selectlanguage{english}L of dNTPs, 3 \selectlanguage{greek}μ\selectlanguage{english}L of 12
bp (10 nmol), 1.5 \selectlanguage{greek}μ\selectlanguage{english}L of M-MLMLV, 1.5 \selectlanguage{greek}μ\selectlanguage{english}L of RRI, and 12 \selectlanguage{greek}μ\selectlanguage{english}L of 5*buffer.
The liquid was mixed, then placed in a 42\selectlanguage{ngerman}°C water bath for 2 to 4 hours.
We used published universal primers and primers designed for different
subtypes in the literature, as well as primers designed based on the
downloaded sequences from the influenza database (www.gisaid.org).

PCR amplification of cDNA was performed in a total volume of 50 \selectlanguage{greek}μ\selectlanguage{english}L and
contained 25 \selectlanguage{greek}μ\selectlanguage{english}L of Premix Taq, 20 \selectlanguage{greek}μ\selectlanguage{english}L of dd H\textsubscript{2}O, 4 \selectlanguage{greek}μ\selectlanguage{english}L of
upstream and downstream primers, and 1 \selectlanguage{greek}μ\selectlanguage{english}L of template cDNA. The PCR
program comprised 34 cycles of 95\selectlanguage{ngerman}°C for 50 s, 55°C for 50 s, and 72°C
for 1 m and 50 s. The PCR products were separated on 1\% agarose gels by
electrophoresis and visualized using a UV transilluminator. The sequence
was measured by generation 1 sequencing technology, and the sequence
result was processed with the Lasergene 7.1 software package.

The sequences of partial H5 subtype AIVs with whole genes from 2016 to
2019 were collected from the Global Initiative on Sharing Avian
Influenza Data (GISAID) database (www.gisaid.org). We used PhyloSuite
data processing software for constructing phylogenetic trees(Dong Zhang
\& Wang, 2019) . MAFF (Katoh,Misawa,Kuma, \& Miyata, 2002), which is
included in the software, was used to compare the nucleotide sequences.
Modefinder was used to find the best model and using maximum likelihood
in IQ-tree (Kalyaanamoorthy,Minh,Wong,von Haeseler, \& Jermiin, 2017;
Nguyen,Schmidt,von Haeseler, \& Minh, 2015), eight phylogenetic trees
were constructed. ITOL was used to beautify the phylogenetic tree
(https://itol.embl.de).

We calculated the timescale of avian influenza virus clades and
evolutionary rates using BEAST 1.1.04 (Suchard et al., 2018). import
eight sequence fragments, tag sequences of each sampling time, chose a
relaxed molecular clock model, set MCMC chain length at 500000000. The
acquisition interval was 50,000 and resulted in 10,000 trees eventually.
The generated XML file was run by BEAST, and the log file was opened by
Tracer1.7.1(Rambaut,Drummond,Xie,Baele, \& Suchard, 2018) to verify that
the ESS value of each parameter was \textgreater{} 200. We opened the
generated tree file using TreeAnnotator v1.10.4 software, burn in 10\%
to generate the tree file, and placed it in Figtree 1.4.4 to
open(http://tree.bio.ed.ac.uk/software/figtree/).

\textbf{Discriminant analysis of principal components (DAPC) analysis}

Year division, sequence comparison, deletion or incomplete fragments of
influenza sequences by MAGE, preparation of a matched sequence file, and
a file containing the classification group names, analysis the sequence
using the DAPC extension package in the R language(Jombart,Devillard, \&
Balloux, 2010).

\textbf{Animal experiment}

BALB/c mice (14-16 g) were divided into five groups, four of which were
the challenge group, and one was the control group, with 12 mice in each
group. The H5 subtype influenza virus was diluted to
10\textsuperscript{6}EID\textsubscript{50}. After anesthesia with
isoflurane, the virus was dripped into the nasal cavity. Twelve mice in
each group were observed, and we recorded the mortality and body weight
for 14 days. On the third and fifth day after the attack, we took three
mice from each group randomly and collected the heart, liver, spleen,
lung, kidney, brain, and turbinate bone to detect the virus titer in
each organ.

