This first fifth-generation scientific journal ranks each article through peer review
1
Large-scale Surveillance and In-depth Evolutionary Analyses of H7N9
2
Avian Influenza Virus
Author(s)
Su-Chun Wang, Shuo Liu, Wen-Ming Jiang, Qing-Ye Zhuang, Kai-Cheng Wang,
Guang-Yu Hou, Jin-Ping Li, Jian-Min Yu, Xiang Du, Zhi-Yuan Yang, Yue-Huan Liu,
Ji-Wang Chen, Ji-Ming Chen
Author Affiliation(s)
China Animal Health and Epidemiology Center, Qingdao, 266032, China (Su-Chun
Wang, Shuo Liu, Wen-Ming Jiang, Qing-Ye Zhuang, Kai-Cheng Wang, Guang-Yu Hou,
Jin-Ping Li, Jian-Min Yu, Xiang Du, Ji-Ming Chen); Institute of Animal Husbandry and
Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing,
100097, China (Zhi-Yuan Yang, Yue-Huan Liu); Department of Medicine, Section of
Pulmonary, Critical Care, Sleep and Allergy Medicine, University of Illinois at Chicago,
Chicago, IL60612, USA (Ji-Wang Chen)
Author Contribution
Conceived and designed the study: Ji-Ming Chen; performed the study: Su-Chun
Wang, Shuo Liu, Wen-Ming Jiang, Qing-Ye Zhuang, Kai-Cheng Wang, Guang-Yu Hou,
Jin-Ping Li, Jian-Min Yu, Xiang Du, Zhi-Yuan Yang, Yue-Huan Liu, Ji-Ming Chen;
contributed reagents/materials/analysis tools: Ji-Ming Chen; wrote the paper: Ji-Ming
Chen, Ji-Wang Chen; contributed equally to this study: Su-Chun Wang, Shuo Liu,
Wen-Ming Jiang
Acknowledgement(s)
The authors of this article thank the researchers and laboratories for originating and
submitting some sequences to EpiFlu database of the Global Initiative on Sharing All
Influenza Data (GISAID) because these sequences partially formed the basis of this
study
Corresponding Author(s)
Ji-Ming Chen (E-mail: [email protected]; [email protected])
Citation
Wang SC, Liu S, Jiang WM, Zhuang QY, Wang KC, Hou GY, et al. Large-scale surveillance
and in-depth evolutionary analyses of H7N9 avian influenza virus. Newpubli. 2015; 1:
e0004
URL and/or DOI
http://www.newpubli.com/article!articleDetail.shtml?articleId=144
Article History
Received: 18-12-2015; preprint published: 10-11-2015; article published: 24-02-2016
PR-Rank
6
Subject Area(s)
Ecology; Epidemiology; Evolution; Genetics; Infectious diseases; Microbiology;
Molecular & cellular biology; Public health; Theoretical biology; Veterinary medicine;
Virology
3
Newpubli, 2015, 1, e0004 ● Page 1
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Abstract
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The novel H7N9 subtype avian influenza virus (AIV) has caused hundreds of human deaths in China since its
6
emergence in 2013. In this study, we conducted large-scale surveillance of AIVs, which demonstrated the
7
prevalence and distribution of the H7N9 AIV and its potential gene-donor viruses (H9N2 subtype AIV) in different
8
species of poultry. We also conducted in-depth phylogenetic analyses of AIVs, which suggested that one genotype
9
of the H9N2 subtype AIV circulating in chickens, pigeons and bramblings could donate six internal genes to the
10
H7N9 AIV, and multiple genotypes of the H7N9 AIV circulated in Henan province. Moreover, by calculating the
11
distribution of the mutations and the nonsynonymous/synonymous rate ratios, we identified five mutations in the
12
viral HA gene specific to the H7N9 AIV, and one of the specific mutations, Q226L (H3 numbering) that confers
13
increased binding to human-like receptors, was probably fixed by positive selection. These results are important for
14
the design of evidence-based measures to control this zoonotic virus, as well as providing novel insights into the
15
distribution, risk and evolution of H7N9 AIVs. Additionally, we proposed a novel hypothesis that the H7N9 AIV may
16
have originated in pigeons through natural selection.
17
Significance
18
This study comprised large-scale surveillance of avian influenza viruses (AIVs) and novel in-depth evolutionary
19
analyses of the H7N9 AIV, which has caused hundreds of human deaths in China. Approximately 15,000 samples
20
were detected and thousands of novel AIV sequences were obtained through the surveillance which demonstrated
21
the prevalence and distribution of the H7N9 AIV and H9N2 subtype AIVs in different species of poultry. Based on
22
evolutionary analyses, all of the early H7N9 AIV and H9N2 subtype AIVs were divided into 43 genotypes. Multiple
23
genotypes of the H7N9 AIV were found exclusively in Henan province and five mutations in the viral HA gene were
24
identified as specific to the H7N9 AIV. The evolutionary analyses also suggested that one of the five specific
25
mutations, Q226L, which confers increased binding to human-like receptors, was probably fixed by positive
26
selection. These results are important for the design of evidence-based measures to control this zoonotic virus, as
27
well as providing novel insights into the distribution, risk and evolution of H7N9 AIVs. Additionally, we propose a
28
novel hypothesis that the H7N9 AIV may have originated in pigeons through natural selection. The hypothesis,
29
although controversial, is a novel explanation regarding the emergency of the H7N9 AIV.
30
Keywords
31
avian influenza virus; distribution; evolution; H7N9; pigeon; receptor; selection; surveillance
32
Abbreviations
33
AIV, avian influenza viruses; GISAID, the Global Initiative on Sharing All Influenza Data; HA, hemagglutinin; LBM, live
34
bird markets; MP, matrix protein; NA, neuraminidase; NP, nucleoprotein; NS, nonstructural protein; PA, acidic
35
polymerase; PB1, basic polymerase 1; PB2, basic polymerase 2; ts/tv, transition/transversion ratios; , the
36
nonsynonymous/synonymous rate ratio
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Introduction
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Influenza A virus causes frequent epidemics and occasional pandemics in various animals, including birds,
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humans, pigs, horses, cattle, marine mammals and bats [1-5]. The viral genome comprises eight segments, which
41
correspond to the viral genes for basic polymerase 2 (PB2), basic polymerase 1 (PB1), acidic polymerase (PA),
42
hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (MP) and nonstructural protein (NS).
43
The viral HA and NA genes encode surface HA and NA glycoproteins. The remaining six internal genes encode PB2,
44
PB1, PA, NP and other internal structural and nonstructural proteins [6-7].
45
Based on differences in the antigenicity of the viral HA and NA glycoproteins, influenza A viruses can be
46
categorized into 18 HA subtypes (H1H18) and 11 NA subtypes (N1N11) [1-4]. Their combinations further
47
generate H1N1, H3N2, H7N7, H9N2 and many other influenza A virus subtypes. Each of the viral genes has evolved
48
into multiple lineages and genomic reassortment of these lineages has generated multiple genotypes for each
49
subtype of influenza A virus [1, 4, 7-8].
50
Avian influenza viruses (AIVs) are influenza A viruses that circulate mainly in birds. They are highly diverse with
51
16 HA subtypes (H1H16) and nine NA subtypes (N1N9). Most AIVs only infect birds, but some can infect humans
52
and other mammals at a low frequency. These zoonotic AIVs continue to present a challenge to human health, such
53
as the H5N1 highly pathogenic AIVs that have circulated in many countries in the past decade [1, 7].
