This first fifth-generation scientific journal aims to spark a revolution in academic publishing
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Large-scale Surveillance and In-depth Evolutionary Analyses of H7N9
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Avian Influenza Virus
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Author(s)
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Su-Chun Wang, Shuo Liu, Wen-Ming Jiang, Qing-Ye Zhuang, Kai-Cheng Wang, Guang-Yu Hou, Jin-Ping Li, Jian-Min Yu,
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Xiang Du, Zhi-Yuan Yang, Yue-Huan Liu, Ji-Wang Chen, Ji-Ming Chen
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Author Affiliation(s)
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China Animal Health and Epidemiology Center, Qingdao, 266032, China (Su-Chun Wang, Shuo Liu, Wen-Ming Jiang,
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Qing-Ye Zhuang, Kai-Cheng Wang, Guang-Yu Hou, Jin-Ping Li, Jian-Min Yu, Xiang Du, Ji-Ming Chen); Institute of
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Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097,
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China (Zhi-Yuan Yang, Yue-Huan Liu); Department of Medicine, Section of Pulmonary, Critical Care, Sleep and Allergy
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Medicine, University of Illinois at Chicago, Chicago, IL60612, USA (Ji-Wang Chen)
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Author Contribution
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Conceived and designed the study: Ji-Ming Chen; performed the study: Su-Chun Wang, Shuo Liu, Wen-Ming Jiang,
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Qing-Ye Zhuang, Kai-Cheng Wang, Guang-Yu Hou, Jin-Ping Li, Jian-Min Yu, Xiang Du, Zhi-Yuan Yang, Yue-Huan Liu,
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Ji-Ming Chen; contributed reagents/materials/analysis tools: Ji-Ming Chen; wrote the paper: Ji-Ming Chen, Ji-Wang
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Chen; contributed equally to this study: Su-Chun Wang, Shuo Liu
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Acknowledgements
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The authors of this article thank the researchers and laboratories for originating and submitting some sequences to
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EpiFlu database of the Global Initiative on Sharing All Influenza Data (GISAID) because these sequences partially
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formed the basis of this study.
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Corresponding Author(s)
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Ji-Ming Chen (E-mail: chenjiming@cahec.cn; jmchen678@qq.com)
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Citation
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Wang SC, Liu S, Jiang WM, Zhuang QY, Wang KC, Hou GY, et al. Large-scale surveillance and in-depth evolutionary
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analyses of H7N9 avian influenza virus. Newpubli. 2015; 1: e0004
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URL & DOI
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Available in coming months
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Article History
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Received: 18-12-2015; accepted: 18-12-2015; preprint published: 10-11-2015
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PR-Rank
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Available before this article is formally published
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Subject Areas
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Veterinary medicine; Ecology; Epidemiology; Evolution; Genetics; Infectious diseases; Microbiology; Molecular &
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cellular biology; Public health; Theoretical biology; Virology
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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
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emergence in 2013. In this study, we conducted large-scale surveillance of AIVs, which demonstrated the
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prevalence and distribution of the H7N9 AIV and its potential gene-donor viruses (H9N2 subtype AIV) in different
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species of poultry. We also conducted in-depth phylogenetic analyses of AIVs, which suggested that one genotype
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of the H9N2 subtype AIV circulating in chickens, pigeons and bramblings could donate six internal genes to the
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H7N9 AIV, and multiple genotypes of the H7N9 AIV circulated in Henan province. Moreover, by calculating the
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distribution of the mutations and the nonsynonymous/synonymous rate ratios, we identified five mutations in the
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viral HA gene specific to the H7N9 AIV, one of which (Q226L, H3 numbering) confers increased binding to
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human-like receptors and was probably fixed by positive selection. These results are important for the design of
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evidence-based measures to control this zoonotic virus, as well as providing novel insights into the distribution, risk
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and evolution of H7N9 AIVs. Additionally, we propose a novel hypothesis that the H7N9 AIV may have originated in
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pigeons through natural selection.
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Significance
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This study comprised large-scale surveillance of avian influenza viruses (AIVs) and novel in-depth evolutionary
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analyses of the H7N9 AIV, which has caused hundreds of human deaths in China. Approximately 15,000 samples
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were detected and thousands of novel AIV sequences were obtained through the surveillance which demonstrated
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the prevalence and distribution of the H7N9 AIV and H9N2 subtype AIVs in different species of poultry. Based on
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evolutionary analyses, all of the early H7N9 AIV and H9N2 subtype AIVs were divided into 43 genotypes. Multiple
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genotypes of the H7N9 AIV were found exclusively in Henan province and five mutations in the viral HA gene were
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identified as specific to the H7N9 AIV. The evolutionary analyses also suggested that one of the five specific
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mutations, Q226L, which confers increased binding to human-like receptors, was probably fixed by positive
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selection. These results are important for the design of evidence-based measures to control this zoonotic virus, as
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well as providing novel insights into the distribution, risk and evolution of H7N9 AIVs. Additionally, we propose a
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novel hypothesis that the H7N9 AIV may have originated in pigeons through natural selection. The hypothesis,
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although controversial, is a novel explanation regarding the emergency of the H7N9 AIV.