\textbf{Results}

\textbf{Sample Collection}

We collected samples of Oropharyngeal and cloacal swabs from live
poultry markets in the Guangdong Province, including Guangzhou,
Shenzhen, Foshan, and Zhanjiang cities. A small number of samples of
clinically suspected avian influenza were collected from other Provinces
(including Jiangsu, Guangdong, Shandong, Henan, Hebei, Guangxi, and
Henan) during 2017-2019, China. All of the samples were inoculated into
9-day-old embryonated chicken eggs for virus isolation individually. The
HA subtype of the viruses was confirmed using the hemagglutinin
inhibition (HI) test. H5N6 strains were isolated from live poultry
markets. In addition, H9N2(Jin et al., 2020), H7N9(Qi et al., 2018),
H6N6, H6N2, H3N2, H3N8(Li et al., 2019), H1N1, and other subtypes were
also isolated. In this study, ten strains of the H5 subtype avian
influenza were selected to sequence the full gene sequence, and four
strains were selected to determine the virulence by developing animal
experiments{[}EPI\_ISL\_402100, EPI\_ISL\_422383-EPI\_ISL\_422396{]}.

\textbf{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.

\textbf{Genetic Reassortment and Virus Evolution}

We performed phylogenetic analyses of the internal genes of H5N6 viruses
and classified them into different lineages according to tree topology
and Bayesian phylogenetic analyses {[}Figure S1{]}{[}Figure S2{]}. Each
internal gene belongs to at least two lineages, one from circulating H5
AIVs and the other from LPAIVs, including H6 and H9N2/H7N9. In general,
the H5/Minor::N6/Major viruses and H5/Major::N6/Major viruses have
nearly all of the internal genes derived from H5N6 viruses, 23 of 34 of
the H5/Major::N6/Minor H5N6 had H9N2/ H7N9-derived internal genes
{[}Figure 1(A){]}. Some H5N6 isolates contained internal gene(s) from
LPAIVs circulating in waterfowl. According to lineage classification of
phylogenetic trees and Bayesian phylogenetic analyses, we classified all
of the H5N6 viruses into 20 genotypes {[}Figure 2{]}{[}Table S1{]}.
These genotypes are G1(53), G1.1(4), G1.2(4), G1.3(1), G1.4(4), G1.5(3),
G2(9), G2.1(30), G2.2(3), G3(1), G3.1(17), G3.2(3), G3.3(3), G3.4(2),
G3.5(4), G3.6(3), G3.7(1), G3.8(1), G2.1.1(6), and G2.1.2(1){[}Figure
2{]}. 81 of 153 comprised 15 genotypes have been isolated since 2016, 39
of the viruses were classified into six genotypes (G1(28), G1.1(2),
G1.2(1), G1.3(1), G2.1(7)) and had been isolated during 2017, and 27 of
153 virus comprised six genotypes (G1(17), G1.1(2), G1.2(3), G1.5(3),
and G2.1(2)) and had been isolated during 2018. Only six viruses (G1(6))
were isolated during 2019 {[}Figure 2{]}. The number of genotypes
decreased between 2016 and 2019 in terms of the genetic diversity of the
viruses, which means that the strain may have evolved to become more
stable. The G1 genotype HA-NA and the combination of internal genes may
be best suited to the current adaptation of the strains.

Bi et al. showed that, from 2013 to 2016, a large number of H5N6
reassortment strains emerged(Bi et al., 2016). Our research confirmed
that the H5N6 reassortment strain types in 2016 were very numerous. By
contrast, the genotypes of this strain gradually decreased from 2017 to
2019 {[}Figure 2{]}. Influenza mainly occurs in winter, and we set July
2015 to June 2016 as one influenza season. It can be seen that all of
the strains of the G3 series mostly (30/33) occurred in this winter.
Many genotypes (G3.1, G3.2, G3.3, G3.4, G3.5, G3.6, G3.7, and G3.8)
emerged by the G3 strain reassortment with H9N2 and other subtypes of
AIVs{[}Table S1{]}. The frequency of G3.1 posed a major advantage. Only
three strains of G3 were isolated during the subsequent influenza
season. The G2 and G2.1 strain appeared in early 2016 and the winter of
2016. In the winter of 2016, two reassortment genotypes, G2.1.1 and
G2.1.2, emerged by the reassortment of G2.1 and H9N2 with other subtype
strains and G2 with H9N2. Four genotypes (G1.1, G1.2, G1.3, and G1.4)
emerged by G1 reassorted with H6 and other subtypes of the AIVs during
July 2017 and June 2018. Only the G1.4 genotype and the new G1.5
genotype were isolated in the winter of 2018 {[}Figure 2{]}{[}Table
S1{]}.