54
A previously unrecognized zoonotic H7N9 AIV that was first identified in China during March 2013, referred to
55
as A/China/2013(H7N9), has since caused >600 human infections with >200 fatalities [9-10]. A/China/2013(H7N9)
56
carries some mutations that confer increased binding to human receptors and enhanced replication in ferrets,
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thereby raising worldwide concerns of a new pandemic [11-16].
58
Multiple studies have been conducted to investigate the origin of A/China/2013(H7N9). These studies suggest
59
that A/China/2013(H7N9) probably resulted from the reassortment of H7N?/H?N9 and H9N2 subtype AIVs, which
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contributed the HA, NA and six internal genes for A/China/2013(H7N9) in eastern China early in 2012 [8, 10, 17-22].
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A/China/2013(H7N9) has evolved into multiple genotypes via further reassortment with other AIVs [17, 22].
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It has been suggested that H7N?/H?N9 AIVs in ducks or other waterfowl probably contributed the HA and NA
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genes to A/China/2013(H7N9), and that the H9N2 subtype AIVs in chickens or wild birds probably contributed the
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six internal genes, but both the original host and the mode of emergence for A/China/2013(H7N9) remain
65
enigmatic [8, 10, 17-22]. In the present study, we conducted large-scale active surveillance and in-depth
66
evolutionary analyses to reveal the host distribution and genetic features of A/China/2013(H7N9) and H9N2 AIVs
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circulating in poultry, as well as exploring the potential role of pigeons in the origin of this zoonotic AIV.
68
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Methods
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Sample collection and virus isolation for AIV surveillance
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We have conducted systematic large-scale active surveillance of AIVs since 2007. During 2012 and 2013, our
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surveillance covered 8–13 provinces, autonomous regions or municipalities. In total, 14,690 swab samples were
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collected by taking smears from the trachea and cloacae of domestic fowl in 2012 and 2013. The samples were
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placed in the transport medium, phosphate-buffered saline containing 10% (v/v) glycerol, and stored at 4°C until
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processing within 2 days. The samples were clarified by centrifugation at 1000 g for 5 min and the supernatants
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were used to inoculate10-day-old specific-pathogen-free chicken embryonated eggs via the allantoic sac route. The
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eggs were further incubated for 4 days and checked twice each day during the incubation period. The dead ones
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were removed and stored in a refrigerator. After the incubation period, the allantoic fluids were collected from the
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live embryos and tested using the hemagglutination assay. All of the hemagglutination-positive samples and the
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allantoic fluids from the dead embryos were investigated further by RT-PCR, as described in the following.
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RT-PCR detection and genomic sequencing
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Viral RNA was extracted from the supernatants using a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany)
83
and stored at –80°C until use. The extracted RNA was analyzed using a RT-PCR assay to amplify and sequence the
84
whole-length genome of influenza A virus, as described previously [23]. The whole-length NA gene of N9 subtype
85
AIVs was amplified using another RT-PCR assay, as described previously [24]. These assays were performed in a
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25-µl reaction system with incubation at 50°C for 30 min and denaturation at 94°C for 2 min, followed by 30 cycles
87
at 94°C for 30 s, 57°C for 30 s and 72°C for 30 s. The amplicons were purified using an agarose gel DNA extraction kit
88
(Takara, Dalian, China) and sequenced using an ABI 3730xl DNA Analyzer. Some amplicons were ligated into the
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pMD19-T Easy vector (Takara) before sequencing.
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Phylogenetic analysis
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Sequences were aligned using the MUSCLE program [25]. The Bayesian information criterion scores of the
92
substitution models and phylogenetic relationships were calculated using the software package MEGA 6.0 [26-27].
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Phylogenetic relationships were calculated using the maximum likelihood model with the lowest Bayesian
94
information criterion score, which was assumed to describe the best substitution pattern. Gaps were handled by
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pairwise deletion and bootstrap values were calculated based on 1000 replicates. Each gene was classified to clades
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according to their phylogenetic relationships and nucleotide sequence identities, as shown in Figure 1. It should be
97
noted that these clades could be divided further into several subclades [8, 10, 18, 22].
98
Structural analysis
99
Structural analysis of the viral HA protein was performed using the Pymol v1.6.x program (www.pymol.org)
100
with the 4ln3 input structural file downloaded from NCBI [15].
101
Calculation of the nonsynonymous/synonymous rate ratios
102
The nonsynonymous/synonymous rate ratio () of each amino acid residue (site) in the viral HA gene was
103
estimated using the PAML 4.4 program [28]. The codon frequencies were set according to the F34 table. The 
104
ratios were analyzed using an unrooted phylogenetic tree under the following models: model M0 (one-ratio)
105
assuming one  for all sites; model M1 (nearly neutral) assuming a class of conserved sites with  = 0 and another
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class of neutral sites with  = 1; model M2 (selection) adding a third class of sites with > 1; model M3 (discrete)
107
assuming a general discrete distribution; model M7 (beta) assuming a beta distribution of , limited in the range (0,
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1); and model M8 (beta &> 1) adding an extra site class with > 1. Models M0, M1 and M7 were set as the null
109
models for comparison with their alternatives [28]. The performance of these models was compared by the
110
likelihood ratio test using the Chi-square test tool in PAML 4.4. The Kappa values (transition/transversion ratios,
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ts/tv) were calculated automatically. The results obtained by Bayes Empirical Bayes analysis were used in this study
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[28-29], except for model M3 where only the Naive Empirical Bayes analysis results were available.
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Nucleotide sequence accession numbers
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The GenBank accession numbers for the 1976 sequences reported in the present study are: GQ166223,
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GQ166224, JN804553, JN804214, JN804405, KP186943–KP187461, KP186146–KP186942, KP185437–KP185518,
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KP185849–KP185930, KP185603–KP185684, KP185685–KP185766, KP185355–KP185436, KP185767–KP185848,
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KP185931–KP186011 and KP185519–KP185602.
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Results
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Prevalence of domestic birds in live bird markets (LBMs)
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Chickens, ducks, geese and pigeons are the first, second, third and fourth most commonly raised domestic
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birds during recent years in China. Among 233 LBMs that we randomly selected in 2012 and 2013 to collect samples
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for AIV surveillance, 50 were selected randomly to estimate the distributions of bird species in LBMs in China.
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Approximately 65.86%, 23.43%, 8.58%, 1.91% and 0.23% of the birds in these 50 LBMs were chickens, pigeons,
124
ducks, geese and other birds, respectively. Thus, chickens and pigeons are the first and second most prevalent birds
125
in LBMs of China in recent years. This is partially because pigeons are mainly sold through LBMs in China, whereas
126
most ducks and geese are not sold through LBMs. Moreover, on multiple occasions, we have observed that pigeons
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stayed for significantly longer in LBMs than other birds, especially in wholesale LBMs.
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Ecology of pigeons in China
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Pigeons were domesticated for meat production over 3000 years ago in China, but large-scale pigeon farms
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were not established until the early 1980s. In recent years, approximately 500 million pigeons have been raised
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annually for meat production in China and the pigeon number has increased annually by 10%–15% [30]. In addition
132
to the pigeons raised for meat production, a huge number of wild pigeons live in cities and the countryside in China.