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Keywords
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avian influenza virus; distribution; evolution; H7N9; pigeon; receptor; selection; surveillance
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Abbreviations
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AIV: avian influenza viruses; GISAID: the Global Initiative on Sharing All Influenza Data; HA: hemagglutinin; LBM: live
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bird markets; MP: matrix protein; NA: neuraminidase; NP: nucleoprotein; NS: nonstructural protein; PA: acidic
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polymerase; PB1: basic polymerase 1; PB2: basic polymerase 2; ts/tv: transition/transversion ratios; : the
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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
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correspond to the viral genes for basic polymerase 2 (PB2), basic polymerase 1 (PB1), acidic polymerase (PA),
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hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (MP) and nonstructural protein (NS).
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The viral HA and NA genes encode surface HA and NA glycoproteins. The remaining six internal genes encode PB2,
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PB1, PA, NP and other internal structural and nonstructural proteins [6-7].
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Based on differences in the antigenicity of the viral HA and NA glycoproteins, influenza A viruses can be
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categorized into 18 HA subtypes (H1H18) and 11 NA subtypes (N1N11) [1-4]. Their combinations further
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generate H1N1, H3N2, H7N7, H9N2 and many other influenza A virus subtypes. Each of the viral genes has evolved
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into multiple lineages and genomic reassortment of these lineages has generated multiple genotypes for each
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subtype of influenza A virus [1, 4, 7-8].
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Avian influenza viruses (AIVs) are influenza A viruses that circulate mainly in birds. They are highly diverse with
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16 HA subtypes (H1H16) and nine NA subtypes (N1N9). Most AIVs only infect birds, but some can infect humans
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and other mammals at a low frequency. These zoonotic AIVs continue to present a challenge to human health, such
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as the H5N1 highly pathogenic AIVs that have circulated in many countries in the past decade [1, 7].
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A previously unrecognized zoonotic H7N9 AIV that was first identified in China during March 2013, referred to
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as A/China/2013(H7N9), has since caused >600 human infections with >200 fatalities [9-10]. A/China/2013(H7N9)
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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].
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Multiple studies have been conducted to investigate the origin of A/China/2013(H7N9). These studies suggest
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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
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enigmatic [8, 10, 17-22]. In the present study, we conducted large-scale active surveillance and in-depth
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evolutionary analyses to reveal the host distribution A/China/2013(H7N9) and H9N2 AIVs in poultry and explore the
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potential role of pigeons in the origin of this zoonotic AIV.
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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)
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and stored at –80°C until use. The extracted RNA was analyzed using a RT-PCR assay to amplify and sequence the
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whole-length genome of influenza A virus, as described previously [23]. The whole-length NA gene of N9 subtype
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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
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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
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(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
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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
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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
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noted that these clades could be divided further into several subclades [8, 10, 18, 22].
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Structural analysis
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Structural analysis of the viral HA protein was performed using the Pymol v1.6.x program (www.pymol.org)
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with the 4ln3 input structural file downloaded from NCBI [15].
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Calculation of the nonsynonymous/synonymous rate ratios
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The nonsynonymous/synonymous rate ratio () of each amino acid residue (site) in the viral HA gene was
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estimated using the PAML 4.4 program [28]. The codon frequencies were set according to the F34 table. The 
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ratios were analyzed using an unrooted phylogenetic tree under the following models: model M0 (one-ratio)
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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)
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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
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models for comparison with their alternatives [28]. The performance of these models was compared by the
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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,
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ducks, geese and other birds, respectively. Thus, chickens and pigeons are the first and second most prevalent birds
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in LBMs of China in recent years. This is partially because pigeons are mainly sold through LBMs in China, whereas
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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
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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
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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
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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).
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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
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significantly less prevalent than H9N2 subtype AIVs, but it was quite difficult to eradicate the zoonotic virus through
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surveillance and culling because the virus had spread to numerous provinces and it did not cause any obvious
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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
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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
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was the dominant genotype in the H9N2 subtype AIVs, comprising nearly half (128/268) of the H9N2 subtype AIVs.
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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
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other genotypes, except Genotype 2, and these H9N2 subtype AIVs had circulated in poultry before the emergence
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of A/China/2013(H7N9) viruses. Therefore, the H9N2 subtype AIVs within Genotype 1 which circulated in multiple
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species of birds, including chickens, pigeons and bramblings, e.g., A/pigeon/Jiangsu/K77/2013(H9N2), possibly
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contributed the six internal genes to the early A/China/2013(H7N9) viruses. The six internal genes of
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A/pigeon/Jiangsu/K77/2013(H9N2) all shared high homology with those of A/China/2013(H7N9),where the
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nucleotide sequence identities ranged from 97.85% to 99.41%.
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Among the 127 early A/China/2013(H7N9) viruses, 21 were isolated from Henan province in China during this
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study. Interestingly, as shown in Attachment 7, these 21 early A/China/2013(H7N9) viruses from Henan could be
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classified into four genotypes, whereas the remaining 106 early A/China/2013(H7N9) viruses from nine provinces,
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autonomous regions or municipalities all belonged to Genotype 1.