\textbf{DAPC and Mutation of Surface Genes}

The HA and NA proteins of AIVs play a critical role in virulence,
transmissibility, and antigenicity. The variation and combinations
between HA and NA also primarily determined the pandemic strain of the
virus. We first divided the 2016-2019 strain into epidemic seasons (that
is, 2015.7\selectlanguage{english}-2016.6 as a popular season, and 2016.7-2017.6 as the next
popular season). We calculated the changes of HA and NA in four popular
seasons from 2016 to 2019 through DAPC analysis. The horizontal axis
represents the first principal component, and the vertical axis
represents the second principal component. It can be seen that the
strains are clustered in each epidemic season {[}Figure 3{]}.

The HA principal component analysis showed a significant difference in
the first principal component in 2016, 2017, and 2018. The 2019 strain
showed a greater distance from the 2018 strain in the second principal
component, while the difference in the first principal component was
smaller {[}Figure 3(A){]}. This means that the variation of the strain
varies significantly from 2016 to 2018 yearly, in contrast to the
smaller variation in 2019. This variation also largely represents the
evolution and antigenicity of the strain. The principal component
analysis of NA was similar to that of HA. The difference in the first
principal component between 2016 and 2017 is increasing, There is no
significant difference in the first principal component between the 2018
strain and the 2019 strain, but there are some significant differences
in the second principal component {[}Figure 3(B){]}.

A total of 22 amino acid sites in the HA gene and 13 amino acid sites in
the NA gene with obvious variation were identified from 2016 to 2019 and
were compared and analyzed {[}Figure 4(A)(B){]}. The results showed that
the amino acid sites changed regularly. The HA amino acid changes were
mainly focused on the HA1 area, positions 83, 115, 120, 123, 126, 127,
138, 140, 141, and 269 have been proven to cause antigenic drift in
other strains of research (Beato et al., 2013; Velkov et al., 2013),
which may cause significant changes in the antigenicity of the virus.

In summary, we believe that the virus has undergone a rapid evolution
from 2016 to 2018, that the amino acid substitutions were basically
completed in 2017\selectlanguage{english}-2018 {[}Figures 3, 4{]}, and that the strain has
basically evolved to adapt itself to the environment. Consequently, the
strain changed considerably less in 2019. We suppose that the reason for
the big difference in strains from 2016 to 2018 may be related to the
current vaccination environment. Vaccines are constantly being updated.
From 2017, the H5-H7 dual vaccine was mainly used for H5 and H7N9.
Research by Shi et al. showed that the vaccine controlled the outbreak
of H7N9 to a large extent and greatly reduced the cases of human
infection with the virus(Shi et al., 2018). We speculate that H5N6 has
evolved to adapt to the current vaccine immune environment.

\textbf{H5N6 Has Become Highly Virulent in Mice}

To understand the pathogenicity of the different epidemic H5N6 of avian
influenza to mice, we selected three H5N6 strains
(A/duck/jiangsu/18012/2018, A/duck/guangdong/18217/2018,
A/chicken/guangdong/18231/2018) and one H5N1 strain
(A/duck/shandong/17771/2017) as control, hereinafter referred to as
18012,18217,18231,17771 respectively) to infect in mice and detect the
EID\textsubscript{50} in the different organs.