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Wild pigeons and many domestic homing pigeons fly freely during the daytime, and thus they may eat or drink
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together with other domestic or wild birds in the same village or on the same wetland. Moreover, many pigeons
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used for meat production are caged close to chickens, ducks and other birds in many LBMs in China, as shown by
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the examples presented in Figure 2.
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Prevalence of H9N2 subtype AIVs and A/China/2013(H7N9) in LBMs during 2012–2013
138
In total, 915 AIVs were detected from the 5051 swab samples that we collected at 87 LBMs in 17 provinces,
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autonomous regions or municipalities during our surveillance study in 2012. Among these, 60.00% (549/915) were
140
H9N2 subtype AIVs distributed in 68.97% of the LBMs and 82.35% of the provinces, autonomous regions or
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municipalities where the samples were collected. As shown in Table 1, the prevalence of H9N2 subtype AIVs was
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significantly higher in chickens (14.53%) and pigeons (8.94%) compared with that in ducks (4.18%) and geese
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(2.56%) (P < 0.01, Chi-square test).
144
H7 subtype AIVs were not detected in the 5051 swab samples that we collected in 2012. We identified only
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one H7 subtype AIV in 2009 based on our large-scale surveillance study from 2007–2012 [31]. By contrast, 31
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A/China/2013(H7N9) viruses were detected from the 6513 swab samples collected at 146 LBMs in 17 provinces,
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autonomous regions or municipalities during 2013. The prevalence of A/China/2013(H7N9) was 0.79% in chickens,
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0.37% in pigeons, 0.00% in ducks, and 0.33% in geese (Table 2). These data suggest that A/China/2013(H7N9) was
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relatively prevalent in chickens and pigeons. It was also relatively prevalent in geese, but not prevalent in ducks,
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which is consistent with a recent report that A/China/2013(H7N9) replicated inefficiently in domestic or wild ducks
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[24]. These results suggest that A/China/2013(H7N9) has become adapted to terrestrial birds.
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Our surveillance study in 2013 demonstrated that H9N2 subtype AIVs were distributed in 77.40% of the LBMs
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and 100% of the provinces, autonomous regions or municipalities in which the samples were collected. In addition,
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H9N2 subtype AIVs were significantly more prevalent in chickens and pigeons compared with ducks and geese
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(Table 2). These results suggest that H9N2 subtype AIVs were highly prevalent in China, and have adapted to
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terrestrial birds.
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A/China/2013(H7N9) viruses were distributed in 4.11% of the LBMs and 11.76% of the provinces, autonomous
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regions or municipalities where the surveillance samples were collected. A/China/2013(H7N9) viruses were
159
significantly less prevalent than H9N2 subtype AIVs, but it was quite difficult to eradicate the zoonotic virus through
160
surveillance and culling because the virus had spread to numerous provinces and it did not cause any obvious
161
symptoms in poultry.
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Analysis of the six internal genes of AIVs
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We performed phylogenetic analyses of the six internal gene sequences of 268 H9N2 subtype AIVs (170 from
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chickens, 36 from pigeons and 62 from other birds) isolated from the samples collected in China during 2010–2013
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(63 of which are reported for the first time in the present study), and 127 early A/China/2013(H7N9) viruses
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isolated from the samples collected before May 1, 2013 (19 of which are reported for the first time in the present
167
study). As shown in Figure 3 and Attachments 1–6, each of the six internal genes in these H7N9 viruses and H9N2
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viruses could be classified into multiple clades. Based on the clade constellation of these six internal genes, the 268
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H9N2 subtype AIVs and 127 early A/China/2013(H7N9) viruses were classified according to 43 genotypes
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(Attachment 7). Among these 43 genotypes, Genotype 1 included both H9N2 subtype AIVs and the early
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A/China/2013(H7N9) viruses; Genotypes 8, 12 and 24 contained only the early A/China/2013(H7N9) viruses; and
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the remaining 39 genotypes contained only H9N2 subtype AIVs.
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As showed in Attachment 7, Genotype 2 was different from Genotype 1 with respect to the viral NS gene and it
174
was the dominant genotype in the H9N2 subtype AIVs, comprising nearly half (128/268) of the H9N2 subtype AIVs.
175
By contrast, Genotype 1 was the dominant genotype in the early A/China/2013(H7N9) viruses, comprising most
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(124/127) of the early A/China/2013(H7N9) viruses. Genotype 1 also included more of the H9N2 subtype AIVs than
177
other genotypes, except Genotype 2, and these H9N2 subtype AIVs had circulated in poultry before the emergence
178
of A/China/2013(H7N9) viruses. Therefore, the H9N2 subtype AIVs within Genotype 1 which circulated in multiple
179
species of birds, including chickens, pigeons and bramblings, e.g., A/pigeon/Jiangsu/K77/2013(H9N2), possibly
180
contributed the six internal genes to the early A/China/2013(H7N9) viruses. The six internal genes of
181
A/pigeon/Jiangsu/K77/2013(H9N2) all shared high homology with those of A/China/2013(H7N9),where the
182
nucleotide sequence identities ranged from 97.85% to 99.41%.
183
Among the 127 early A/China/2013(H7N9) viruses, 21 were isolated from Henan province in China during this
184
study. Interestingly, as shown in Attachment 7, these 21 early A/China/2013(H7N9) viruses from Henan could be
185
classified into four genotypes, whereas the remaining 106 early A/China/2013(H7N9) viruses from nine provinces,
186
autonomous regions or municipalities all belonged to Genotype 1.
187
Analysis of the mutations specific to A/China/2013(H7N9)
188
We downloaded and analyzed the HA gene sequences ( 500 bp) of 1261 H7 subtype influenza viruses isolated
189
in the eastern hemisphere, including Africa, Europe, Asia and Oceania, from the Global Initiative on Sharing All
190
Influenza Data (GISAID) database on June 1, 2014. Among these, 207 were A/China/2013(H7N9) viruses detected in
191
2013–2014 and 1054 were other H7 viruses (176 circulating in 2010–2013 and 878 circulating before 2010). As
192
shown in Table 3, three mutations, i.e., I179V (H3 numbering throughout), T189A and N289D, were prevalent
193
(prevalence > 85%) in the 207 A/China/2013(H7N9) viruses and not rare (prevalence  20%) in the 176 other H7
194
viruses circulating in 2010–2013. Five other mutations, i.e., D174S, G186V, Q226L, E312R and N445D, were
195
prevalent (prevalence > 85%) in the 207 A/China/2013(H7N9) viruses but rare (prevalence < 10%) in the two groups
196
of other H7 viruses. Therefore, these five mutations were considered to be specific to A/China/2013(H7N9) viruses
197
and no other mutations in the viral HA genes were identified as specific to A/China/2013(H7N9) viruses.
198
Two of the five mutations specific to A/China/2013(H7N9) viruses, i.e., G186V and Q226L, were located in the
199
viral HA protein motif responsible for receptor binding (Figure 2). It is known that these two specific mutations,
200
especially Q226L, confer increased binding to human-like receptors. Various studies have demonstrated that
201
although A/China/2013(H7N9) retains its tight binding to avian-like receptors and weak binding to human-like
202
receptors, its binding to human-like receptors increased, which is assumed to be crucial for the causation of human
203
infections [11-16, 32].