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Analysis of the mutations specific to A/China/2013(H7N9)
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We downloaded and analyzed the HA gene sequences ( 500 bp) of 1261 H7 subtype influenza viruses isolated
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in the eastern hemisphere, including Africa, Europe, Asia and Oceania, from the Global Initiative on Sharing All
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Influenza Data (GISAID) database on June 1, 2014. Among these, 207 were A/China/2013(H7N9) viruses detected in
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2013–2014 and 1054 were other H7 viruses (176 circulating in 2010–2013 and 878 circulating before 2010). As
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shown in Table 3, three mutations, i.e., I179V (H3 numbering throughout), T189A and N289D, were prevalent
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(prevalence > 85%) in the 207 A/China/2013(H7N9) viruses and not rare (prevalence  20%) in the 176 other H7
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viruses circulating in 2010–2013. Five other mutations, i.e., D174S, G186V, Q226L, E312R and N445D, were
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prevalent (prevalence > 85%) in the 207 A/China/2013(H7N9) viruses but rare (prevalence < 10%) in the two groups
226
of other H7 viruses. Therefore, these five mutations were considered to be specific to A/China/2013(H7N9) viruses
227
and no other mutations in the viral HA genes were identified as specific to A/China/2013(H7N9) viruses.
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Two of the five mutations specific to A/China/2013(H7N9) viruses, i.e., G186V and Q226L, were located in the
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viral HA protein motif responsible for receptor binding (Figure 2). It is known that these two specific mutations,
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especially Q226L, confer increased binding to human-like receptors. Various studies have demonstrated that
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although A/China/2013(H7N9) retains its tight binding to avian-like receptors and weak binding to human-like
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receptors, its binding to human-like receptors increased, which is assumed to be crucial for the causation of human
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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
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population, lineage or species levels. Thus, only a small proportion of random mutations can be fixed at the
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population, lineage or species levels through the effects of random factors (i.e., random genetic drift), selective
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factors (i.e., natural selection) or hitchhiking (i.e., fixation of a mutation by natural selection leading to the fixation
238
of another mutation linked to the naturally selected mutation). The mutations fixed through random drift or
239
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
241
receptor-binding property of a protein [33-34]. Less than 20 of the approximately 560 amino acid residues in the
242
viral HA gene confer increased binding to human-like receptors [11-16], so the possibility should be less than:
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(20×5/560)×(19×5/560) = 3.0%, for two of the only five specific mutations (G186V and Q226L) to occur at the
244
residues conferring increased binding to human-like receptors through random genetic drift or hitchhiking.
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Therefore, at least one of the two mutations that confer increased binding to human-like receptors was probably
246
fixed by positive selection rather than random genetic drift. We did not exclude the conserved stalk region of the
247
HA gene because at least two of the five specific mutations, i.e., E312R and N445D, occur in the stalk region (Figure
248
4), and the receptor-binding motif in the head region of the viral HA gene is also highly conserved [35].
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Calculation of the  value for each site in the viral HA gene
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Among the aforementioned 207 H7 subtype influenza viruses, 104 belonged to the early A/China/2013(H7N9)
251
viruses detected before May 1, 2013 without ambiguous nucleotides in their HA gene sequences. Among the
252
aforementioned 1054 other H7 viruses, 53 had no ambiguous nucleotides in their HA gene sequences and they
253
were most closely related in phylogenetics to the 104 early A/China/2013(H7N9) viruses according to their
254
phylogenetic relationships (see Figure 5) and HA gene sequence identities (> 96.8%). Based on the HA gene
255
sequences of the 104 early A/China/2013(H7N9) viruses and the 53 other H7 viruses, we calculated the  value for
256
each site (namely amino acid residue) in the viral HA gene of the H7 subtype AIVs using PAML 4.4 to further
257
examine whether any of the two amino acid mutations that confer increased binding to human-like receptors were
258
fixed through positive selection, because sites in a gene with the  ratio > 1 have frequently been identified as
259
under positive selection [33-34]. These 157 viruses were selected to calculate the  values because they were the
260
most suitable for reflecting the selection pressure on each site during the origin of the virus.
261
The likelihood ratio tests based on the calculation of the  ratio suggested that model M3 (discrete) was
262
significantly more suitable than the other models, including M0 (one-ratio), M1 (nearly neutral), M2 (positive
263
selection), M7 (beta) and M8 (beta and  > 1) (P < 0.01, Chi-square test) (Table 4). As shown in Table 4, although
264
the sites with  ratios > 1 had a little variation according to the three models allowing positive selection (M2, M3
265
and M8), site 226 had a probability > 95% to be of the  ratio > 1 calculated using all the three models. This
266
suggests that Q226L, one of the five mutations specific to A/China/2013(H7N9) viruses and conferring increased
267
binding to human-like receptors, was likely fixed through positive selection rather than random genetic drift or
268
hitchhiking. Therefore, this specific crucial mutation was probably selected in a host population that favored
269
mutations conferring increased binding to human-like receptors.