Three strains of H5N6 showed different virulence in mice.18012 had the
strongest virulence among the four strains as 50\% of the mice died
within three days of challenge, and all of the mice died in four days of
challenge {[}Figure 5 A, B{]}. 18012 maintained a high virus
concentration in the lungs and brain in the 3-5 dpi period. The strain
17771 was slightly less virulent than 18012,all of the mice died in 5
days of post-challenge.18217 showed a moderate virulence in mice, which
caused partial death at the 7 dpi, 30\% survived in 14 dpi. 18231 showed
a weak virulence in mice, as 100\% were alive 14 dpi {[}Figure 5A, B{]}
and only showed decreased body weight. Four strains had detected
different viral levels in different organs. And the concentration was
highest in the lungs. Strains 17772 and 18012 were detected in the brain
at 3 dpi, whereas strains 17772, 18012, and 18217 were detected in the
brain at 5 dpi {[}Figure 5C, D{]}. This suggests that 17772, 18012, and
18217 can cross the blood\selectlanguage{english}-brain barrier.

\textbf{Discussion}

Highly pathogenic AIVs have been affecting the world's poultry industry
and also brought enormous challenges to the public health security since
they accidentally moved from animals to human, causing a first outbreak
in Guangdong, China, in 1996(Shortridge et al., 1998; Webster \&
Govorkova, 2014; Xu et al., 1999). Fortunately, there is no evidence of
human-to-human transmission of the avian influenza virus. The H5 avian
influenza virus has evolved rapidly from Clade 0 to the present Clade
2.3.4.4 and Clade 2.3.2.1(Group, 2012), and frequent reassortments of
AIVs have been the leading cause of several epidemics(Bi et al., 2016;
Karo-karo et al., 2019; Qi et al., 2018; Shi et al., 2018). The highly
pathogenic avian influenza virus prevalence in China is an
extraordinarily complicated problem to solve. We focused on the
prevalence, gene differentiation, and reassortment of H5N6 in China,
based on the available data for 2016\selectlanguage{english}-2019. We gathered evidence on a new
reassortment virus complex diversity produced by frequent reassortment
of different subtypes AIVs. Cases of both poultry and human infections
are largely related to the complexity of the reassortment.

The rate of evolution of avian influenza also deserves close attention.
The rate of evolution of H5N6 HA and NA genes is 7.3262E-03 and
6.9073E-03, respectively. And the rate of evolution of H9N2 HA and NA is
2.19E-03-2.83E-03, and is 3.3E-03-3.7E-03, respectively(Jin et al.,
2020). H5N6 compared to H9N2 has a faster rate of evolution in HA and NA
genes.This has a lot to do with the clade of HA and NA genes rapid
differentiation is closely linked. And internal gene is relatively
complicated, due to the restructuring of the LAIVs, especially the part
of basic internal genes from H6N6, H6N2, H9N2, most notably the two H6N6
viruses from which the immune pressure is relatively small. According to
the study of Bi et al(Bi et al., 2016), the PB1 gene of H5N6 is
basically derived from H5N1, and PB2 is partly derived from H6N6, which
may be the reason why our study showed that PB1 has significant gene
differentiation compared with other genes.

In this study, a total of 16 gene sequences from humans {[}Figure 6
A{]}, including eight strains isolated during 2016 (six isolates from
January to April and two from November and December), two isolated since
November, seven of eight strains were new reassortment H5N6 viruses that
contain the internal genes fragments from H9N2 {[}Figure 6 A{]}, two
strains isolated during 2017 which are of the G1 genotype, and six
strains isolated during 2018. These last six strains comprise three
strains with the G1 genotype and three with the G1.4 genotype, and
differed from G1 due to a PA gene from other AIVs. We propose that the
virus can move from poultry to humans after new reassortment. The LPAIV
provides the gene segments for the reassortment event, which is a
problem that cannot be ignored. The current epidemic strain is of the G1
genotype, which is of great importance because G1 can also infect
humans. From our mice experimental data, all of the strains are H5N1,
except one, three H5N6 belong to the G1, G1.2, and G1.4 genotypes, and
show differences virulence in mice. Apparently, one strain is unable to
represent all of the genotype fully, but what caught our attention is
that H5N6 shows high virulence, which potentially increases the risk of
infection to humans.