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During evolution, most random mutations occur only in some individuals and they cannot be fixed at the
205
population, lineage or species levels. Thus, only a small proportion of random mutations can be fixed at the
206
population, lineage or species levels through the effects of random factors (i.e., random genetic drift), selective
207
factors (i.e., natural selection) or hitchhiking (i.e., fixation of a mutation by natural selection leading to the fixation
208
of another mutation linked to the naturally selected mutation). The mutations fixed through random drift or
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hitchhiking are probably in random distribution, whereas the random mutations fixed by natural selection are
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probably distributed in specific motifs with biological significance, e.g., those determining the antigenicity or
211
receptor-binding property of a protein [33-34]. Less than 20 of the approximately 560 amino acid residues in the
212
viral HA gene confer increased binding to human-like receptors [11-16], so the possibility should be less than:
213
(20×5/560)×(19×5/560) = 3.0%, for two of the only five specific mutations (G186V and Q226L) to occur at the
214
residues conferring increased binding to human-like receptors through random genetic drift or hitchhiking.
215
Therefore, at least one of the two mutations that confer increased binding to human-like receptors was probably
216
fixed by positive selection rather than random genetic drift. We did not exclude the conserved stalk region of the
217
HA gene because at least two of the five specific mutations, i.e., E312R and N445D, occur in the stalk region (Figure
218
4), and the receptor-binding motif in the head region of the viral HA gene is also highly conserved [35].
219
Calculation of the  value for each site in the viral HA gene
220
Among the aforementioned 207 H7 subtype influenza viruses, 104 belonged to the early A/China/2013(H7N9)
221
viruses detected before May 1, 2013 without ambiguous nucleotides in their HA gene sequences. Among the
222
aforementioned 1054 other H7 viruses, 53 had no ambiguous nucleotides in their HA gene sequences and they
223
were most closely related in phylogenetics to the 104 early A/China/2013(H7N9) viruses according to their
224
phylogenetic relationships (see Figure 5) and HA gene sequence identities (> 96.8%). Based on the HA gene
225
sequences of the 104 early A/China/2013(H7N9) viruses and the 53 other H7 viruses, we calculated the  value for
226
each site (namely amino acid residue) in the viral HA gene of the H7 subtype AIVs using PAML 4.4 to further
227
examine whether any of the two amino acid mutations that confer increased binding to human-like receptors were
228
fixed through positive selection, because sites in a gene with the  ratio > 1 have frequently been identified as
229
under positive selection [33-34]. These 157 viruses were selected to calculate the  values because they were the
230
most suitable for reflecting the selection pressure on each site during the origin of the virus.
231
The likelihood ratio tests based on the calculation of the  ratio suggested that model M3 (discrete) was
232
significantly more suitable than the other models, including M0 (one-ratio), M1 (nearly neutral), M2 (positive
233
selection), M7 (beta) and M8 (beta and  > 1) (P < 0.01, Chi-square test) (Table 4). As shown in Table 4, although
234
the sites with  ratios > 1 had a little variation according to the three models allowing positive selection (M2, M3
235
and M8), site 226 had a probability > 95% to be of the  ratio > 1 calculated using all the three models. This
236
suggests that Q226L, one of the five mutations specific to A/China/2013(H7N9) viruses and conferring increased
237
binding to human-like receptors, was likely fixed through positive selection rather than random genetic drift or
238
hitchhiking. Therefore, this specific crucial mutation was probably selected in a host population that favored
239
mutations conferring increased binding to human-like receptors.
240
Discussion
241
In this study, we conducted large-scale surveillance of AIVs to determine the distribution of the H7N9 AIV and
242
its potential gene-donor viruses (H9N2 subtype AIVs) in different species of poultry. We also conducted in-depth
243
evolutionary analyses of thousands of AIV sequences, which showed that some H9N2 subtype AIVs from chickens,
244
pigeons and bramblings could donate six internal genes to the H7N9 AIV. In addition, we identified five mutations in
245
the viral HA gene specific to the H7N9 AIV, where one that confers increased binding to human-like receptors, i.e.,
246
Q226L (H3 numbering), was probably fixed through positive selection, according to the calculated site distribution
247
and the nonsynonymous/synonymous rate ratios.
248
The ability of an influenza virus to replicate efficiently in a host depends on multiple factors [35]. Clearly,
249
receptor matching is necessary but not sufficient for efficient replication of the virus, which explains why
250
A/China/2013(H7N9) replicated inefficiently in ducks and it was rare in ducks [24], although it bound efficiently to
251
duck avian-like receptors.
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It has been reported many times that pigeons are naturally resistant to infection of most AIVs [36-37]. In part,
253
this may be because pigeons uniquely carry abundant human-like receptors and few avian-like receptors in their
254
respiratory tracts, and thus most AIVs cannot replicate efficiently in pigeons [38-40]. In fact, it has been found that
255
A/China/2013(H7N9) replicated inefficiently in pigeons, but efficiently in chickens, and our surveillance study and
256
those published previously all suggest that A/China/2013(H7N9) was most prevalent in chickens [9, 16, 41]. This is
257
consistent with previously reported epidemiological findings that chickens were the major source for human
258
infections and pigeons were the probable source for only a few human cases [9, 42].
259
Thus, why did A/China/2013(H7N9) replicate inefficiently in pigeons even though the virus exhibited increased
260
binding to human-like receptors in pigeons? It is possible that A/China/2013(H7N9) remains to bind weakly to
261
human-like receptors and tightly to avian-like receptors [11-16]. Similar to this suggested scenario, common AIVs
262
bind to avian-like and human-like receptors with 100 and 5 units of avidity, respectively, whereas
263
A/China/2013(H7N9) binds to avian-like and human-like receptors with 100 and 15 units of avidity, respectively.
264
Our surveillance data suggest that H9N2 subtype AIVs were relatively prevalent in pigeons on LBMs in China;
265
however, this does not contradict the fact that pigeons are resistant to AIVs infections for the following three
266
reasons. First, the replication of a low pathogenic virus in the respiratory and alimentary systems of a host might
267
not lead to an infection with clinical signs. Second, unlike many other AIVs, most of the H9N2 subtype AIVs that
268
circulated in China during recent years carried the Q226L mutation in their HA gene, which confers increased
269
binding to pigeon human-like receptors [13, 16, 43], thereby facilitating viral replication in pigeons. Third, many
270
pigeons are kept in bad conditions on LBMs, which could weaken the pigeons and facilitate the replication of H9N2
271
subtype AIVs in pigeons.
272
Our surveillance data also suggest that A/China/2013(H7N9) was relatively prevalent in pigeons on LBMs in
273
China. This is consistent with the emergent disease surveillance conducted by Harbin Veterinary Research Institute
274
in April and May 2013, which showed that the prevalence of A/China/2013(H7N9) was 1.74% (3/172) in pigeons on
275
LBMs [16]. In addition, they identified A/China/2013(H7N9) only on one pigeon farm among 253 poultry farms
276
where they collected samples, and only in one wild pigeon sample among the 739 wild bird samples that they
277
detected through that emergent disease surveillance [16]. Another research group also isolated two strains of
278
A/China/2013(H7N9) from pigeons in April 2013, with GISAID accession numbers of 162,874 and 162,875. The
279
relatively high prevalence of A/China/2013(H7N9) in pigeons on LBMs indicates that the virus can replicate in
280
pigeons. This does not contradict the fact that pigeons are naturally resistant to AIV infection and that
281
A/China/2013(H7N9) replicated inefficiently in pigeons due to the same reasons given above to explain the
282
relatively high prevalence of H9N2 subtype AIVs in pigeons, including the increased binding to human-like receptors
283
in pigeons by A/China/2013(H7N9) [13, 16, 43]. Moreover, many pigeons are kept in bad conditions on LBMs and
284
farms, which could facilitate viral replication in pigeons.