270
Discussion
271
In this study, we conducted large-scale surveillance of AIVs to determine the distribution of the H7N9 AIV and
272
its potential gene-donor viruses (H9N2 subtype AIVs) in different species of poultry. We also conducted in-depth
273
evolutionary analyses of thousands of AIV sequences, which showed that some H9N2 subtype AIVs from chickens,
274
pigeons and bramblings could donate six internal genes to the H7N9 AIV. In addition, we identified five mutations in
275
the viral HA gene specific to the H7N9 AIV, where one that confers increased binding to human-like receptors, i.e.,
276
Q226L (H3 numbering), was probably fixed through positive selection, according to the calculated site distribution
277
and the nonsynonymous/synonymous rate ratios.
278
The ability of an influenza virus to replicate efficiently in a host depends on multiple factors [35]. Clearly,
279
receptor matching is necessary but not sufficient for efficient replication of the virus, which explains why
280
A/China/2013(H7N9) replicated inefficiently in ducks and it was rare in ducks [24], although it bound efficiently to
281
duck avian-like receptors.
282
It has been reported many times that pigeons are naturally resistant to infection of most AIVs [36-37]. In part,
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this may be because pigeons uniquely carry abundant human-like receptors and few avian-like receptors in their
284
respiratory tracts, and thus most AIVs cannot replicate efficiently in pigeons [38-40]. In fact, it has been found that
285
A/China/2013(H7N9) replicated inefficiently in pigeons, but efficiently in chickens, and our surveillance study and
286
those published previously all suggest that A/China/2013(H7N9) was most prevalent in chickens [9, 16, 41]. This is
287
consistent with previously reported epidemiological findings that chickens were the major source for human
288
infections and pigeons were the probable source for only a few human cases [9, 42].
289
Thus, why did A/China/2013(H7N9) replicate inefficiently in pigeons even though the virus exhibited increased
290
binding to human-like receptors in pigeons? It is possible that A/China/2013(H7N9) remains to bind weakly to
291
human-like receptors and tightly to avian-like receptors [11-16]. Similar to this suggested scenario, common AIVs
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292
bind to avian-like and human-like receptors with 100 and 5 units of avidity, respectively, whereas
293
A/China/2013(H7N9) binds to avian-like and human-like receptors with 100 and 15 units of avidity, respectively.
294
Our surveillance data suggest that H9N2 subtype AIVs were relatively prevalent in pigeons on LBMs in China;
295
however, this does not contradict the fact that pigeons are resistant to AIVs infections for the following three
296
reasons. First, the replication of a low pathogenic virus in the respiratory and alimentary systems of a host might
297
not lead to an infection with clinical signs. Second, unlike many other AIVs, most of the H9N2 subtype AIVs that
298
circulated in China during recent years carried the Q226L mutation in their HA gene, which confers increased
299
binding to pigeon human-like receptors [13, 16, 43], thereby facilitating viral replication in pigeons. Third, many
300
pigeons are kept in bad conditions on LBMs, which could weaken the pigeons and facilitate the replication of H9N2
301
subtype AIVs in pigeons.
302
Our surveillance data also suggest that A/China/2013(H7N9) was relatively prevalent in pigeons on LBMs in
303
China. This is consistent with the emergent disease surveillance conducted by Harbin Veterinary Research Institute
304
in April and May 2013, which showed that the prevalence of A/China/2013(H7N9) was 1.74% (3/172) in pigeons on
305
LBMs [16]. In addition, they identified A/China/2013(H7N9) only on one pigeon farm among 253 poultry farms
306
where they collected samples, and only in one wild pigeon sample among the 739 wild bird samples that they
307
detected through that emergent disease surveillance [16]. Another research group also isolated two strains of
308
A/China/2013(H7N9) from pigeons in April 2013, with GISAID accession numbers of 162,874 and 162,875. The
309
relatively high prevalence of A/China/2013(H7N9) in pigeons on LBMs indicates that the virus can replicate in
310
pigeons. This does not contradict the fact that pigeons are naturally resistant to AIV infection and that
311
A/China/2013(H7N9) replicated inefficiently in pigeons due to the same reasons given above to explain the
312
relatively high prevalence of H9N2 subtype AIVs in pigeons, including the increased binding to human-like receptors
313
in pigeons by A/China/2013(H7N9) [13, 16, 43]. Moreover, many pigeons are kept in bad conditions on LBMs and
314
farms, which could facilitate viral replication in pigeons.
315
Multiple research entities have isolated other AIV subtypes from pigeons on LBMs or farms in recent years [8,
316
16, 44-52], which are similar to our surveillance data (Tables 1 and 2). Most reports of animal experimental data
317
suggest that pigeons are naturally resistant to AIV infection but they also showed that AIVs can replicate in pigeons
318
for a short time, although inefficiently [37, 40, 53-58]. Two experimental studies showed that A/China/2013(H7N9)
319
could replicate in pigeons, but only inefficiently [24, 59].
320
According to many previous reports, A/China/2013(H7N9) probably originated in birds rather than mammals [8,
321
10, 17-22]. Among the known bird species that harbor AIVs, only pigeons possess abundant human-like receptors
322
and few avian-like receptors in their respiratory tracts [38-39], so pigeons can potentially provide a favorable
323
environment for the selection of the crucial specific mutation in A/China/2013(H7N9), i.e., Q226L, which confers
324
increased binding to human-like receptors.