Waterfowl have long been considered the natural reservoir of avian
influenza, and our research results show that the duck, goose, and wild
bird waterfowl contain numerous genotypes, respectively 8, 3, and 3
{[}Figure 6B{]}. Two major epidemic genotypes, G1 and G2.1, which are
distributed in the three kinds of waterfowl. There were no G2.1
genotypes in human and chicken sources, and we only found H9N2 gene
reassortment strains isolated from chicken, people, and the environment.
We speculate that the virus could have spread from ducks to chickens
with H9N2 reassorting and the production of the G2.2, G3.1, and G3.3 new
genotypes. The virus could then have spread to people through chickens
or the environment. G1.4 and G3.2 are distributed only in ducks, humans,
and the environment, and from ducks or the environment can spread to
people {[}Figure 6B{]}. The pandemic genotypes G1 and G2 are distributed
in wild birds, which probably means that the virus continues to
circulate in wild birds and waterfowl. The contact between waterfowl and
landfowl mainly occurs in LPMs, which are the main place for virus
reassortment and cross-host transmission of avian influenza. Both
previous research and the work of our group confirm this scenario.

H5N1 and H5N6 can be highly pathogenic and induce high mortality in
chicken. Vaccination is one of our main measures against the highly
pathogenic avian influenza(Alarcon et al., 2018; Arai et al., 2019; Shi
et al., 2018). The Chinese vaccine is upgraded continuously, following
the evolution of the influenza virus, and the vaccine can protect
poultry effectively, reducing the virus isolation rate greatly. However,
there are still outbreaks of HPAIV every year, according to statement
from the Ministry of Agriculture. Our laboratory has received from the
farm of clinically suspected cases of AIV. Through analysis, we
confirmed that the part of the clinically suspected case really is
HPAIV, and most isolated strains from the clinically suspected cases are
reassortment viruses, which may explained why there are still bits of
outbreaks of HPAIV. Some may unuseed vaccines,and these reassortment
strains may escape the vaccine's immune response. A large number of
reassortment viruses can be detected each year. And the vaccine
obviously cannot resist all of the new reassortment viruses. Even so,
occasional outbreaks can be addressed by rapid culling, disinfection,
and landfills, and the effect of preventing the spread of the virus is
also significant. Thus, we need to raise enough awareness about the
occasional outbreak, and only quick and effective prevention measures
can prevent a possible outbreak.

\textbf{Conflict of interest}

The authors declare not conflict of interest.

\textbf{Acknowledgments}

We acknowledge the authors, originating and submitting laboratories of
the sequences from GISAID's EpiFlu Database. This work was supported by
China National Natural Science Foundation (31830097, 31972709), China
Agriculture Research System (CARS-41-G16\selectlanguage{english}), China National Animal
Disease Surveillance and Epidemiological Survey Program (No.201947).

We thank LetPub (www.letpub.com) for its linguistic assistance during
the preparation of this manuscript.

\textbf{Author Contributions}

Xiao Li, Jianglin Chen, Jing liu, Lezi Yin, Shumin Xie, Mengmeng Zhang,
Siru lin, Yiqi liao, Meifeng liang, Xuanjiang Jin, Jingkai Hu, Jinfeng
Wang and Yifan Wu. conducted the experiments. Xiao Li and Weixin Jia
analyzed the data; Xiao Li, Wenbao Qi, Ming Liao and Weixin Jia wrote
the paper.

\textbf{Data Availability Statement}

Used in this study data are derived from the Global Initiative on
Sharing Avian Influenza Data (GISAID's EpiFlu Database), Moreover, the
sequence data generated in this study has been uploaded to GISAID's
EpiFlu Database{[}EPI\_ISL\_402100,
EPI\_ISL\_422383-EPI\_ISL\_422396{]}.

\textbf{Ethics Statement}

The authors confirm that the ethical policies of the journal, as noted
on the journal's author guidelines page, have been adhered to and the
appropriate ethical review committee approval has been received. The
South China agricultural university's guidelines for the Care and Use of
Laboratory Animals were followed.