285
Multiple research entities have isolated other AIV subtypes from pigeons on LBMs or farms in recent years [8,
286
16, 44-52], which are similar to our surveillance data (Tables 1 and 2). Most reports of animal experimental data
287
suggest that pigeons are naturally resistant to AIV infection but they also showed that AIVs can replicate in pigeons
288
for a short time, although inefficiently [37, 40, 53-58]. Two experimental studies showed that A/China/2013(H7N9)
289
could replicate in pigeons, but only inefficiently [24, 59].
290
According to many previous reports, A/China/2013(H7N9) probably originated in birds rather than mammals [8,
291
10, 17-22]. Among the known bird species that harbor AIVs, only pigeons possess abundant human-like receptors
292
and few avian-like receptors in their respiratory tracts [38-39], so pigeons can potentially provide a favorable
293
environment for the selection of the crucial specific mutation in A/China/2013(H7N9), i.e., Q226L, which confers
294
increased binding to human-like receptors.
295
Based on all of these findings, we consider that A/China/2013(H7N9) may have originated in pigeons through
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natural selection for the following reasons. First, pigeons are populous in China and they are frequently kept close
297
to other birds, especially on LBMs. Therefore, there are numerous opportunities for AIVs to replicate in pigeons and
298
spread between pigeons and other birds. Second, A/China/2013(H7N9) was relatively prevalent in pigeons during
299
2013. Third, H9N2 subtype AIVs were also relatively prevalent in pigeons during 2012 and 2013, and some H9N2
300
subtype AIVs in pigeons, such as A/pigeon/Jiangsu/K77/2013(H9N2), could have provided their six internal genes to
301
A/China/2013(H7N9). Fourth and most importantly, the crucial specific mutation found in A/China/2013(H7N9) that
302
confers increased binding to human-like receptors was probably fixed in the viral genome through positive selection,
303
and pigeons are the only known birds that may exert pressure on the selection of this specific mutation because
304
they uniquely possess abundant human-like receptors and a few avian-like receptors in their respiratory tracts. We
305
also explain this hypothesis in Attachment 8 as well as suggesting that multiple species of birds were possibly
306
involved in the emergence of A/China/2013(H7N9) (Figure 6). More evidence is needed to support this hypothesis,
307
but for the first time, this controversial hypothesis suggests a novel potential mechanism for the origin of zoonotic
308
AIVs, including A/China/2013(H7N9), without the involvement of pigs.
309
The ecological and evolutionary results reported in the present study are important for the control of the
310
zoonotic virus and its risk analysis. For example, more efforts should be directed to chickens rather than ducks on
311
LBMs to control the zoonotic virus, and we should not ignore the potentially important role of pigeons in the
312
circulation and evolution of AIVs, although they are naturally resistant to infection by AIVs.
313
In summary, we obtained many novel results in this study related to the ecology and evolution of
314
A/China/2013(H7N9), which are important for the design of evidence-based measures to control the zoonotic virus,
315
and shed novel insights into the distribution, risk and evolution of A/China/2013(H7N9).
316
Attachments
317
Attachment 1. Clades of some H9N2 and H7N9 viruses based on their PB2 gene (png, 1.1Mb).
318
Attachment 2. Clades of some H9N2 and H7N9 viruses based on their PB1 gene (png, 1.1Mb).
319
Attachment 3. Clades of some H9N2 and H7N9 viruses based on their PA gene (png, 1.1Mb).
320
Attachment 4. Clades of some H9N2 and H7N9 viruses based on their NP gene (png, 1.1Mb).
321
Attachment 5. Clades of some H9N2 and H7N9 viruses based on their MP gene (png, 1.1Mb).
322
Attachment 6. Clades of some H9N2 and H7N9 viruses based on their NS gene (png, 1.1Mb).
323
Attachment 7. Genotypes of 268 H9N2 and 127 early A/China/H7N9/2013 viruses based on the clade
324
constellation of their six internal genes (docx, 27 Kb).
325
Attachment 8. Explanation of the hypothesis regarding the possible origin of A/China/2013(H7N9) in pigeons
326
through natural selection (docx, 32 Kb).
327
References
328
1.
Liu S, Ji K, Chen J, Tai D, Jiang W, Hou G, et al. Panorama phylogenetic diversity and distribution of Type A
329
influenza virus. PLoS One. 2009; 4(3): e5022. PMID: 19325912.
330
http://dx.doi.org/10.1371/journal.pone.0005022.
331
2.
Pathog. 2013; 9(10): e1003657. PMID: 24130481. http://dx.doi.org/10.1371/journal.ppat.1003657.
332
333
3.
4.
Yoon SW, Webby RJ, Webster RG. Evolution and ecology of influenza A viruses. Curr Top Microbiol Immunol.
2014. PMID: 24990620. http://dx.doi.org/10.1007/82_2014_396.
336
337
Wu Y, Tefsen B, Shi Y, Gao GF. Bat-derived influenza-like viruses H17N10 and H18N11. Trends Microbiol. 2014;
22(4): 183-191. PMID: 24582528. http://dx.doi.org/10.1016/j.tim.2014.01.010.
334
335
Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, et al. New world bats harbor diverse influenza A viruses. PLoS
5.
Jiang WM, Wang SC, Peng C, Yu JM, Zhuang QY, Hou GY, et al. Identification of a potential novel type of
Newpubli, 2015, 1, e0004 ● Page 9
H7N9 surveillance & evolution
338
influenza virus in Bovine in China. Virus Genes. 2014; 49(3): 493-496. PMID: 25142163.
339
http://dx.doi.org/10.1007/s11262-014-1107-3.
340
6.
Kageyama T, Fujisaki S, Takashita E, Xu H, Yamada S, Uchida Y, et al. Genetic analysis of novel avian A(H7N9)
341
influenza viruses isolated from patients in China, February to April 2013. Euro Surveill. 2013; 18(15): 20453.
342
PMID: 23594575.
343
7.
Chen JM, Sun YX, Chen JW, Liu S, Yu JM, Shen CJ, et al. Panorama phylogenetic diversity and distribution of
344
type A influenza viruses based on their six internal gene sequences. Virol J. 2009; 6: 137. PMID: 19737421.
345
http://dx.doi.org/10.1186/1743-422X-6-137.
346
8.
Lam TT, Wang J, Shen Y, Zhou B, Duan L, Cheung CL, et al. The genesis and source of the H7N9 influenza viruses
347
causing human infections in China. Nature. 2013; 502(7470): 241-244. PMID: 23965623.
348
http://dx.doi.org/10.1038/nature12515.
349
9.
Li Q, Zhou L, Zhou M, Chen Z, Li F, Wu H, et al. Epidemiology of human infections with avian influenza A(H7N9)
350
virus in China. N Engl J Med. 2014; 370(6): 520-532. PMID: 23614499.
351
http://dx.doi.org/10.1056/NEJMoa1304617.
352
10. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, et al. Human infection with a novel avian-origin influenza A (H7N9)
353
virus. N Engl J Med. 2013; 368(20): 1888-1897. PMID: 23577628. http://dx.doi.org/10.1056/NEJMoa1304459.