325
Based on all of these findings, we consider that A/China/2013(H7N9) may have originated in pigeons through
326
natural selection for the following reasons. First, pigeons are populous in China and they are frequently kept close
327
to other birds, especially on LBMs. Therefore, there are numerous opportunities for AIVs to replicate in pigeons and
328
spread between pigeons and other birds. Second, A/China/2013(H7N9) was relatively prevalent in pigeons during
329
2013. Third, H9N2 subtype AIVs were also relatively prevalent in pigeons during 2012 and 2013, and some H9N2
330
subtype AIVs in pigeons, such as A/pigeon/Jiangsu/K77/2013(H9N2), could have provided their six internal genes to
331
A/China/2013(H7N9). Fourth and most importantly, the crucial specific mutation found in A/China/2013(H7N9) that
332
confers increased binding to human-like receptors was probably fixed in the viral genome through positive selection,
333
and pigeons are the only known birds that may exert pressure on the selection of this specific mutation because
334
they uniquely possess abundant human-like receptors and a few avian-like receptors in their respiratory tracts. We
335
also explain this hypothesis in Attachment 8 as well as suggesting that multiple species of birds were possibly
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336
involved in the emergence of A/China/2013(H7N9) (Figure 6). More evidence is needed to support this hypothesis,
337
but for the first time, this controversial hypothesis suggests a novel potential mechanism for the origin of zoonotic
338
AIVs, including A/China/2013(H7N9), without the involvement of pigs.
339
The ecological and evolutionary results reported in the present study are important for the control of the
340
zoonotic virus and its risk analysis. For example, more efforts should be directed to chickens rather than ducks on
341
LBMs to control the zoonotic virus, and we should not ignore the potentially important role of pigeons in the
342
circulation and evolution of AIVs, although they are naturally resistant to infection by AIVs.
343
In summary, we obtained many novel results in this study related to the ecology and evolution of
344
A/China/2013(H7N9), which are important for the design of evidence-based measures to control the zoonotic virus,
345
and shed novel insights into the distribution, risk and evolution of A/China/2013(H7N9).
346
Attachments
347
Attachment 1. Clades of some H9N2 and H7N9 viruses based on their PB2 gene (png, 1.1Mb).
348
Attachment 2. Clades of some H9N2 and H7N9 viruses based on their PB1 gene (png, 1.1Mb).
349
Attachment 3. Clades of some H9N2 and H7N9 viruses based on their PA gene (png, 1.1Mb).
350
Attachment 4. Clades of some H9N2 and H7N9 viruses based on their NP gene (png, 1.1Mb).
351
Attachment 5. Clades of some H9N2 and H7N9 viruses based on their MP gene (png, 1.1Mb).
352
Attachment 6. Clades of some H9N2 and H7N9 viruses based on their NS gene (png, 1.1Mb).
353
Attachment 7. Genotypes of 268 H9N2 and 127 early A/China/H7N9/2013 viruses based on the clade
354
constellation of their six internal genes (docx, 27 Kb).
355
Attachment 8. Explanation of the hypothesis regarding the possible origin of A/China/2013(H7N9) in pigeons
356
through natural selection (docx, 32 Kb).
357
References
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359
360
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362
363
364
365
366
367
368
369
370
371
372
373
374
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376
377
378
1.
Liu S, Ji K, Chen J, Tai D, Jiang W, Hou G, et al. Panorama phylogenetic diversity and distribution of Type A influenza
virus. PLoS One. 2009; 4(3): e5022. PubMed PMID: 19325912. http://dx.doi.org/10.1371/journal.pone.0005022.
2.
Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, et al. New world bats harbor diverse influenza A viruses. PLoS
Pathog. 2013; 9(10): e1003657. PubMed PMID: 24130481. http://dx.doi.org/10.1371/journal.ppat.1003657.
3.
Wu Y, Tefsen B, Shi Y, Gao GF. Bat-derived influenza-like viruses H17N10 and H18N11. Trends Microbiol. 2014; 22(4):
183-191. PubMed PMID: 24582528. http://dx.doi.org/10.1016/j.tim.2014.01.010.
4.
Yoon SW, Webby RJ, Webster RG. Evolution and ecology of influenza A viruses. Curr Top Microbiol Immunol. 2014.
PubMed PMID: 24990620. http://dx.doi.org/10.1007/82_2014_396.
5.
Jiang WM, Wang SC, Peng C, Yu JM, Zhuang QY, Hou GY, et al. Identification of a potential novel type of influenza
virus in Bovine in China. Virus Genes. 2014; 49(3): 493-496. PubMed PMID: 25142163.
http://dx.doi.org/10.1007/s11262-014-1107-3.
6.
Kageyama T, Fujisaki S, Takashita E, Xu H, Yamada S, Uchida Y, et al. Genetic analysis of novel avian A(H7N9) influenza
viruses isolated from patients in China, February to April 2013. Euro Surveill. 2013; 18(15): 20453. PubMed PMID:
23594575.
7.
