\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,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Mutation of D201G near the receptor binding site significantly drive
antigenic drift of circulating H9N2 subtype avian influenza virus}



\author[1]{Jing Xia}%
\author[1]{Yu-Wen Luo}%
\author[1]{Meng-Yi Dong}%
\author[1]{Yong-Xin Li}%
\author[1]{An-Dong Wang}%
\author[1]{Nian-Ling Li}%
\author[1]{Yu-Xi Shen}%
\author[1]{Shu-Yun Li}%
\author[1]{Min Cui}%
\author[1]{Xinfeng Han}%
\author[2]{Song-Cheng Yu}%
\author[3]{Min Li}%
\author[1]{Yong  Huang}%
\affil[1]{Sichuan Agricultural University - Chengdu Campus}%
\affil[2]{Meishan Dongpo District Agricultural and Rural Burean Livestock station Meishan 620010 Sichuan China}%
\affil[3]{Animal disease prevention and control center of Chengdu city Chengdu 610041 Sichuan China}%


\vspace{-1em}



  
  \date{May 21, 2024}


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





\selectlanguage{english}
\begin{abstract}
The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused
significant losses in chicken flocks throughout China. Our previous
research has showed that field isolates of H9N2 underwent antigenic
drift to evolve into distinct groups with significant antigenic
divergence from the commercially available vaccines. The present study
sought to identify which single mutations that have naturally appeared
in isolates from the past 5 years has driven antigenic drift. Six
high-frequency mutation sites in/near the receptor binding site (RBS)
region were screened by comparing amino acid alignments of the H9N2 AIVs
isolated from China between 2014 and 2019. Two substitutions, (A168N and
D201G) were demonstrated to have a significant impact on the
antigenicity, but did not change the growth kinetics and cell tropism of
the virus. It is worth noting that the D201G substitution not only
significantly changed the antigenicity, but also caused immune escape of
the parental virus. In conclusion, A168N and D201G substitution are
newly discovered determinants that can significantly change the
antigenicity of H9N2 AIV, which should be tracked during outbreaks.%
\end{abstract}%



\sloppy


Mutation of D201G near the receptor binding site significantly drive
antigenic drift of circulating H9N2 subtype avian influenza virus

Short title: Antigenic drift site of H9N2 AIV

Jing Xia\selectlanguage{ngerman}\textsuperscript{1,2¶}, Yu-Wen Luo\textsuperscript{1,2¶},
Meng-Yi Dong\textsuperscript{1,2}, Yong-Xin Li\textsuperscript{1,2},
An-Dong Wang\textsuperscript{1,2}, Nian-Ling Li\textsuperscript{1,2},
Yu-Xi Shen\textsuperscript{1,2}, Shu-Yun Li\textsuperscript{1,2}, Min
Cui\textsuperscript{1,2}, Xin-Feng Han\textsuperscript{1,2}, Song-Cheng
Yu\textsuperscript{3}, Min Li\textsuperscript{4*}, Yong
Huang\textsuperscript{1,2*}

1. Preventive of Veterinary Medicine, College of Veterinary Medicine,
Sichuan Agricultural University, Chengdu 611130, Sichuan, China.

2. Key Laboratory of Animal Disease and Human Health of Sichuan
Province, Sichuan Agricultural University, Chengdu 611130, Sichuan,
China.

3. Meishan Dongpo District Agricultural and Rural Burean, Livestock
station, Meishan 620010, Sichuan, China

4. Animal disease prevention and control center of Chengdu city, Chengdu
610041, Sichuan, China

\textsuperscript{¶} These authors contributed equally to this work

* Correspondence:

Yong Huang, E-mail: hyong601@163.com

Min Li, E-mail: 350360849@qq.com

Jing Xia: 14551@sicau.edu.cn xiajing1028@163.com

Yu-Wen Luo: Yuwen\_Luo@126.com

Meng-Yi Dong: 18200356764@163.com

Yong-Xin Li: 41984561@qq.com

An-Dong Wang: antone@stu.sicau.edu.cn

Nian-Ling Li: linianling123@163.com

Yu-Xi Shen: shenyuxi1019@163.com

Shu-Yun Li: lishuyun607@163.com

Min Cui: cuimintracy@163.com

Xin-Feng Han: hanxinf@163.com

Song-Cheng Yu: 853769133@qq.com

\textbf{Summary:}

The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused
significant losses in chicken flocks throughout China. Our previous
research has showed that field isolates of H9N2 underwent antigenic
drift to evolve into distinct groups with significant antigenic
divergence from the commercially available vaccines. The present study
sought to identify which single mutations that have naturally appeared
in isolates from the past 5 years has driven antigenic drift. Six
high-frequency mutation sites in/near the receptor binding site (RBS)
region were screened by comparing amino acid alignments of the H9N2 AIVs
isolated from China between 2014 and 2019. Two substitutions, (A168N and
D201G) were demonstrated to have a significant impact on the
antigenicity, but did not change the growth kinetics and cell tropism of
the virus. It is worth noting that the D201G substitution not only
significantly changed the antigenicity, but also caused immune escape of
the parental virus. In conclusion, A168N and D201G substitution are
newly discovered determinants that can significantly change the
antigenicity of H9N2 AIV, which should be tracked during outbreaks.