354
11. Belser JA, Gustin KM, Pearce MB, Maines TR, Zeng H, Pappas C, et al. Pathogenesis and transmission of avian
355
influenza A (H7N9) virus in ferrets and mice. Nature. 2013; 501(7468): 556-559. PMID: 23842497.
356
http://dx.doi.org/10.1038/nature12391.
357
12. Richard M, Schrauwen EJ, de Graaf M, Bestebroer TM, Spronken MI, van Boheemen S, et al. Limited airborne
358
transmission of H7N9 influenza A virus between ferrets. Nature. 2013; 501(7468): 560-563. PMID: 23925116.
359
http://dx.doi.org/10.1038/nature12476.
360
13. Tharakaraman K, Jayaraman A, Raman R, Viswanathan K, Stebbins NW, Johnson D, et al. Glycan receptor
361
binding of the influenza A virus H7N9 hemagglutinin. Cell. 2013; 153(7): 1486-1493. PMID: 23746830.
362
http://dx.doi.org/10.1016/j.cell.2013.05.034.
363
14. Xiong X, Martin SR, Haire LF, Wharton SA, Daniels RS, Bennett MS, et al. Receptor binding by an H7N9 influenza
364
virus from humans. Nature. 2013; 499(7459): 496-499. PMID: 23787694.
365
http://dx.doi.org/10.1038/nature12372.
366
15. Yang H, Carney PJ, Chang JC, Villanueva JM, Stevens J. Structural analysis of the hemagglutinin from the recent
367
2013 H7N9 influenza virus. J Virol. 2013; 87(22): 12433-12446. PMID: 24027325.
368
http://dx.doi.org/10.1128/JVI.01854-13.
369
16. Zhang Q, Shi J, Deng G, Guo J, Zeng X, He X, et al. H7N9 influenza viruses are transmissible in ferrets by
370
respiratory droplet. Science. 2013; 341(6144): 410-414. PMID: 23868922.
371
http://dx.doi.org/10.1126/science.1240532.
372
17. Cui L, Liu D, Shi W, Pan J, Qi X, Li X, et al. Dynamic reassortments and genetic heterogeneity of the
373
human-infecting influenza A (H7N9) virus. Nat Commun. 2014; 5: 3142. PMID: 24457975.
374
http://dx.doi.org/10.1038/ncomms4142.
375
18. Feng Y, Mao H, Xu C, Jiang J, Chen Y, Yan J, et al. Origin and characteristics of internal genes affect infectivity of
376
the novel avian-origin influenza A (H7N9) virus. PLoS One. 2013; 8(11): e81136. PMID: 24278391.
377
http://dx.doi.org/10.1371/journal.pone.0081136.
378
19. Lee RT, Gunalan V, Van TD, Le LT, Eisenhaber F, Maurer-Stroh S. A new piece in the puzzle of the novel
379
avian-origin influenza A (H7N9) virus. Biol Direct. 2013; 8: 26. PMID: 24160334.
380
http://dx.doi.org/10.1186/1745-6150-8-26.
381
20. Van Ranst M, Lemey P. Genesis of avian-origin H7N9 influenza A viruses. Lancet. 2013; 381(9881): 1883-1885.
Newpubli, 2015, 1, e0004 ● Page 10
H7N9 surveillance & evolution
382
383
PMID: 23643449. http://dx.doi.org/10.1016/S0140-6736(13)60959-9.
21. Wang Y, Dai Z, Cheng H, Liu Z, Pan Z, Deng W, et al. Towards a better understanding of the novel avian-origin
384
H7N9 influenza A virus in China. Sci Rep. 2013; 3: 2318. PMID: 23897131.
385
http://dx.doi.org/10.1038/srep02318.
386
22. Wu A, Su C, Wang D, Peng Y, Liu M, Hua S, et al. Sequential reassortments underlie diverse influenza H7N9
387
genotypes in China. Cell Host Microbe. 2013; 14(4): 446-452. PMID: 24055604.
388
http://dx.doi.org/10.1016/j.chom.2013.09.001.
389
390
391
23. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. Universal primer set for the full-length amplification of all
influenza A viruses. Arch Virol. 2001; 146(12): 2275-2289. PMID: 11811679.
24. Pantin-Jackwood MJ, Miller PJ, Spackman E, Swayne DE, Susta L, Costa-Hurtado M, et al. Role of poultry in the
392
spread of novel H7N9 influenza virus in China. J Virol. 2014; 88(10): 5381-5390. PMID: 24574407.
393
http://dx.doi.org/10.1128/JVI.03689-13.
394
395
396
397
25. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res.
2004; 32(5): 1792-1797. PMID: 15034147. http://dx.doi.org/10.1093/nar/gkh340.
26. Hall BG. Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol. 2013; 30(5): 1229-1235.
PMID: 23486614. http://dx.doi.org/10.1093/molbev/mst012.
398
27. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version
399
6.0. Mol Biol Evol. 2013; 30(12): 2725-2729. PMID: 24132122. http://dx.doi.org/10.1093/molbev/mst197.
400
401
402
403
404
405
28. Yang Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007; 24(8): 1586-1591. PMID:
17483113. http://dx.doi.org/10.1093/molbev/msm088.
29. Yang Z, Wong WS, Nielsen R. Bayes empirical bayes inference of amino acid sites under positive selection. Mol
Biol Evol. 2005; 22(4): 1107-1118. PMID: 15689528. http://dx.doi.org/10.1093/molbev/msi097.
30. Chen Z. The current status, investment profit and development trends of raising meat in China. China Poultry.
2012; 19(8): 8-9.
406
31. Jiang WM, Wang SC, Liu HL, Yu JM, Du X, Hou GY, et al. Evaluation of avian influenza virus isolated from ducks
407
as a potential live vaccine candidate against novel H7N9 viruses. Vaccine. 2014; 32(48): 6433-6439. PMID:
408
25285880. http://dx.doi.org/10.1016/j.vaccine.2014.09.050.
409
32. Dortmans JC, Dekkers J, Wickramasinghe IN, Verheije MH, Rottier PJ, van Kuppeveld FJ, et al. Adaptation of
410
novel H7N9 influenza A virus to human receptors. Sci Rep. 2013; 3: 3058. PMID: 24162312.
411
http://dx.doi.org/10.1038/srep03058.
412
33. Chen J, Sun Y. Variation in the analysis of positively selected sites using nonsynonymous/synonymous rate
413
ratios: an example using influenza virus. PLoS One. 2011; 6(5): e19996. PMID: 21629696.
414
http://dx.doi.org/10.1371/journal.pone.0019996.
415
34. Yang Z. Maximum likelihood estimation on large phylogenies and analysis of adaptive evolution in human
416
influenza virus A. J Mol Evol. 2000; 51(5): 423-432. PMID: 11080365.
417
http://dx.doi.org/10.1007/s002390010105.
418
35. Klenk HD, Matrosovich MN, Stech J, editors. Avian influenza. Basel: S. Karger AG; 2008.
419
36. Kaleta EF, Honicke A. Review of the literature on avian influenza A viruses in pigeons and experimental studies
420
on the susceptibility of domestic pigeons to influenza A viruses of the haemagglutinin subtype H7. Dtsch
421
Tierarztl Wochenschr. 2004; 111(12): 467-472. PMID: 15648616.