Chen JM, Sun YX, Chen JW, Liu S, Yu JM, Shen CJ, et al. Panorama phylogenetic diversity and distribution of type A
influenza viruses based on their six internal gene sequences. Virol J. 2009; 6: 137. PubMed PMID: 19737421.
http://dx.doi.org/10.1186/1743-422X-6-137.
8.
Lam TT, Wang J, Shen Y, Zhou B, Duan L, Cheung CL, et al. The genesis and source of the H7N9 influenza viruses
causing human infections in China. Nature. 2013; 502(7470): 241-244. PubMed PMID:
23965623.http://dx.doi.org/10.1038/nature12515.
9.
Li Q, Zhou L, Zhou M, Chen Z, Li F, Wu H, et al. Epidemiology of human infections with avian influenza A(H7N9) virus
Newpubli, 2015, 1, e0004 ● Page 9
H7N9 surveillance & evolution
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
in China. N Engl J Med. 2014; 370(6): 520-532. PubMed PMID: 23614499.
http://dx.doi.org/10.1056/NEJMoa1304617.
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) virus.
N Engl J Med. 2013; 368(20): 1888-1897. PubMed PMID: 23577628. http://dx.doi.org/10.1056/NEJMoa1304459.
11. Belser JA, Gustin KM, Pearce MB, Maines TR, Zeng H, Pappas C, et al. Pathogenesis and transmission of avian
influenza A (H7N9) virus in ferrets and mice. Nature. 2013; 501(7468): 556-559. PubMed PMID: 23842497.
http://dx.doi.org/10.1038/nature12391.
12. Richard M, Schrauwen EJ, de Graaf M, Bestebroer TM, Spronken MI, van Boheemen S, et al. Limited airborne
transmission of H7N9 influenza A virus between ferrets. Nature. 2013; 501(7468): 560-563. PubMed PMID:
23925116. http://dx.doi.org/10.1038/nature12476.
13. Tharakaraman K, Jayaraman A, Raman R, Viswanathan K, Stebbins NW, Johnson D, et al. Glycan receptor binding of
the influenza A virus H7N9 hemagglutinin. Cell. 2013; 153(7): 1486-1493. PubMed PMID:
23746830.http://dx.doi.org/10.1016/j.cell.2013.05.034.
14. Xiong X, Martin SR, Haire LF, Wharton SA, Daniels RS, Bennett MS, et al. Receptor binding by an H7N9 influenza virus
from humans. Nature. 2013; 499(7459): 496-499. PubMed PMID: 23787694.
http://dx.doi.org/10.1038/nature12372.
15. Yang H, Carney PJ, Chang JC, Villanueva JM, Stevens J. Structural analysis of the hemagglutinin from the recent 2013
H7N9 influenza virus. J Virol. 2013; 87(22): 12433-12446. PubMed PMID: 24027325.
http://dx.doi.org/10.1128/JVI.01854-13.
16. Zhang Q, Shi J, Deng G, Guo J, Zeng X, He X, et al. H7N9 influenza viruses are transmissible in ferrets by respiratory
droplet. Science. 2013; 341(6144): 410-414. PubMed PMID: 23868922. http://dx.doi.org/10.1126/science.1240532.
17. Cui L, Liu D, Shi W, Pan J, Qi X, Li X, et al. Dynamic reassortments and genetic heterogeneity of the human-infecting
influenza A (H7N9) virus. Nat Commun. 2014; 5: 3142. PubMed PMID: 24457975.
http://dx.doi.org/10.1038/ncomms4142.
18. Feng Y, Mao H, Xu C, Jiang J, Chen Y, Yan J, et al. Origin and characteristics of internal genes affect infectivity of the
novel avian-origin influenza A (H7N9) virus. PLoS One. 2013; 8(11): e81136. PubMed PMID: 24278391.
http://dx.doi.org/10.1371/journal.pone.0081136.
19. Lee RT, Gunalan V, Van TD, Le LT, Eisenhaber F, Maurer-Stroh S. A new piece in the puzzle of the novel avian-origin
influenza A (H7N9) virus. Biol Direct. 2013; 8: 26. PubMed PMID: 24160334.
http://dx.doi.org/10.1186/1745-6150-8-26.
20. Van Ranst M, Lemey P. Genesis of avian-origin H7N9 influenza A viruses. Lancet. 2013; 381(9881): 1883-1885.
PubMed 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 H7N9
influenza A virus in China. Sci Rep. 2013; 3: 2318. PubMed PMID: 23897131. http://dx.doi.org/10.1038/srep02318.
22. Wu A, Su C, Wang D, Peng Y, Liu M, Hua S, et al. Sequential reassortments underlie diverse influenza H7N9
genotypes in China. Cell Host Microbe. 2013; 14(4): 446-452. PubMed PMID: 24055604.
http://dx.doi.org/10.1016/j.chom.2013.09.001.
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. PubMed PMID: 11811679.
24. Pantin-Jackwood MJ, Miller PJ, Spackman E, Swayne DE, Susta L, Costa-Hurtado M, et al. Role of poultry in the
spread of novel H7N9 influenza virus in China. J Virol. 2014; 88(10): 5381-5390. PubMed PMID: 24574407.
http://dx.doi.org/10.1128/JVI.03689-13.
25. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;
32(5): 1792-1797. PubMed 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.
PubMed PMID: 23486614. http://dx.doi.org/10.1093/molbev/mst012.
Newpubli, 2015, 1, e0004 ● Page 10
H7N9 surveillance & evolution
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
27. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0.
Mol Biol Evol. 2013; 30(12): 2725-2729. PubMed PMID: 24132122. http://dx.doi.org/10.1093/molbev/mst197.
28. Yang Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007; 24(8): 1586-1591. PubMed 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. PubMed 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.
31. Jiang WM, Wang SC, Liu HL, Yu JM, Du X, Hou GY, et al. Evaluation of avian influenza virus isolated from ducks as a
potential live vaccine candidate against novel H7N9 viruses. Vaccine. 2014; 32(48): 6433-6439. PubMed PMID:
25285880. http://dx.doi.org/10.1016/j.vaccine.2014.09.050.
32. Dortmans JC, Dekkers J, Wickramasinghe IN, Verheije MH, Rottier PJ, van Kuppeveld FJ, et al. Adaptation of novel
H7N9 influenza A virus to human receptors. Sci Rep. 2013; 3: 3058. PubMed PMID: 24162312.
http://dx.doi.org/10.1038/srep03058.
33. Chen J, Sun Y. Variation in the analysis of positively selected sites using nonsynonymous/synonymous rate ratios: an
example using influenza virus. PLoS One. 2011; 6(5): e19996. PubMed PMID: 21629696.
http://dx.doi.org/10.1371/journal.pone.0019996.
34. Yang Z. Maximum likelihood estimation on large phylogenies and analysis of adaptive evolution in human influenza
virus A. J Mol Evol. 2000; 51(5): 423-432. PubMed PMID: 11080365. http://dx.doi.org/10.1007/s002390010105.
35. Klenk HD, Matrosovich MN, Stech J, editors. Avian influenza. Basel: S. Karger AG; 2008.
36. Kaleta EF, Honicke A. Review of the literature on avian influenza A viruses in pigeons and experimental studies on
the susceptibility of domestic pigeons to influenza A viruses of the haemagglutinin subtype H7. Dtsch Tierarztl
Wochenschr. 2004; 111(12): 467-472. PubMed PMID: 15648616.
37. Abolnik C. A current review of avian influenza in pigeons and doves (Columbidae). Vet Microbiol. 2014; 170(3-4):
181-196. PubMed 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. PubMed PMID: 19937510.
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
different tissues of chickens, ducks and turkeys. Virol J. 2010; 7: 5. PubMed PMID: 20067630.
http://dx.doi.org/10.1186/1743-422X-7-5.
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. PubMed 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 Influenza
Virus H7N9 in Guangdong, China. J Virol. 2014; 88(15): 8297-8306. PubMed PMID: 24829356.
http://dx.doi.org/10.1128/JVI.00630-14.
42. Han J, Jin M, Zhang P, Liu J, Wang L, Wen D, et al. Epidemiological link between exposure to poultry and all influenza
A(H7N9) confirmed cases in Huzhou city, China, March to May 2013. Euro Surveill. 2013; 18(20). PubMed PMID:
23725866.
43. Jiang W, Liu S, Hou G, Li J, Zhuang Q, Wang S, et al. Chinese and global distribution of H9 subtype avian influenza
viruses. PLoS One. 2012; 7(12): e52671. PubMed PMID: 23285143.
http://dx.doi.org/10.1371/journal.pone.0052671.
44. Hayashi T, Hiromoto Y, Chaichoune K, Patchimasiri T, Chakritbudsabong W, Prayoonwong N, et al. Host cytokine
responses of pigeons infected with highly pathogenic Thai avian influenza viruses of subtype H5N1 isolated from
wild birds. PLoS One. 2011; 6(8): e23103. PubMed PMID: 21826229.
http://dx.doi.org/10.1371/journal.pone.0023103.
Newpubli, 2015, 1, e0004 ● Page 11
H7N9 surveillance & evolution
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
45. Guan Y, Peiris JS, Lipatov AS, Ellis TM, Dyrting KC, Krauss S, et al. Emergence of multiple genotypes of H5N1 avian
influenza viruses in Hong Kong SAR. Proc Natl Acad Sci U S A. 2002; 99(13): 8950-8955. PubMed PMID: 12077307.
http://dx.doi.org/10.1073/pnas.132268999
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. PubMed PMID: 22416344.
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
central china. Virology. 2003; 305(2): 267-275. PubMed PMID: 12573572. http://dx.doi.org/S0042682202917629.
48. Mansour SM, ElBakrey RM, Ali H, Knudsen DE, Eid AA. Natural infection with highly pathogenic avian influenza virus
H5N1 in domestic pigeons (Columba livia) in Egypt. Avian Pathol. 2014: 1-6. PubMed PMID:
24861170.http://dx.doi.org/10.1080/03079457.2014.926002.
49. Wan XF, Nguyen T, Davis CT, Smith CB, Zhao ZM, Carrel M, et al. Evolution of highly pathogenic H5N1 avian influenza
viruses in Vietnam between 2001 and 2007. PLoS One. 2008; 3(10): e3462. PubMed PMID: 18941631.
http://dx.doi.org/10.1371/journal.pone.0003462.