\textbf{References:}

Alarcon, P., Brouwer, A., Venkatesh, D., Duncan, D., Dovas, C. I.,
Georgiades, G.,\ldots{} Brown, I. H. (2018). Comparison of 2016--17 and
Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong
Lineage in Europe. \emph{Emerging Infectious Diseases,}
24(12)\textbf{,}2270-2283.doi:10.3201/eid2412.171860

Arai, Y., Ibrahim, M. S., Elgendy, E. M., Daidoji, T., Ono, T., Suzuki,
Y.,\ldots{} Watanabe, Y. (2019). Genetic Compatibility of Reassortants
between Avian H5N1 and H9N2 Influenza Viruses with Higher Pathogenicity
in Mammals.\emph{Journal of Virology,} 93(4).doi:10.1128/JVI.01969-18

Beato, M. S., Mancin, M., Yang, J., Buratin, A., Ruffa, M., Maniero,
S.,\ldots{} Capua, I. (2013). Antigenic characterization of recent H5N1
highly pathogenic avian influenza viruses circulating in Egyptian
poultry.\emph{Virology,}
435(2)\textbf{,}350-356.doi:10.1016/j.virol.2012.09.016

Bi, Y., Chen, J., Zhang, Z., Li, M., Cai, T., Sharshov, K.,\ldots{} Gao,
G. F. (2016). Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c
virus in migratory birds, 2014-2015. \emph{Virologica Sinica,}
31(4)\textbf{,}300-305.doi:10.1007/s12250-016-3750-4

Bi, Y., Chen, J., Zhang, Z., Li, M., Cai, T., Sharshov, K.,\ldots{} Gao,
G. F. (2016). Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c
virus in migratory birds, 2014--2015. \emph{Virologica Sinica,}
31(4)\textbf{,}300-305.doi:10.1007/s12250-016-3750-4

Bi, Y., Chen, Q., Wang, Q., Chen, J., Jin, T., Wong, G.,\ldots{} Gao, G.
F. (2016). Genesis, Evolution and Prevalence of H5N6 Avian Influenza
Viruses in China. \emph{Cell Host \& Microbe,}
20(6)\textbf{,}810-821.doi:10.1016/j.chom.2016.10.022

Dong Zhang, F. G. I. J., \& Wang, W. X. L. G. (2019). PhyloSuite: An
integrated and scalable desktop platform for streamlined molecular
sequence data management and evolutionary phylogenetics
studies.doi:10.1111/1755

Duan, L., Bahl, J., Smith, G. J. D., Wang, J., Vijaykrishna, D., Zhang,
L. J.,\ldots{} Guan, Y. (2008). The development and genetic diversity of
H5N1 influenza virus in China, 1996--2006. \emph{Virology,}
380(2)\textbf{,}243-254.doi:10.1016/j.virol.2008.07.038

Fouchier, R. A. M., Munster, V., Wallensten, A., Bestebroer, T. M.,
Herfst, S., Smith, D.,\ldots{} Infektionssjukdomar. (2005).
Characterization of a novel influenza A virus hemagglutinin subtype
(H16) obtained from black-headed gulls. \emph{Journal of Virology,}
79(5)\textbf{,}2814.doi:10.1128/JVI.79.5.2814

Gao, G. F. (2014). Influenza and the Live Poultry Trade.
\emph{Science,}344(6181)\textbf{,} 235.doi:10.1126/science.1254664

Group, W. O. F. H. (2012). Continued evolution of highly pathogenic
avian influenza A (H5N1): updated nomenclature. \emph{Influenza and
Other Respiratory Viruses,}
6(1)\textbf{,}1-5.doi:10.1111/j.1750-2659.2011.00298.x

Jin, X., Zha, Y., Hu, J., Li, X., Chen, J., Xie, S.,\ldots{} Jia, W.
(2020). New molecular evolutionary characteristics of H9N2 avian
influenza virus in Guangdong Province, China. \emph{Infection, Genetics
and Evolution,}77\textbf{,} 104064.doi:10.1016/j.meegid.2019.104064

Jombart, T., Devillard, S., \& Balloux, F. (2010). Discriminant analysis
of principal components: a new method for the analysis of genetically
structured populations. \emph{Bmc Genetics,}
11\textbf{,}94.doi:10.1186/1471-2156-11-94

Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., \&
Jermiin, L. S. (2017). ModelFinder: fast model selection for accurate
phylogenetic estimates. \emph{Nature Methods,}
14(6)\textbf{,}587-589.doi:10.1038/nmeth.4285