\textbf{Keywords:} Avian influenza virus, H9N2, Antigenic drift,
Evolution, RBS, Antigenicity

\textbf{Introduction}

Chickens natural infected with the H9N2 subtype of low pathogenic avian
influenza (LPAI) exhibited mild respiratory signs and decreased egg
production. Co-infection with other pathogenic microorganisms will
aggravate the clinical signs. Although the H9N2 subtype of avian
influenza virus (H9N2-AIV) is of low pathogenicity to birds, the actual
threat lies in its broad host range. The virus not only infects birds,
but has been reported to jump species to infect humans and other
mammals. More serious H9N2-AIV frequently donates gene segments to
facilitate the generation of novel reassortants, causing epidemics or
even pandemics in poultry (Gerloff et al., 2014). An analysis of the
hemagglutinin (HA) gene sequence database of the National Center for
Biotechnology Information (NCBI) in 2016 revealed that \textless{}90\%
of the H9N2-AIV isolates came from Asia, of which 78\% originated in
China (Li et al., 2017). Another dataset showed that the AIV positivity
rate was 12.73\% between 2016 and 2019 in China, of which H9N2 accounted
for 72.75\% of cases (Bi et al., 2020). This shows that H9N2-AIV has
become the dominant AIV in recent years within China, which seriously
threatens the public health of humans as well as the livestock and
poultry industries.

Vaccination of poultry is a key element of disease control in endemic
countries. Influenza A virus mutates rapidly, resulting in antigenic
drift and poor year-to-year vaccine efficacy. Commercial vaccine strains
of H9N2 in China, including A/chicken/Shandong/6/96 (SD696),
A/chicken/Guangdong/SS/94 (SS) and A/chicken/Shandong/F/98 (F98), were
all isolated prior to 2000. Previously, we demonstrated that H9N2 virus
isolated from 2013 to 2016 in China underwent antigenic drift to evolve
into distinct antigenic groups, and accumulated significant antigenic
differences compared with the commercial vaccines (Xia, Cui, et al.,
2017). A growing body of research supports these observations (Sun \&
Liu, 2015). The identification of antigenic sites for monitoring of
variants for the development effective vaccines is crucial. More than 46
HA amino acid antigenic sites were identified in H9N2-AIV (T. P. Peacock
et al., 2017; Song et al., 2020; Su et al., 2020; Wan et al., 2014; Zhu
et al., 2015). Some of those positions are multifunctional, such as the
D200N substitution, which also increases replication in chicken embryo
fibroblast cells and embryonated chicken eggs (Song et al., 2020). It
was reported that N166D also affects the pathogenicity (Jin et al.,
2019; T. P. Peacock et al., 2017), and the 220 loop deletion could arise
in the field due to immune selection pressure, which also reduces HA
stability (T. P. Peacock et al., 2017). However, it is still unknown
which single substitution of recent isolates is responsible for the
observed antigenic drift.

Although the epitopes of H9N2-AIV are not well characterized, it is
known that not all substitutions affect viral antigenicity (T. P.
Peacock, Harvey, Sadeyen, Reeve, \& Iqbal, 2018). For example, mutations
in H3N2 and H1N1 viruses near the receptor binding site (RBS) determine
major antigenic changes, but are also affected by mutations to adjacent
sites as well (Koel et al., 2013; Lewis et al., 2014; Santos et al.,
2019). Interestingly, our previous analyses established a link between
high-frequency substitutions and those key antigenic sites of H3N2
viruses (Xia et al., 2020). Therefore, the high-frequency mutation sites
near the H9N2-AIV HA RBS protein may be related to the key amino acid
sites producing antigenic variation. In this study, we aimed to
demonstrate that the single high-frequency mutation site near the RBS
could drive antigenic drift of H9N2-AIV circulating in 3 recent years in
China.

\textbf{Material and methods}

\textbf{Viruses, cells, eggs and plasmids}

Human embryonic kidney 293T (HEK-293T) cells were obtained from the
research center for swine diseases of Sichuan Agricultural University,
and Madin-Darby canine kidney (MDCK) cells were obtained from Zigong
Center for Disease Control and Prevention of Sichuan province (Zigong
CDC). Both cell lines were maintained in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10 \% fetal calf serum at
37\textsuperscript{o}C with 5\% CO\textsubscript{2}. Specific pathogen
free (SPF) chicken embryos were obtained from the Beijng Merial Vital
Laboratory Animal Technology Co., Ltd (Beijing, China). Avian origin
H9N2 virus A/Chicken/Sichuan/CQY/2014 (abbreviated as CQY-2014) was
isolated by the author's group in 2014. Viruses were propagated in
allantoic cavities of 10-day-old SPF embryonated chicken eggs for 48
hours and stored at -80 \textsuperscript{o}C. The dual-promoter plasmid,
pHW2000 was provided by Shanghai Veterinary Research Institute, Chinese
Academy of Agricultural Sciences.

\textbf{Phylogenetic and antigenic site analysis of H9N2 HA
gene/protein}

\textbf{Collection of sequence data:} The HA gene sequences of H9N2-AIV
isolated in recent years (2014-2020) were collected from the Global
Initiative on Sharing All Influenza Data (GISAID) platform. A total of
4935 sequences were downloaded. A total of 312 records were removed that
were duplicates or had a sequence length \textless{} 1600 bp. The
remaining sequences used for the subsequent analyses covered 4623 taxa.

\textbf{Phylogenetic analysis} : Due to the large amount of data, we cut
down the number of sequence for the construction of phylogenetic tree.
Next, up to 70 sequences from collection years with greater than 70
sequences per year were randomly sampled while maintaining all sequences
from collection years with less than 70 sequences per year. This was
performed to prevent over representation of certain years.