422
423
424
425
37. Abolnik C. A current review of avian influenza in pigeons and doves (Columbidae). Vet Microbiol. 2014;
170(3-4): 181-196. PMID: 24667061. http://dx.doi.org/10.1016/j.vetmic.2014.02.042.
38. Liu Y, Han C, Wang X, Lin J, Ma M, Shu Y, et al. Influenza A virus receptors in the respiratory and intestinal tracts
of pigeons. Avian Pathol. 2009; 38(4): 263-266. PMID: 19937510.
Newpubli, 2015, 1, e0004 ● Page 11
H7N9 surveillance & evolution
426
427
http://dx.doi.org/10.1080/03079450903055363.
39. Pillai SP, Lee CW. Species and age related differences in the type and distribution of influenza virus receptors in
428
different tissues of chickens, ducks and turkeys. Virol J. 2010; 7: 5. PMID: 20067630.
429
http://dx.doi.org/10.1186/1743-422X-7-5.
430
431
432
40. Fang TH, Lien YY, Cheng MC, Tsai HJ. Resistance of immune-suppressed pigeons to subtypes H5N2 and H6N1
low pathogenic avian influenza virus. Avian Dis. 2006; 50(2): 269-272. PMID: 16863079.
41. Lu J, Wu J, Zeng X, Guan D, Zou L, Yi L, et al. Continuing Reassortment Leads to the Genetic Diversity of
433
Influenza Virus H7N9 in Guangdong, China. J Virol. 2014; 88(15): 8297-8306. PMID: 24829356.
434
http://dx.doi.org/10.1128/JVI.00630-14.
435
42. Han J, Jin M, Zhang P, Liu J, Wang L, Wen D, et al. Epidemiological link between exposure to poultry and all
436
influenza A(H7N9) confirmed cases in Huzhou city, China, March to May 2013. Euro Surveill. 2013; 18(20).
437
PMID: 23725866.
438
43. Jiang W, Liu S, Hou G, Li J, Zhuang Q, Wang S, et al. Chinese and global distribution of H9 subtype avian
439
influenza viruses. PLoS One. 2012; 7(12): e52671. PMID: 23285143.
440
http://dx.doi.org/10.1371/journal.pone.0052671.
441
44. Hayashi T, Hiromoto Y, Chaichoune K, Patchimasiri T, Chakritbudsabong W, Prayoonwong N, et al. Host cytokine
442
responses of pigeons infected with highly pathogenic Thai avian influenza viruses of subtype H5N1 isolated
443
from wild birds. PLoS One. 2011; 6(8): e23103. PMID: 21826229.
444
http://dx.doi.org/10.1371/journal.pone.0023103.
445
45. Guan Y, Peiris JS, Lipatov AS, Ellis TM, Dyrting KC, Krauss S, et al. Emergence of multiple genotypes of H5N1
446
avian influenza viruses in Hong Kong SAR. Proc Natl Acad Sci U S A. 2002; 99(13): 8950-8955. PMID: 12077307.
447
http://dx.doi.org/10.1073/pnas.132268999
448
449
46. Li SC, Li XH, Zhong SG, Sun HL, Pan JJ, Chen SJ, et al. Genome sequencing and phylogenetic analysis of avian
influenza viruses subtype H9N2. Bing Du Xue Bao. 2012; 28(1): 7-14. PMID: 22416344.
450
47. Liu M, He S, Walker D, Zhou N, Perez DR, Mo B, et al. The influenza virus gene pool in a poultry market in South
451
central china. Virology. 2003; 305(2): 267-275. PMID: 12573572. http://dx.doi.org/S0042682202917629.
452
48. Mansour SM, ElBakrey RM, Ali H, Knudsen DE, Eid AA. Natural infection with highly pathogenic avian influenza
453
virus H5N1 in domestic pigeons (Columba livia) in Egypt. Avian Pathol. 2014: 1-6. PMID:
454
24861170.http://dx.doi.org/10.1080/03079457.2014.926002.
455
49. Wan XF, Nguyen T, Davis CT, Smith CB, Zhao ZM, Carrel M, et al. Evolution of highly pathogenic H5N1 avian
456
influenza viruses in Vietnam between 2001 and 2007. PLoS One. 2008; 3(10): e3462. PMID: 18941631.
457
http://dx.doi.org/10.1371/journal.pone.0003462.
458
50. Wang Q, Ju L, Liu P, Zhou J, Lv X, Li L, et al. Serological and Virological Surveillance of Avian Influenza A Virus
459
H9N2 Subtype in Humans and Poultry in Shanghai, China, Between 2008 and 2010. Zoonoses Public Health.
460
2014. PMID: 24803167. http://dx.doi.org/10.1111/zph.12133.
461
51. Gronesova P, Mizakova A, Betakova T. Determination of hemagglutinin and neuraminidase subtypes of avian
462
influenza A viruses in urban pigeons by a new nested RT-PCR. Acta Virol. 2009; 53(3): 213-216. PMID:
463
19941404. http://dx.doi.org/10.4149/av_2009_03_213.
464
52. Perk S, Panshin A, Shihmanter E, Gissin I, Pokamunski S, Pirak M, et al. Ecology and molecular epidemiology of
465
H9N2 avian influenza viruses isolated in Israel during 2000-2004 epizootic. Dev Biol (Basel). 2006; 124: 201-209.
466
PMID: 16447512.
467
53. Jia B, Shi J, Li Y, Shinya K, Muramoto Y, Zeng X, et al. Pathogenicity of Chinese H5N1 highly pathogenic avian
468
influenza viruses in pigeons. Arch Virol. 2008; 153(10): 1821-1826. PMID: 18779923.
469
http://dx.doi.org/10.1007/s00705-008-0193-8.
Newpubli, 2015, 1, e0004 ● Page 12
H7N9 surveillance & evolution
470
54. Klopfleisch R, Werner O, Mundt E, Harder T, Teifke JP. Neurotropism of highly pathogenic avian influenza virus
471
A/chicken/Indonesia/2003 (H5N1) in experimentally infected pigeons (Columbia livia f. domestica). Vet Pathol.
472
2006; 43(4): 463-470. PMID: 16846988. http://dx.doi.org/10.1354/vp.43-4-463.
473
55. Petersen H, Matrosovich M, Pleschka S, Rautenschlein S. Replication and adaptive mutations of low pathogenic
474
avian influenza viruses in tracheal organ cultures of different avian species. PLoS One. 2012; 7(8): e42260.
475
PMID: 22912693. http://dx.doi.org/10.1371/journal.pone.0042260.
476
56. Smietanka K, Minta Z, Wyrostek K, Jozwiak M, Olszewska M, Domanska-Blicharz AK, et al. Susceptibility of
477
pigeons to clade 1 and 2.2 high pathogenicity avian influenza H5N1 virus. Avian Dis. 2011; 55(1): 106-112.
478
PMID: 21500645. http://dx.doi.org/10.1637/9514-090110-resnote.1.
479
57. Werner O, Starick E, Teifke J, Klopfleisch R, Prajitno TY, Beer M, et al. Minute excretion of highly pathogenic
480
avian influenza virus A/chicken/Indonesia/2003 (H5N1) from experimentally infected domestic pigeons
481
(Columbia livia) and lack of transmission to sentinel chickens. J Gen Virol. 2007; 88(Pt 11): 3089-3093. PMID:
482
17947534. http://dx.doi.org/10.1099/vir.0.83105-0.