Karo-karo, D., Bodewes, R., Wibawa, H., Artika, M., Pribadi, E. S.,
Diyantoro, D.,\ldots{} Koch, G. (2019). Reassortments among Avian
Influenza A(H5N1) Viruses Circulating in Indonesia, 2015--2016.
\emph{Emerging Infectious Diseases,} 25(3)\textbf{,}
465-472.doi:10.3201/eid2503.180167

Katoh, K., Misawa, K., Kuma, K., \& Miyata, T. (2002). MAFFT: a novel
method for rapid multiple sequence alignment based on fast Fourier
transform.\emph{Nucleic Acids Research,}
30(14)\textbf{,}3059-3066.doi:10.1093/nar/gkf436

Kim, Y., Biswas, P. K., Giasuddin, M., Hasan, M., Mahmud, R., Chang, Y.
M.,\ldots{} Fournie, G. (2018). Prevalence of Avian Influenza A(H5) and
A(H9) Viruses in Live Bird Markets, Bangladesh. \emph{Emerging
Infectious Diseases,} 24(12)\textbf{,}
2309-2316.doi:10.3201/eid2412.180879

Lee, D. H., Bertran, K., Kwon, J. H., \& Swayne, D. E. (2017).
Evolution, global spread, and pathogenicity of highly pathogenic avian
influenza H5Nx clade 2.3.4.4. \emph{Journal of Veterinary
Science,}18(S1)\textbf{,} 269-280.doi:10.4142/jvs.2017.18.S1.269

Li, X., Chen, J., Jin, X., Hu, J., Xie, S., Dai, Y.,\ldots{} Jia, W.
(2019). Characterization of three H3N2 and one new reassortant H3N8
avian influenza virus in South China. \emph{Infection, Genetics and
Evolution,} 75\textbf{,} 104016.doi:10.1016/j.meegid.2019.104016

Nguyen, L., Schmidt, H. A., von Haeseler, A., \& Minh, B. Q. (2015).
IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating
Maximum-Likelihood Phylogenies. \emph{Molecular Biology and
Evolution,}32(1)\textbf{,} 268-274.doi:10.1093/molbev/msu300

Qi, W., Jia, W., Liu, D., Li, J., Bi, Y., Xie, S.,\ldots{} Liao, M.
(2018). Emergence and Adaptation of a Novel Highly Pathogenic H7N9
Influenza Virus in Birds and Humans from a 2013 Human-Infecting
Low-Pathogenic Ancestor. \emph{Journal of Virology,} 92(2

).doi:10.1128/JVI.00921-17

Rambaut, A., Drummond, A. J., Xie, D., Baele, G., \& Suchard, M. A.
(2018). Posterior Summarization in Bayesian Phylogenetics Using Tracer
1.7.\emph{Systematic Biology,}
67(5)\textbf{,}901-904.doi:10.1093/sysbio/syy032

Shi, J., Deng, G., Ma, S., Zeng, X., Yin, X., Li, M.,\ldots{} Chen, H.
(2018). Rapid Evolution of H7N9 Highly Pathogenic Viruses that Emerged
in China in 2017. \emph{Cell Host \& Microbe,}
24(4)\textbf{,}558-568.doi:10.1016/j.chom.2018.08.006

Shi, J., Deng, G., Ma, S., Zeng, X., Yin, X., Li, M.,\ldots{} Chen, H.
(2018). Rapid Evolution of H7N9 Highly Pathogenic Viruses that Emerged
in China in 2017. \emph{Cell Host \& Microbe,}
24(4)\textbf{,}558-568.doi:10.1016/j.chom.2018.08.006

Shortridge, K. F., Zhou, N. N., Guan, Y., Gao, P., Ito, T., Kawaoka,
Y.,\ldots{} Webster, R. G. (1998). Characterization of Avian H5N1
Influenza Viruses from Poultry in Hong Kong. \emph{Virology,}
252(2)\textbf{,}331-342.doi:https://doi.org/10.1006/viro.1998.9488

Suchard, M. A., Lemey, P., Baele, G., Ayres, D. L., Drummond, A. J., \&
Rambaut, A. (2018). Bayesian phylogenetic and phylodynamic data
integration using BEAST 1.10. \emph{Virus Evol,} 4(1)\textbf{,}
y16.doi:10.1093/ve/vey016