Each taxa was aligned using MUSCLE (v3.8.4) (Edgar, 2004) and the
proportion of unique mutations were identified by subclade using
Geneious Prime. A time-scaled phylogenetic tree was generated using
BEAST (v1.8.4) (Drummond, Nicholls, Rodrigo, \& Solomon, 2002; Pybus \&
Rambaut, 2002; Suchard et al., 2018). Parameter setting of the
evolutionary model was performed as previously reported (Xia et al.,
2020). Briefly, a nucleotide GTR + I + \selectlanguage{greek}Γ\selectlanguage{english}4 substitution model was
selected, with an uncorrelated log-normal prior molecular clock model
over a strict clock, and a non-parametric Bayesican skyline demographic
tree (Baele et al., 2012; Baele, Li, Drummond, Suchard, \& Lemey, 2013;
Drummond, Rambaut, Shapiro, \& Pybus, 2005). A total of 50 million
Markov Chain Monte Carlo generations were specified for sampling every
10,000 steps, and assessed for sufficient mixing and convergence using
tracer (v1.6) after considering the first 10\% of samples as burn-in
(Rambaut, Drummond, Xie, Baele, \& Suchard, 2018). A maximum clade
credibility (MCC) tree was generated in treeannotator (v1.8.4) and
visualized in figtree (v1.4.3). The specific amino acid mutations of
each subclade were also counted by comparing the amino acid sequences in
Geneious Prime.

\textbf{Antigenic site counting} : Keyword searches in PubMed, Google
Scholar, and China national knowledge internet (CNKI) databases were
used to count the number of reported H9N2-AIV antigenic sites (Table
S1). The mutation and frequency of antigenic sites in years 2014, 2016,
2018 and 2019-2020 were analyzed using BioAider (v1.334) (Zhou, Qiu, Pu,
Huang, \& Ge, 2020). To demonstrate that the single high-frequency
mutation site near the RBS could drive antigenic drift of H9N2-AIV
circulating in three recent years in China, the high-frequency mutation
sites in/near the RBS, and the substitution accounting for [?] 20\% were
selected as the pre-selection substitutions for subsequent antigenicity
verification.

\textbf{Generation of single mutant H9N2-AIVs by reverse genetics}

Total RNA extraction and reverse transcription (RT) reactions of
CQY-2014 were performed as previously reported (Xia et al., 2016). PCR
amplification of the 8 gene fragments (PB2, PB1, PA, NP, NS, M, NA and
HA) were carried out using the primers presented in Table S2. PCR
products were sequenced by Shanghai Sangong Biological Engineering
Technology \& Services Co., Ltd. (Shanghai, China). Complete genome
sequences were submitted to GenBank under the accession numbers
MW493190-MW493196 and MW493229.

For the construction of recombinant plasmids, a classical cloning method
was used as previously described (Hoffmann, Stech, Guan, Webster, \&
Perez, 2001). In brief, the 8 purified complete genome fragments were
cloned into the dual-promoter plasmid pHW2000 by homologous
recombination, and transformed into \emph{E.coli} TOP10 competent cells.
Homologous recombinant primers are presented in Table S2. The linearized
pHW2000 plasmid was prepared by PCR amplification using the primers
pHW2000F: CCCCCCCAACTTCGGAGGTC and pHW2000R: AATAACCCGGCGGCCCAAAA.
Recombination was performed according to the SE seamless cloning and
assembly kit instructions (Beijing Zoman Biotechnology, China). The
single pre-selection substitutions were separately introduced into the
HA1 gene of the CQY-2014 virus using TaKaRa MutanBEST kit (TaKaRa,
Japan) according to the manufacturer's instructions. At least 4 clones
were picked for each transformation pool of recombinant plasmid, and was
sequenced to ensure the absence of unwanted mutations. Viral rescue was
performed by transfecting 293T cells with plasmid prepared using the
Lipofectamine\textsuperscript{TM} 3000 Reagent Protocol kit (Thermo
Fisher Scientific, China). The supernatant and cell mixture were
harvested after 48 h of culture and blind passaged in MDCK cells for 3
generations. Hemagglutination test and RT-PCR were used to identify the
rescued viruses.

\textbf{Replication kinetics in MDCK cells and chicken embryos}

Growth properties of rescued mutant and parental viruses were compared
in MDCK cells and chicken embryos. Each virus was inoculated onto MDCK
monolayers and nine-day old chicken embryos at a multiplicity of
infection (MOI) of 0.001 and 10\textsuperscript{3}EID\textsubscript{50},
respectively. The supernatants of the infected cells and chicken embryos
were collected in triplicate at 2, 4, 6, 12, 24, 36, 48 and 72 h post
inoculation and stored at -80\textsuperscript{o}C. The titer of each
sample was determined using Quantitative real time PCR (qRT-PCR) and HA
tests.

\textbf{Antigenic analysis of mutants}

To determine whether high-frequency mutations near the RBS could drive
antigenic drift of H9N2-AIV, antiserum preparation and reciprocal HI
tests were performed as previously reported (Xia, Cui, et al., 2017).
Here, three criteria (antigenic relatedness values (ARV, r) of HI and
microneutralization titer, and antigenic map) were used to judge the
difference of virul antigenicity.

The HI and microneutralization titers were used to calculate the ``r''
by the method of Archetti \& Horsfall (1950):\(\text{AVR}\left(r\right)=\sqrt{r1\times r2}\times 100\%\) where r1
represents the ratio of the heterologous HI titer of virus 1 to the
homologous titer of virus 1, and r2 represents the ratio of heterologous
titer of virus 2 to the homologous titer of virus 2. Mutants where 0.67
[?] r < \selectlanguage{english}1.5 were considered to be antigenically similar, whereas those
with 0.5 [?] r < \selectlanguage{english}0.67 were considered to possess small antigenic
differences, and r < \selectlanguage{english}0.5 and r [?] 1.5 were considered to contain
significant antigenic differences. Antigen mapping were also performed
by using the program AntigenMap
(http://sysbio.cvm.msstate.edu/AntigenMap) as previously reported (Xia,
Cui, et al., 2017).

