\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{Emergence of a Novel Reassortant H5N6 Highly Pathogenic Avian Influenza
Virus of Clade 2.3.2.1c from domestic poultry in China}



\author[1]{Wenming Jiang}%
\author[2]{Yang Li}%
\author[1]{Xiao Lin}%
\author[1]{Shuo Liu}%
\author[2]{Cheng Peng}%
\author[2]{Jingjing Wang}%
\author[1]{Xiaohui Yu}%
\author[2]{Hualei Liu}%
\affil[1]{Affiliation not available}%
\affil[2]{China Animal Health and Epidemiology Center}%


\vspace{-1em}



  
  \date{May 14, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
H5N6 avian influenza virus (AIV) has posed a threat to poultry and human
health. Here, we isolated a new clade 2.3.2.1 H5N6 virus, not like most
reported H5N6 AIVs of clade 2.3.4.4. Our analysis revealed a complex
pattern of its evolution in which the virus was derived as a result of
genetic reassortment among clade 2.3.2.1 and clade 2.3.4.4 H5 and H6N6
AIVs. Moreover, the results suggested that H5N6 clade 2.3.2.1 AIVs had
multiple reassortment pattern. It is necessary that to strengthen
continuing surveillance to prevent infection in human and poultry.%
\end{abstract}%



\sloppy


\textbf{Emergence of a Novel Reassortant H5N6 Highly Pathogenic Avian
Influenza Virus of Clade 2.3.2.1c from domestic poultry in China}

Jiang Wenming\textsuperscript{a,1}, Li Yang\textsuperscript{a,1}, Lin
Xiao\textsuperscript{c}, Liu Shuo\textsuperscript{a}, Peng
Cheng\textsuperscript{a}, Wang Jingjing\textsuperscript{a}, Yu
Xiaohui\textsuperscript{a}, Liu Hualei\textsuperscript{a,}*

\textsuperscript{1} These authors contributed equally to this work.

\textsuperscript{a} China Animal Health and Epidemiology Center,
Qingdao, China.

\textsuperscript{b} Nanning Mootek Intelligent Technology Co.,Ltd,
Nanning, China

Correspondence author: Liu Hualei, liuhualei@cahec.cn.

\textbf{Abstract:}

H5N6 avian influenza virus (AIV) has posed a threat to poultry and human
health. Here, we isolated a new clade 2.3.2.1 H5N6 virus, not like most
reported H5N6 AIVs of clade 2.3.4.4. Our analysis revealed a complex
pattern of its evolution in which the virus was derived as a result of
genetic reassortment among clade 2.3.2.1 and clade 2.3.4.4 H5 and H6N6
AIVs. Moreover, the results suggested that H5N6 clade 2.3.2.1 AIVs had
multiple reassortment pattern. It is necessary that to strengthen
continuing surveillance to prevent infection in human and poultry.

H5 subtype highly pathogenic avian influenza (HPAI) viruses has become
endemic among domestic poultry in China since the first detection in
1996(Chen, 2009; Swayne, 2012). Based on the phylogenetic analysis of HA
sequences, H5 avian influenza viruses (AIVs) has evolved into clades
0\textasciitilde{}9(Zhao et al., 2012). Clades 2.3.4.4 and 2.3.2.1 of
H5N1 subtype HPAIVs have become dominant clade in China(J. Li et al.,
2018). Currently, in the process of constant evolution, H5 subtypes AIVs
of clade 2.3.4.4 are now reasserted with various NA subtypes such as the
emerging reassortant H5N2, H5N5, H5N6, and H5N8 viruses. Notably, H5N6
AIVs are now one of the major subtype reassortant viruses and pose a
potential threat to human health. In the reports of AIV surveillance in
China from 2014 to 2016, Bi et al found the H5N6 has replaced H5N1 as a
dominant AIV subtype in southern China(Bi et al., 2016). Thus far, 24
human H5N6 cases have been reported(Y. Li et al., 2020). Importantly,
nearly all of the H5N6 HA sequences mentioned in these previous reports
fell within clade 2.3.4.4. They arose from reassortments with the main
pattern identified: H5 HA genes, H6N6 NA genes, and various internal
genes of low pathogenic AIVs (eg.H5-origin, H6-origin, H9-origin,
H3-origin)(Sun et al., 2018).