Sun, H., Pu, J., Hu, J., Liu, L., Xu, G., Gao, G. F.,\ldots{} Liu, J.
(2016). Characterization of clade 2.3.4.4 highly pathogenic H5 avian
influenza viruses in ducks and chickens. \emph{Veterinary
Microbiology,}182\textbf{,} 116-122.doi:10.1016/j.vetmic.2015.11.001

Tong, S., Li, Y., Rivailler, P., Conrardy, C., Castillo, D. A. A., Chen,
L. M.,\ldots{} Donis, R. O. (2012). A distinct lineage of influenza A
virus from bats. \emph{Proceedings of the National Academy of
Sciences,}109(11)\textbf{,} 4269-4274.doi:10.1073/pnas.1116200109

Velkov, T., Ong, C., Baker, M. A., Kim, H., Li, J., Nation, R.
L.,\ldots{} Rockman, S. (2013). The antigenic architecture of the
hemagglutinin of influenza H5N1 viruses. \emph{Molecular Immunology,}
56(4)\textbf{,}705-719.doi:10.1016/j.molimm.2013.07.010

Webster, R. G., \& Govorkova, E. A. (2014). Continuing challenges in
influenza.\emph{Annals of the New York Academy of Sciences,}
1323(1)\textbf{,}115-139.doi:10.1111/nyas.12462

Wille, M., \& Holmes, E. C. (2019). The Ecology and Evolution of
Influenza Viruses. \emph{Cold Spring Harbor Perspectives in Medicine}
\textbf{,}a38489.doi:10.1101/cshperspect.a038489

Xu, X., Subbarao, Cox, N. J., \& Guo, Y. (1999). Genetic
characterization of the pathogenic influenza A/Goose/Guangdong/1/96
(H5N1) virus: similarity of its hemagglutinin gene to those of H5N1
viruses from the 1997 outbreaks in Hong Kong. \emph{Virology,}
261(1)\textbf{,}15-19.doi:10.1006/viro.1999.9820

Figure 1. Phylogenetic analysis of H5N6 Influenza Viruses.

\begin{enumerate}
\tightlist
\item
  Phylogenetic tree of hemagglutinin genes of H5N6 viruses sequenced in
  this study; the clade origins of each gene segment are indicated by
  different colored bars.
\item
  Phylogenetic tree of neuraminidase genes of H5N6 viruses sequenced in
  this study.
\end{enumerate}

Figure 2. Reassortment of the H5N6 AIV.

The eight gene segments are PB2, PB1, PA, HA, NP, NA, M, and NS
(horizontal bars starting from top to bottom of the virion). Different
colors represent different virus lineages.

Figure 3. Scatterplots resulting from the DAPC.

Individual isolates from the same AVIs season are depicted as unique
color shapes and surrounded by 95\% inertia ellipses. The PCA and DA
eigenvalues inset panels show the overall variability among individuals
and the relative capture of variance for each discriminant function,
respectively. The y- and x-axes indicate the first and second
discriminant principal components, respectively, which best summarize
the differences between clusters while neglecting within-cluster
variation. (A) and (B) represent HA and NA genes, respectively.

Figure 4. SeqLogo analysis of amino acid substitutions in H5N6 viruses.

, (B) HA and NA (H5 numbering) are shown.(A) The first three sites (9,
12, 16) in HA signal peptide,The last site (169) in HA2,others in HA1.

Figure 5. Replication and virulence of the H5N6 viruses in mice.

Weight changes in mice after challenge, (B) Survival rate of mice after
challenge. (C) The virus content in different organs in the mice on the
third day of the challenge. (D) The virus content in various organs in
mice on the fifth day of the challenge.

Figure 6. Genotype of H5N6 viruses in different hosts.

Schematic for the genetic Source of human H5N6 isolates. (B) Genotypes
of H5N6 viruses isolated in humans, domestic ducks, domestic chickens,
domestic geese, environment, and wild birds. The eight gene segments are
PB2, PB1, PA, HA, NP, NA, M, and NS (horizontal bars starting from top
to bottom of the virion). Different colors represent different virus
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