Sites between non-adjacent unit were considered to have antigenic
differences. The farther the distance, the greater the antigenicity
difference. The mutation was been considered as significant if they were
supported by both of the r value and antigen map.

\textbf{Analysis of immune protection conferred by the parental strain
against single mutants}

To identify whether the key antigenic single mutations can break through
the immune protection induced by the parental strain, the parental virus
CQY-2014 was selected as the vaccine strain and prepared as an
oil-adjuvant inactivated vaccine, as previously reported (Xia, Yao, et
al., 2017). Commercial un-immunized 28-day-old Partridge Shank broilers
(n = 80) were randomly divided into 8 groups, 4 groups were
subcutaneously immunized with 10\textsuperscript{5}EID\textsubscript{50}
CQY-2014 inactivated vaccine. At 21 day-post-immunization (d.p.i), birds
of 3 immune-challenge groups and 3 challenge control groups were
challenged with 10\textsuperscript{6}EID\textsubscript{50} of CQY-2014,
CQY-A168N, or CQY-D201G virus by eye and nose drip, respectively. The
birds of immune control group were received only the CQY-2014 vaccine.
Birds of blank control group were mock infected with phosphate-buffered
saline (PBS) buffer at the corresponding time points.

Chicken feeding and sample collection were carried out as previously
described (Xia, Cui, et al., 2017). Briefly, birds in each group were
held in separate biosafety level 2+ (BSL2+) isolators and monitored
daily for appetite, activity, fecal output, conjunctivitis, cyanosis of
the comb, ruffled feathers and dyspnea. Tracheal swabs from each group
were collected at 3, 5 and 7 day-post-challenge (d.p.c) for virus
isolation. At 14 d.p.c, all remaining birds were euthanized and
necropsied for pathological observation.

\textbf{Ethical compliance}

All animal experiments were conducted in compliance with protocols
approved by the Sichuan provincial Laboratory Animal Management
Committee {[}Permit Number SYXK (Sichuan) 2019-187{]}, and the Ethics
and Animal Welfare Committee (EAWC) of the Sichuan Agricultural
University. Humane endpoints were strictly adhered to over the entire
experimental period. Birds that were unable or unwilling to eat and/or
drink during the study period were immediately euthanized. All birds
were euthanized via cervical dislocation by a trained technician, as
approved by the EAWC.

\textbf{Results}

\textbf{Phylogenetic analysis}

H9N2 AIV isolates evolved at steady mean evolutionary rates, 8.036 \selectlanguage{ngerman}×
10\textsuperscript{-3} (95\% HPD: 6.058 ×
10\textsuperscript{-3}\textasciitilde{} 9.702 × 10\textsuperscript{-3})
in recent years in China. All randomly down-sampling sequences belonged
to clade 15 of H9N2-AIV. The sequences were diversified into 3
additional subclades (A, B and C) based on significant nucleotide
differences, with subclade C comprising the predominant branch (Figure
1).

\textbf{Seven high-frequency mutation sites were located near/in RBS}

A total of 46 reported antigenic sites were curated form research papers
in three databases (Table S1). A total of 2,927 full-length HA sequences
were analyzed in this part of the study. Only 43.48\% (20/46) of the
sequences were hypervariable under the natural selection, most of which
were conservative (Table S1). Interestingly, 14 of the 20 mutant
antigenic sites (70\%) are located in/near the HA RBS.

Figure 1 shows the specific mutations of each additional subclade,
consisting of a total of 14 amino acid sites, which were also
distributed in/near the RBS. Of those, 6 positions, including 164, 168,
171, 198, 200 and 201 (H9 numbering) near/in the RBS of H9N2-AIV were
selected as high-frequency mutations sites. All of those seven sites
were located on the surface of the HA protein head domain (Figure 2A).
The seven mutations (R164Q, A168N, I171T, T198A, R200T, D201G and D201A)
were selected as the pre-selection substitutions for subsequent
antigenicity verification.

\textbf{Comparison of growth characteristics of the mutants}

The virus, CQY-2014, was generated from a wild-type H9N2-AIV using
reverse genetics. Each of the high-frequency mutation sites (R164Q,
A168N, I171T, T198A, R200T, D201G and D201A) were introduced into the
CQY-2014 backbone individually, and there were no unexpected mutations
in the sequencing of each virus (Figure 3 A). The titer of the parental
CQY-2014 virus in chicken embryos reached 8.5log10
EID\textsubscript{50}/mL. Observed titers in MDCK cells were slightly
lower at 6.5log10 TCID\textsubscript{50}/mL. Compared with the parental
virus, titers of the seven mutants were slightly lower in both CEK cells
and chicken embryos (Table 1). However, no significant differences among
growth curves were observed (Figure 3 B).

\textbf{Two single substitutions significantly change the antigenicity
of H9N2-AIV}

A total of seven substitutions (including six high-frequency mutation
sites) in/near the RBS were tested using reciprocal HI and
microneutralization test to support the hypothesis that the single
high-frequency mutation site near the RBS drives antigenic drift of
H9N2-AIV. The original data were suppored in Table S3 and Figure S1, and
the Integration results were shown in Table 2. As the Table 2 showed,
mutations that drive antigen drift were different, whether based on the
r-values of HI and neutralizing titers, or the antigen maps. Only two
mutations, A168N and D201G were strong supported by all criteria, and
those two mutations were considered to significantly cause the antigenic
differences. The other 5 mutations, which are only supported by partial
criteria, are thought to exerte mild effects on antigenicity. Notably,
at the 201 site, two single substitutions (either D to G and D to A)
produced drastic phenotypic differences, with glycine (G) had a
significant effect on antigenicity, while the substitution of alanine
(A) had no significant effect. The visualizations in Figure S1 were even
more obvious. The antigen distance between the two single substitutions
at site 201 was more than 2 units in antigen map, with more than 4-fold
HI and microneutralization titer change. By predicting the 3D structure
of the HA protein, it was observed that the substitution of glycine at
position 201 did not change the \selectlanguage{greek}α \selectlanguage{english}helix structure, but significantly
changed the surface structure (Figure 2 B). In conclusion, the A168N and
D201G mutations near the RBS significantly affected the antigenicity of
circulating H9N2-AIV.