In the previous phylogenetic analysis of 505 H5N6 AIVs performed by Bi
et al, the HA gene of one H5N6 strain, A/duck/Vietnam/LBM360c1-4-1/2013,
belonged to clade 2.3.2.1(Bi et al., 2016). A novel reasortment H5N6
AIVs of clade 2.3.2.1c, A/Streptopelia decaocto/Jiangxi/G6/2016, was
detected from a wild bird in China(Zhang, Li, Zhu, Chang, \& Xu, 2019).
Here, we isolated one HPAI H5N6 virus (A/chicken/Guangxi/16/2019, GX16)
from chicken in China in 2019, with the HA gene divided into clade
2.3.2.1c. Remarkable, not all clade 2.3.2.1 viruses belong to H5N1, and
it also involves several H5N6 reassortments. Moreover, the diverse host
range of these variants detected in poultry and wild bird species, and
the distinct geographic regions, demonstrate the current expansion of
H5N6 reassortments of clade 2.3.2.1.

Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen,
Gmbh, Hilden, Germany) according to the manufacturer's instructions. The
complete genome sequences of the virus GX16 was amplified using RT-PCR
with SuperScript-III One-Step RT-PCR System with Platinum\selectlanguage{ngerman}® Taq DNA
Polymerase (Invitrogen, Waltham, MA, USA) with primers described
previously(Hoffmann, Stech, Guan, Webster, \& Perez, 2001). The
gene-specific RT-PCR amplicons were purified from gels using the
QIAquick Gel Extraction Kit (Qiagen, Gmbh, Hilden, Germany) and
sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied
Biosystems, Foster City, CA, USA). Sequences generated in this study
were deposited in GenBank (accession nos.--).

Nucleotide identity analysis showed that the GX16 virus was closely
related, according to its hemagglutinin (HA) gene segment, to
A/Streptopelia decaocto/China/4/2016(H5N6), with a nucleotide sequence
identity of 98.36\%; the identity of 98.30\% with
A/pigeon/Zhejiang/112090/2014(H5N1) virus was also recorded. The
neuraminidase (NA) gene of the GX16 virus shared 98.70\% nucleotide
identity with the H5N6 viruses isolated from Kumamoto and Korea
(Supplementary file). Six internal segments---polymerase basic (PB2),
PB1, polymerase acidic (PA), nucleoprotein (NP), matrix protein (MP),
nonstructural protein (NS)---revealed a nucleotide identity of
98.42\%--99.49\% with the H5N6 and H5N1 viruses (Supplementary file).

To understand the evolutionary pattern of clade 2.3.2.1 H5N6 viruses,
the phylogenetic tree was constructed based on the HA sequences of H5
AIVs from reference sequences of WHO and from GenBank Influenza Virus
Database and GISAID database. The GX16/H5N6 virus belongs to the clade
2.3.2.1 with other six H5N6 viruses in these databases. As shown in
Figure, there were three clusters in clade 2.3.2.1c: Nanjing-like,
including the Hunan(HN232)/2015, Hunan(HN234)/2015, Yunnan/2015, and
Hubei/2015; Vietnam-like, including the Vietnam/2013; Alberta-like,
including GX16/2019 isolate and China non-duck originated isolate
A/Streptopelia decaocto/China/4/2016. There were 7 kinds of possible
evolutionary approaches of the novel clade 2.3.2.1 H5N6 viruses found,
in which the pattern of GX16/2019 was obviously different from other 5
isolates in China.

Interestingly, the genetic distance between the seven viruses was
disproportionate to the date of the virus was isolated. The Alberta-like
was more closed to Vietnam-like, compared to Nanjing-like in which most
of H5N6 isolates clustered (Supplementary file). It is important that
each cluster has isolate can infect human in clade 2.3.2.1.

The phylogenetic tree of N6 genes showed that there were 2 groups: Group
1 of H5N6 2.3.4.4d with aa58--68 deletion and Group 2 of clade 2.3.4.4
H5N6 and H6N6 with no deletion. Isolates that infected human mainly came
from Group 1. Among above seven clade 2.3.2.1 H5N6 isolates, the NA
genes of Vietnam/2013 and Hubei/2015 were derived from H6N6 with no
deletion in their regions respectively. The NA gene of GX16/2019 isolate
was originated from Japan-like clade 2.3.4.4 H5N6 (Supplementary
Figure).

The remaining six internal genes of the GX16/2019 virus were reassorted
with clade 2.3.2.1c Alberta-like H5N1 viruses (NP, MP, and NS) and clade
2.3.4.4d Japan-like H5N6 viruses (PB2, PB1, and PA), and revealed a
close phylogenetic relatedness with these viruses frequently found in
Asia in 2014--2019 (Supplementary Figure). However, the NP gene of
Hunan(HN234)/2015 which was reported previously originated from the H7N3
virus.