483
58. Yamamoto Y, Nakamura K, Yamada M, Mase M. Limited susceptibility of pigeons experimentally inoculated
484
with H5N1 highly pathogenic avian influenza viruses. J Vet Med Sci. 2012; 74(2): 205-208. PMID: 21921436.
485
http://doi.org/10.1292/jvms.11-0312.
486
59. Kalthoff D, Bogs J, Grund C, Tauscher K, Teifke JP, Starick E, et al. Avian influenza H7N9/13 and H7N7/13: A
487
comparative virulence study in chickens, pigeons, and ferrets. J Virol. 2014. PMID: 24899194.
488
http://dx.doi.org/10.1128/JVI.01241-14.
489
490
Newpubli, 2015, 1, e0004 ● Page 13
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Table(s)
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493
Table 1. Prevalence of different subtypes of AIVs in different birds on LBMs
detected by surveillance during 2012
Bird species
Subtype#1 Chicken
Pigeon
Duck
Goose
(n = 3201)
(n = 246)
(n = 1291)
(n = 313)
H9
H7
Others
14.53%
0.00%
2.06%
8.94%
0.00%
3.25%
4.18%
0.00%
20.45%
2.56%
0.00%
8.95%
#1
All of the H7 and H9 subtypes of AIVs were A/China/2013(H7N9) viruses
and H9N2 subtype AIVs, respectively.
Table 2. Prevalence of different subtypes of AIVs in different birds on LBMs
detected by surveillance during 2013
Bird species
Subtype#1
Chicken
(n = 3299)
Pigeon
(n = 1083)
Duck
(n = 1656)
Goose
(n = 301)
H9
H7
Others
19.49%
0.79%
1.61%
7.29%
0.37%
1.66%
4.71%
0.00%
16.55%
5.32%
0.33%
16.94%
#1
All of the H7 and H9 subtypes of AIVs were A/China/2013(H7N9) viruses
and H9N2 subtype AIVs, respectively.
494
495
496
Table 3. Prevalence of eight mutations in the HA genes of three groups of H7 subtype AIVs
Virus group
A/China/2013(H7N9) viruses
(n = 207)
Other H7 AIVs circulating in
2010–2013 (n = 176)
Other H7 AIVs circulating
before 2010 (n = 878)
Prevalence of mutations (%)
D174S
I179V
G186V
T189A
Q226L
N289D
E312R
N445D
98.49
100.00
98.99
100.00
88.83
100.00
98.48
100.00
0.00
24.00
1.14
49.71
0.00
35.80
5.68
0.57
0.00
12.73
5.62
5.62
0.00
0.11
0.23
3.00
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H7N9surveillance & evolution
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498
Table 4. Calculation  ratios for each site in the viral HA gene
Model
Np#1
Loglikelihood
Kappa
(ts/tv)
Sites with  > 1#2
M0 (one ratio)
M1 (nearly neutral)
M2 (positive selection)
M3 (discrete)
M7 (beta)
313
314
316
317
314
−4041.977
−4015.628
−4015.830
−4009.564
−4018.824
5.814
5.662
5.662
5.872
5.931
M8 ( beta &  > 1)
316
−4018.082
5.734
None
None
57R, 119D, 164M, 174S, 226L, 541N, 542G
57R, 119D, 164M, 174S, 214V, 226L, 541N, 542G
None
57R, 119D, 135A, 164M, 174S, 186V, 214V, 226L,
276N, 541N, 542G
499
#1
500
501
502
#2
Np, number of free parameters.
Amino acids refer to the HA gene sequence of A/Anhui/1/2013(H7N9), where the sites shown in bold had 
values > 1 with a probability > 95%.
503
504
505
Figure(s)
506
507
Figure 1. Simulated example showing the classification of the clades in this study. The nucleotide sequence
identities between the viruses in Clade A and the viruses in Clade B were all < 97.0% except for a small
proportion (less than 10%) of intermediate viruses marked with asterisks. The nucleotide sequence identities
between the viruses within Clade A or between the strains within Clade B wereall  97.0% except for a small
proportion (less than 10%) of strains marked with circles, which accumulated more mutations than the others.
508
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H7N9 surveillance & evolution
509
510
511
512
Figure 2. Pictures of several typical LBMs in China. These pictures show that pigeons were prevalent and caged closely with other birds on LBMs.
513
514
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H7N9 surveillance & evolution
515
516
517
518
Figure 3. Phylogenetic relationships among 127 A/China/2013(H7N9) viruses and 268 H9N2 subtype AIVs based on
519
the sequences of their six internal genes. The clades of each internal gene of the viruses are designated
520
alphabetically and shown in deep red, navy blue, violet, cyan, red, light green, blue, purple red, light blue, olive,
521
light red and deep green, respectively, from the top down. The same clade (e.g., Clade A) with different genes could
522
include different viruses. Genotype 1 harbors AIVs where all of the internal genes belong to clade A, including most
523
of the H7N9 AIVs and some H9N2 subtype AIVs, e.g., A/pigeon/Jiangsu/K77/2013(H9N2), which is represented by
524
the triangles in the figure.
525
526
527
Figure 4. Locations of the five amino acid mutations in
the viral HA protein specific to A/China/2013(H7N9)
illuminated using PyMol 1.6.x.
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H7N9 surveillance & evolution
528
529
530
531
Figure 5. Phylogenetic relationships among 445 H7 AIVs isolated in Asia during 2008–2013. The A/China/2013(H7N9)
532
viruses and other H7 viruses selected for calculating the  ratios are marked with triangles and circles, respectively.
533
534
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H7N9 surveillance & evolution
535
Figure 6. A possible pathway toward the origin and development of
A/China/2013(H7N9) in China.
536
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H7N9 surveillance & evolution
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Statements
538
Ethics
The authors declare that they have not conducted plagiarism, falsification or dual submission with
respect to this article, and that they have been aware of and complied with the ethical
requirements of Newpubli regarding authorship, human rights, animal welfare, biosecurity and
dual use of research.
The authors also declare that this study was conducted in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory Animals of China Animal Health
and Epidemiology Center. The feces samples, drinking-water samples and swab samples from
poultry farms, backyard flocks and live bird markets were all collected with permission given by
various relevant parties, including the Ministry of Agriculture of China, China Animal Health and
Epidemiology Center, the relevant veterinary section in the provincial and county or city
government, and the owners of the relevant birds.
Competing
The authors declare that no competing interests exist with respect to this article, except that some
Interests
authors are editors of Newpubli. Newpubli has established a mechanism using software to ensure
that the rating of each peer reviewer regarding the value of each article is blind to everyone and
cannot be changed by anyone.
Data Sharing
The authors declare that all of the data underlying the findings or conclusions of this article and its
preprint are fully available without restriction.
Funding
This study was supported by the Avian Influenza Surveillance Program of the Ministry of Agriculture
and the Sci-tech Basic Work Project of the Ministry of Science and Technology (SQ2012FY3260033)
in China. The funders had no role in the study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Copyright
The copyright of this article and its preprint completely belongs to its authors who allow anyone to
read, download, save, copy and print this article or its preprint, as well as using the metadata of
this article related to indexing, searching and citation, without any restriction. The authors require
that any part of this article and its preprint cannot be used without appropriate citation.
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540
541
542
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