\textbf{Immune protection analysis}

Next, the ability of the two mutants (A168N and D201G) to break through
the immune protection induced by the parent strain was analyzed by
avaccinal protection test. There were no unexpected deaths observed
during the study in all groups. There was also no infection in immunized
control chickens (Vaccine\_control group) and blank control group. No
significant difference in the proportion of sick chickens with
respiratory signs were observed (including cough and mouth breathing)
among the 3 challenge control groups (CQY-2014, A168N and D201G group)
at 3 \textasciitilde{} 6 day-post-challenge (d.p.c), which were between
50\% \textasciitilde{} 70\%. At 14 d.p.c, the pathological lesions of
chickens in each of the 3 challenge and control groups were also
similar, and about 20\% \textasciitilde{} 30\% of chickens had
intestinal and tracheal congestion or hemorrhage. The results suggest
that the 2 key mutations (A168N and D201G) influencing antigenicity did
not exert a significant effect on H9N2-AIV pathogenicity.

Comparing the data between the 3 immune-challenge groups, it can be
observed that the D201G mutation {[}Vaccine\_(D201G) group{]} has the
potential ability to break through the immune protection conferred by
the parental virus. Recovery of challenge virus remained at 60\% and
20\% at 5 and 7 d.p.c, respectively (Figure 4 B and C). During the study
30\% of chickens presented with signs of respiratory disease in
Vaccine\_(D201G) group. Groups challenged with other mutants had a
significant decrease in re-isolation rate (20\% \textasciitilde{}30\%)
at 5 d.p.c. At 7 d.p.c., no virus was isolated and no clinical signs or
pathological impairments were observed (Figure 4). The results suggested
that the D201G mutation not only changes the antigenicity of H9N2-AIV,
but also confers the ability to escape the host immune responses.

\textbf{Discussion}

Avian influenza viruses have posed a significant threat to the global
poultry industry, in addition to public health. Due to the wide host
range, some H9N2 AIVs not only circulate in poultry and wild bird
population, but have also been detected in mammals, and which elevates
the risk of transmission to humans. Another concern is the extensive
genetic reassortment with other influenza serotypes, with H9N2 AIV
considered to be the most common and destructive LPAIV subtype. While
vaccination is an effective method to control the spread of influenza
viruses, the efficacy of the H9N2 AIV vaccine has been challenged by the
perpetual antigenic drift. Our previous research demonstrated that H9N2
field isolates have undergone antigenic drift to evolve into distinct
antigenic groups, which also resulted in significant antigenic
differences with the commercial vaccines. Unfortunately, the key amino
acids associated with those antigenic drift events remain elusive. Two
discrete antigenic sites ``H9-A'' and ``H9-B'' (or group I and II) which
include at least 46 amino acid sites have been identified (T. Peacock et
al., 2016). We also analyzed the evolution of these 46 aa in isolates
collected over the most recent five years and found that most of these
sites were completely conserved among circulating H9N2 viruses. To
identify which single mutations of these viruses are driving the
antigenic drift, 6 high-frequency mutations including those at aa sites
164, 168, 171, 198, 200 and 201 were screened by comparing amino acid
alignments of the H9N2 AIVs isolated from China in 2014-2019. It should
be noted that 5 of these (164,168,198,200 and 201) are located both
in the antigenic sites and RBS region (Kaverin et al., 2004). Three of
them (164, 168 and 201) were under positive selection (data not shown)
pressure (Su et al., 2020). Therefore, these sites were likely
responsible for the significant antigenic drift observed in recent
years.

A variety of mechanisms, namely changes to epitope structure,
acquisition of additional glycosylation sites and modulation of
receptor-binding avidity, can contribute to both actual and apparent
antigenic change (Abe et al., 2004; Das et al., 2011; Hensley et al.,
2009). In the present study, 2 sites containing a high-frequency of
mutations were identified that contribute to the viral antigenic drift.
The A168N and D201G substitutions resulted in significant antigenic
changes.

The HA protein of influenza is a highly glycosylated. N-linked
glycosylation of HA has been reported to contribute to immune escape and
virulence of influenza (Gao et al., 2021), and obtaining new
glycosylation sites is also an important mechanism of viral antigenic
drift. None of the mutations characterized here introduced novel
glycosylation sites into the virus. In response to escape under
neutralizing antibody pressure of the virus, influenza A virus could
evolve by regulating HA receptor avidity via amino acid substitutions in
the HA1 globular head domain (Das et al., 2011), many of which
simultaneously alter the antigenicity (Hensley et al., 2009; Wu \&
Wilson, 2020). Some studies have shown that receptor avidity may be a
more important factor than antigenicity in avoiding neutralizing
antibodies (Crowe, 2012). Here, the effects of two mutations at
antigenic site II of residue 201 (D201G and D201A) on antigenicity were
significantly altered, in which the glycine substitution had a
significant effect both on antigenicity and immune escape, while alanine
had no effect. In addition, a serine substitution at residue 201 can
also lead to antibody escape (Wan et al., 2014). It was observed that
the change of surface structure was a result of the D201G substitution
(G: hydrophobic, D: hydrophilic). This in turn may be due to the change
of hydrophilicity, thus leading to the change of receptor avidity and
antigenicity of the virus. It is important to note that changes in
receptor avidity were not validated at residue 201 in this study. Of
note, 201G mainly exists in predominant subclade C (Figure 1), which may
result in immune escape in chicken flocks in the future. The other sites
that may cause a similar phenomenon are at residues 201 and 168, which
are both in the antigenic epitopes and RBS region. It was observed that
asparagine (N, 37.1\%) and alanine (A, 35.7\%) accounted for the largest
proportion at residue 168 in 2019-2020, which was significantly
different from the major site (aspartic acid, D, 52.9\%, and A, 33.0\%)
in 2014 by amino acid comparisons (Table S1). The substitution of those
three amino acids can affect the antigenicity of H9N2 viruses (T.
Peacock et al., 2016).