The HA gene of the GX16/2019 virus possessed multiple basic amino acids,
``PQRERRRKR/GLF'', in the cleavage site of the HA indicating high
pathogenicity of this virus. The GX16 virus exhibited D101N, S137A,
S158N, S159N, T160A, S114R, and T151I amino acid substitutions (H3
numbering) at its HA protein have been reported previously to be related
to virulence, transmission, and host specificity. The NA coded protein
of the GX16 virus was found 11aa deletion in position 58--68, indicating
that the virus could exhibit increased virulence in mice. Further, the
GX16 possessed several mutations, like L89V and G309D in PB2, D3V and
D622G in PB1, N383D in PA, N30D, I43M and T215A in M1, or. P42S in NS1,
suggesting that the virus could exhibit increased virulence and
transmission in mammals.

Summary, we have isolated a new clade 2.3.2.1 H5N6 virus and revealed a
complex pattern of its evolution. The evidence demonstrates that clade
2.3.2.1 H5N1 AIVs could reassort with circulating H5N6 and H6N6 AIVs.
Clearly, clade 2.3.2.1 H5N6 viruses are capable of receiving gene
segments from different subtypes and different clades.

Avian influenza viruses are a threat to human health, as they could
cross the species barrier and infect humans occasionally with severe
outcome. The genetic diversity of H5 viruses is increasing, due to
continued circulation and reassortment in poultry, posing a constant
risk for public health and requiring regular risk assessments. Our study
underlines the importance of active surveillance in the timely detection
of new AIV reassortants, including influenza itself and its hosts from
the perspective of epidemiology, virology and ecology.

\textbf{Conflict of Interest:} All authors have no conflict of interest.

References

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

Chen, H. (2009). H5N1 avian influenza in China. \emph{Sci China C Life
Sci, 52} (5), 419-427. doi:10.1007/s11427-009-0068-6

Hoffmann, E., Stech, J., Guan, Y., Webster, R. G., \& Perez, D. R.
(2001). Universal primer set for the full-length amplification of all
influenza A viruses. \emph{Arch Virol, 146} (12), 2275-2289.
doi:10.1007/s007050170002

Li, J., Hou, G., Wang, Y., Wang, S., Cheng, S., Peng, C., \& Jiang, W.
(2018). Protective efficacy of an inactivated chimeric H5 avian
influenza vaccine against H5 highly pathogenic avian influenza virus
clades 2.3.4.4 and 2.3.2.1. \emph{J Gen Virol, 99} (12), 1600-1607.
doi:10.1099/jgv.0.001140

Li, Y., Li, M., Li, Y., Tian, J., Bai, X., Yang, C., . . . Chen, H.
(2020). Outbreaks of Highly Pathogenic Avian Influenza (H5N6) Virus
Subclade 2.3.4.4h in Swans, Xinjiang, Western China, 2020. \emph{Emerg
Infect Dis, 26} (12), 2956-2960. doi:10.3201/eid2612.201201

Sun, W., Li, J., Hu, J., Jiang, D., Xing, C., Zhan, T., \& Liu, X.
(2018). Genetic analysis and biological characteristics of different
internal gene origin H5N6 reassortment avian influenza virus in China in
2016. \emph{Vet Microbiol, 219} , 200-211.
doi:10.1016/j.vetmic.2018.04.023

Swayne, D. E. (2012). Impact of vaccines and vaccination on global
control of avian influenza.\emph{Avian Dis, 56} (4 Suppl), 818-828.
doi:10.1637/10183-041012-Review.1

Zhang, T., Li, R., Zhu, G., Chang, J., \& Xu, B. (2019). First Detection
of a Novel Reassortant Avian Influenza A(H5N6) Clade 2.3.2.1c Virus,
Isolated from a Wild Bird in China.\emph{Microbiol Resour Announc, 8}
(36). doi:10.1128/MRA.00797-19

Zhao, G., Gu, X., Lu, X., Pan, J., Duan, Z., Zhao, K., . . . Liu, X.
(2012). Novel reassortant highly pathogenic H5N2 avian influenza viruses
in poultry in China. \emph{PLoS One, 7} (9), e46183.
doi:10.1371/journal.pone.0046183

Figure Legend

Figure. Genesis of H5N6 AIVs. 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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure/Figure}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