Comprised of a weak genetic basis of preferences for alternative avian
receptors and human-like receptors, residue 198 may be a multifunctional
site which could function to modulate polyclonal antisera binding and
receptor-binding avidity at the same time (T. P. Peacock et al., 2020).
In the present study, T198A was capable of immune escape in a reciprocal
HI assay using polyclonal antibodies, while escape was not observed in a
microneutralization assay. One possible explanation for this result is
that the HI receptor-binding avidity and neutralization antibody
epitopes are different. Thus, the molecular basis requires an in depth
analysis, which is beyond the scope of this report.

In conclusion, key amino acid substitutions that may drive antigenic
drift of H9N2-AIV in a recent five year periods were identified. Two
predominant substitutions, including A168N and D201G were demonstrated
to significantly affect antigenicity, but did not change growth
characteristics and cell tropism. It is worth noting that the D201G
substitution not only changed the antigenicity, but also produced an
immune escape variant of the parental virus. The data presented here
provide a reference for the prediction of the evolutionary direction of
H9N2-AIV, and the development of effective vaccines.

\textbf{Acknowledgements}

This work was financially supported by the Program of ``Shuangzhi plan''
of Sichuan Agriculture University.

\textbf{Conflict of interest statement}

All authors have no conflict of interest to declare.

\textbf{Reference}

Abe, Y., Takashita, E., Sugawara, K., Matsuzaki, Y., Muraki, Y., \&
Hongo, S. (2004). Effect of the addition of oligosaccharides on the
biological activities and antigenicity of influenza A/H3N2 virus
hemagglutinin. J Virol, 78(18), 9605-9611.
doi:10.1128/JVI.78.18.9605-9611.2004

Baele, G., Lemey, P., Bedford, T., Rambaut, A., Suchard, M. A., \&
Alekseyenko, A. V. (2012). Improving the accuracy of demographic and
molecular clock model comparison while accommodating phylogenetic
uncertainty. Mol Biol Evol, 29(9), 2157-2167. doi:10.1093/molbev/mss084

Baele, G., Li, W. L., Drummond, A. J., Suchard, M. A., \& Lemey, P.
(2013). Accurate model selection of relaxed molecular clocks in bayesian
phylogenetics. Mol Biol Evol, 30(2), 239-243. doi:10.1093/molbev/mss243

Bi, Y., Li, J., Li, S., Fu, G., Jin, T., Zhang, C., . . . Shi, W.
(2020). Dominant subtype switch in avian influenza viruses during
2016-2019 in China. Nat Commun, 11(1), 5909.
doi:10.1038/s41467-020-19671-3

Crowe, J. E., Jr. (2012). Influenza virus resistance to human
neutralizing antibodies. mBio, 3(4), e00213-00212.
doi:10.1128/mBio.00213-12

Das, S. R., Hensley, S. E., David, A., Schmidt, L., Gibbs, J. S.,
Puigbo, P., . . . Yewdell, J. W. (2011). Fitness costs limit influenza A
virus hemagglutinin glycosylation as an immune evasion strategy. Proc
Natl Acad Sci U S A, 108(51), E1417-1422. doi:10.1073/pnas.1108754108

Drummond, A. J., Nicholls, G. K., Rodrigo, A. G., \& Solomon, W. (2002).
Estimating mutation parameters, population history and genealogy
simultaneously from temporally spaced sequence data. Genetics, 161(3),
1307-1320. doi:10.1093/genetics/161.3.1307

Drummond, A. J., Rambaut, A., Shapiro, B., \& Pybus, O. G. (2005).
Bayesian coalescent inference of past population dynamics from molecular
sequences. Mol Biol Evol, 22(5), 1185-1192. doi:10.1093/molbev/msi103

Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high
accuracy and high throughput. Nucleic Acids Res, 32(5), 1792-1797.
doi:10.1093/nar/gkh340

Gao, R., Gu, M., Shi, L., Liu, K., Li, X., Wang, X., . . . Liu, X.
(2021). N-linked glycosylation at site 158 of the HA protein of H5N6
highly pathogenic avian influenza virus is important for viral
biological properties and host immune responses. Vet Res, 52(1), 8.
doi:10.1186/s13567-020-00879-6

Gerloff, N. A., Khan, S. U., Balish, A., Shanta, I. S., Simpson, N.,
Berman, L., . . . Davis, C. T. (2014). Multiple reassortment events
among highly pathogenic avian influenza A(H5N1) viruses detected in
Bangladesh. Virology, 450-451, 297-307. doi:10.1016/j.virol.2013.12.023

Hensley, S. E., Das, S. R., Bailey, A. L., Schmidt, L. M., Hickman, H.
D., Jayaraman, A., . . . Yewdell, J. W. (2009). Hemagglutinin receptor
binding avidity drives influenza A virus antigenic drift. Science,
326(5953), 734-736. doi:10.1126/science.1178258

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. Arch Virol, 146(12), 2275-2289.
doi:10.1007/s007050170002

Jin, F., Dong, X., Wan, Z., Ren, D., Liu, M., Geng, T., . . . Ye, J.
(2019). A Single Mutation N166D in Hemagglutinin Affects Antigenicity
and Pathogenesis of H9N2 Avian Influenza Virus. Viruses, 11(8).
doi:10.3390/v11080709

Kaverin, N. V., Rudneva, I. A., Ilyushina, N. A., Lipatov, A. S.,
Krauss, S., \& Webster, R. G. (2004). Structural differences among
hemagglutinins of influenza A virus subtypes are reflected in their
antigenic architecture: analysis of H9 escape mutants. J Virol, 78(1),
240-249. doi:10.1128/jvi.78.1.240-249.2004

Koel, B. F., Burke, D. F., Bestebroer, T. M., van der Vliet, S., Zondag,
G. C., Vervaet, G., . . . Smith, D. J. (2013). Substitutions near the
receptor binding site determine major antigenic change during influenza
virus evolution. Science, 342(6161), 976-979.
doi:10.1126/science.1244730

Lewis, N. S., Anderson, T. K., Kitikoon, P., Skepner, E., Burke, D. F.,
\& Vincent, A. L. (2014). Substitutions near the hemagglutinin
receptor-binding site determine the antigenic evolution of influenza A
H3N2 viruses in U.S. swine. J Virol, 88(9), 4752-4763.
doi:10.1128/JVI.03805-13

Li, X., Tian, B., Jianfang, Z., Yongkun, C., Xiaodan, L., Wenfei, Z., .
. . Shu, Y. (2017). A comprehensive retrospective study of the
seroprevalence of H9N2 avian influenza viruses in occupationally exposed
populations in China. PLoS One, 12(6), e0178328.
doi:10.1371/journal.pone.0178328

Okamatsu, M., Sakoda, Y., Kishida, N., Isoda, N., \& Kida, H. (2008).
Antigenic structure of the hemagglutinin of H9N2 influenza viruses. Arch
Virol, 153(12), 2189-2195. doi:10.1007/s00705-008-0243-2

Peacock, T., Reddy, K., James, J., Adamiak, B., Barclay, W., Shelton,
H., \& Iqbal, M. (2016). Antigenic mapping of an H9N2 avian influenza
virus reveals two discrete antigenic sites and a novel mechanism of
immune escape. Sci Rep, 6, 18745. doi:10.1038/srep18745

Peacock, T. P., Benton, D. J., James, J., Sadeyen, J. R., Chang, P.,
Sealy, J. E., . . . Iqbal, M. (2017). Immune Escape Variants of H9N2
Influenza Viruses Containing Deletions at the Hemagglutinin Receptor
Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic
Characteristics. J Virol, 91(14). doi:10.1128/JVI.00218-17

Peacock, T. P., Harvey, W. T., Sadeyen, J. R., Reeve, R., \& Iqbal, M.
(2018). The molecular basis of antigenic variation among A(H9N2) avian
influenza viruses. Emerg Microbes Infect, 7(1), 176.
doi:10.1038/s41426-018-0178-y

Peacock, T. P., Sealy, J. E., Harvey, W. T., Benton, D. J., Reeve, R.,
\& Iqbal, M. (2020). Genetic determinants of receptor-binding preference
and zoonotic potential of H9N2 avian influenza viruses. J Virol.
doi:10.1128/JVI.01651-20

Poh, Z. W., Wang, Z., Kumar, S. R., Yong, H. Y., \& Prabakaran, M.
(2020). Modification of neutralizing epitopes of hemagglutinin for the
development of broadly protective H9N2 vaccine. Vaccine, 38(6),
1286-1290. doi:10.1016/j.vaccine.2019.11.080

Pybus, O. G., \& Rambaut, A. (2002). GENIE: estimating demographic
history from molecular phylogenies. Bioinformatics, 18(10), 1404-1405.
doi:10.1093/bioinformatics/18.10.1404

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

Santos, J. J. S., Abente, E. J., Obadan, A. O., Thompson, A. J.,
Ferreri, L., Geiger, G., . . . Perez, D. R. (2019). Plasticity of Amino
Acid Residue 145 Near the Receptor Binding Site of H3 Swine Influenza A
Viruses and Its Impact on Receptor Binding and Antibody Recognition. J
Virol, 93(2). doi:10.1128/JVI.01413-18

Song, J., Wang, C., Gao, W., Sun, H., Jiang, Z., Wang, K., . . . Pu, J.
(2020). A D200N hemagglutinin substitution contributes to antigenic
changes and increased replication of avian H9N2 influenza virus. Vet
Microbiol, 245, 108669. doi:10.1016/j.vetmic.2020.108669

Su, H., Zhao, Y., Zheng, L., Wang, S., Shi, H., \& Liu, X. (2020).
Effect of the selection pressure of vaccine antibodies on evolution of
H9N2 avian influenza virus in chickens. AMB Express, 10(1), 98.
doi:10.1186/s13568-020-01036-0

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. Virus Evol, 4(1), vey016.
doi:10.1093/ve/vey016

Sun, Y., Cong, Y., Yu, H., Ding, Z., \& Cong, Y. (2021). Assessing the
effects of a two-amino acid flexibility in the Hemagglutinin 220-loop
receptor-binding domain on the fitness of Influenza A(H9N2) viruses.
Emerg Microbes Infect, 10(1), 822-832. doi:10.1080/22221751.2021.1919566

Sun, Y., \& Liu, J. (2015). H9N2 influenza virus in China: a cause of
concern. Protein Cell, 6(1), 18-25. doi:10.1007/s13238-014-0111-7

Wan, Z., Ye, J., Xu, L., Shao, H., Jin, W., Qian, K., . . . Qin, A.
(2014). Antigenic mapping of the hemagglutinin of an H9N2 avian
influenza virus reveals novel critical amino acid positions in antigenic
sites. J Virol, 88(7), 3898-3901. doi:10.1128/JVI.03440-13

Wu, N. C., \& Wilson, I. A. (2020). Structural Biology of Influenza
Hemagglutinin: An Amaranthine Adventure. Viruses, 12(9).
doi:10.3390/v12091053

Xia, J., Adam, D. C., Moa, A., Chughtai, A. A., Barr, I. G., Komadina,
N., \& MacIntyre, C. R. (2020). Comparative epidemiology, phylogenetics,
and transmission patterns of severe influenza A/H3N2 in Australia from
2003 to 2017. Influenza Other Respir Viruses, 14(6), 700-709.
doi:10.1111/irv.12772

Xia, J., Cui, J. Q., He, X., Liu, Y. Y., Yao, K. C., Cao, S. J., . . .
Huang, Y. (2017). Genetic and antigenic evolution of H9N2 subtype avian
influenza virus in domestic chickens in southwestern China, 2013-2016.
PLoS One, 12(2), e0171564. doi:10.1371/journal.pone.0171564

Xia, J., He, X., Yao, K. C., Du, L. J., Liu, P., Yan, Q. G., . . .
Huang, Y. (2016). Phylogenetic and antigenic analysis of avian
infectious bronchitis virus in southwestern China, 2012-2016. Infect
Genet Evol, 45, 11-19. doi:10.1016/j.meegid.2016.08.011

Xia, J., Yao, K. C., Liu, Y. Y., You, G. J., Li, S. Y., Liu, P., . . .
Huang, Y. (2017). Isolation and molecular characterization of prevalent
Fowl adenovirus strains in southwestern China during 2015-2016 for the
development of a control strategy. Emerg Microbes Infect, 6(11), e103.
doi:10.1038/emi.2017.91

Zhou, Z. J., Qiu, Y., Pu, Y., Huang, X., \& Ge, X. Y. (2020). BioAider:
An efficient tool for viral genome analysis and its application in
tracing SARS-CoV-2 transmission. Sustain Cities Soc, 63, 102466.
doi:10.1016/j.scs.2020.102466

Zhu, Y., Yang, D., Ren, Q., Yang, Y., Liu, X., Xu, X., . . . Liu, X.
(2015). Identification and characterization of a novel antigenic epitope
in the hemagglutinin of the escape mutants of H9N2 avian influenza
viruses. Vet Microbiol, 178(1-2), 144-149.
doi:10.1016/j.vetmic.2015.04.012

\textbf{Table 1} The titer of mutant CQY-2014 H9N2-AIVs in MDCK cells
and chicken embryos\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
Virus\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
TCID\textsubscript{50}/mL (log10)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
EID\textsubscript{50}/mL (log10)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Parental virus CQY-2014\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
6.50\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
8.50\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
R164Q\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
6.0\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
A168N\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
5.33\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
I171T\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
5.0\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
8.0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
T198A\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
5.0\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7.75\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
R200T\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
5.0\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
D201G\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
5.33\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7.25\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
D201A\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
5.33\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7.0\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2} Analysis of mutants with antigenicity differences
compared with parental virus CQY-2014\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Influence extent & Judgment method & Judgment method & Judgment method &
Judgment method\tabularnewline
\midrule
\endhead
& r value of HI titer & Antigen map of HI titer & r value of
microneutralization titer & Antigen map of microneutralization
titer\tabularnewline
Significant antigenic differences & \textbf{A168N,} T198A,
\textbf{D201G} & \textbf{A168N}, \textbf{D201G} & R164Q, \textbf{D201G},
\textbf{A168N}, D201A & \textbf{A168N,} \textbf{D201G}, T198A,
I171T\tabularnewline
Small antigenic differences & D201A, R164Q & / & I171T, R200T &
/\tabularnewline
Antigenically similar & I171T, R200T & T198A, I171T, R200T, D201A, R164Q
& T198A & R200T, R164Q, D201A, R200T\tabularnewline
\bottomrule
\end{longtable}

Note: The mutants in bold were those that meet the all criteria\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} HA maximum clade credibility (MCC) phylogenetic tree
and specific antigenic sites for each subclade of H9N2 AIVs isolated
between 2014 and 2020 in China. Branches were colored by height. Only
specific antigenic mutations accounting for more than 30\% of each
subclade are presented.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2} Antigenic sites on the surface of the HA protein of
H9N2 AIV. The HA amino acid sequence of CQY-2014 isolate and mutations
were uploaded to the SWISS-MODEL website for model prediction, and the
predicted trimeric proteins were annotated using the ChimeraX 1.3
software. \textbf{A:} Forty-six antigenic sites were labeled gray in the
\selectlanguage{greek}α-\selectlanguage{english}subunits, and the six preselected high-frequency mutation sites were
labeled yellow in the \selectlanguage{greek}β-\selectlanguage{english}subunits.\textbf{B:} When asparagine was
substituted with glycine at site 201 (D201G), the \selectlanguage{greek}α \selectlanguage{english}helix structure
remained unchanged, but the surface structure changed significantly (red
boxes).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} Nucleotide alignment of mutant fragments and growth
kinetics of CQY-2014 mutants. \textbf{A} : There were no unexpected
mutations in the sequencing of each virus. \textbf{B} : growth kinetics
of CQY-2014 mutants in MDCK cells and chicken embryos.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4} Virus recovery from chickens at different times post
challenge.

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