50. Wang Q, Ju L, Liu P, Zhou J, Lv X, Li L, et al. Serological and Virological Surveillance of Avian Influenza A Virus H9N2
Subtype in Humans and Poultry in Shanghai, China, Between 2008 and 2010. Zoonoses Public Health. 2014. PubMed
PMID: 24803167. http://dx.doi.org/10.1111/zph.12133.
51. Gronesova P, Mizakova A, Betakova T. Determination of hemagglutinin and neuraminidase subtypes of avian
influenza A viruses in urban pigeons by a new nested RT-PCR. Acta Virol. 2009; 53(3): 213-216. PubMed PMID:
19941404. http://dx.doi.org/10.4149/av_2009_03_213.
52. Perk S, Panshin A, Shihmanter E, Gissin I, Pokamunski S, Pirak M, et al. Ecology and molecular epidemiology of H9N2
avian influenza viruses isolated in Israel during 2000-2004 epizootic. Dev Biol (Basel). 2006; 124: 201-209. PubMed
PMID: 16447512.
53. Jia B, Shi J, Li Y, Shinya K, Muramoto Y, Zeng X, et al. Pathogenicity of Chinese H5N1 highly pathogenic avian influenza
viruses in pigeons. Arch Virol. 2008; 153(10): 1821-1826. PubMed PMID: 18779923.
http://dx.doi.org/10.1007/s00705-008-0193-8.
54. Klopfleisch R, Werner O, Mundt E, Harder T, Teifke JP. Neurotropism of highly pathogenic avian influenza virus
A/chicken/Indonesia/2003 (H5N1) in experimentally infected pigeons (Columbia livia f. domestica). Vet Pathol. 2006;
43(4): 463-470. PubMed PMID: 16846988. http://dx.doi.org/10.1354/vp.43-4-463.
55. Petersen H, Matrosovich M, Pleschka S, Rautenschlein S. Replication and adaptive mutations of low pathogenic
avian influenza viruses in tracheal organ cultures of different avian species. PLoS One. 2012; 7(8): e42260. PubMed
PMID: 22912693. http://dx.doi.org/10.1371/journal.pone.0042260.
56. Smietanka K, Minta Z, Wyrostek K, Jozwiak M, Olszewska M, Domanska-Blicharz AK, et al. Susceptibility of pigeons to
clade 1 and 2.2 high pathogenicity avian influenza H5N1 virus. Avian Dis. 2011; 55(1): 106-112. PubMed PMID:
21500645. http://dx.doi.org/10.1637/9514-090110-resnote.1.
57. Werner O, Starick E, Teifke J, Klopfleisch R, Prajitno TY, Beer M, et al. Minute excretion of highly pathogenic avian
influenza virus A/chicken/Indonesia/2003 (H5N1) from experimentally infected domestic pigeons (Columbia livia)
and lack of transmission to sentinel chickens. J Gen Virol. 2007; 88(Pt 11): 3089-3093. PubMed PMID: 17947534.
http://dx.doi.org/10.1099/vir.0.83105-0.
58. Yamamoto Y, Nakamura K, Yamada M, Mase M. Limited susceptibility of pigeons experimentally inoculated with
H5N1 highly pathogenic avian influenza viruses. J Vet Med Sci. 2012; 74(2): 205-208. PubMed PMID: 21921436.
http://doi.org/10.1292/jvms.11-0312.
59. Kalthoff D, Bogs J, Grund C, Tauscher K, Teifke JP, Starick E, et al. Avian influenza H7N9/13 and H7N7/13: A
comparative virulence study in chickens, pigeons, and ferrets. J Virol. 2014. PubMed PMID: 24899194.
http://dx.doi.org/10.1128/JVI.01241-14.
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Tables
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
14.53%
8.94%
4.18%
2.56%
H7
0.00%
0.00%
0.00%
0.00%
Others
2.06%
3.25%
20.45%
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
Pigeon
Duck
Goose
= 3299)
(n = 1083)
(n = 1656)
(n = 301)
H9
19.49%
7.29%
4.71%
5.32%
H7
0.79%
0.37%
0.00%
0.33%
Others
1.61%
1.66%
16.55%
16.94%
#1
Chicken
(n
All of the H7 and H9 subtypes of AIVs were A/China/2013(H7N9) viruses and
H9N2 subtype AIVs, respectively.
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519
520
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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523
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Table 4. Calculation  ratios for each site in the viral HA gene
Kappa
Sites with  > 1#2
Model
Np#1
Log- likelihood
M0 (one ratio)
313
−4041.977
5.814
None
M1 (nearly neutral)
314
−4015.628
5.662
None
M2 (positive selection)
316
−4015.830
5.662
57R, 119D, 164M, 174S, 226L, 541N, 542G
M3 (discrete)
317
−4009.564
5.872
57R, 119D, 164M, 174S, 214V, 226L, 541N, 542G
M7 (beta)
314
−4018.824
5.931
None
M8 ( beta &  > 1)
316
−4018.082
5.734
(ts/tv)
57R, 119D, 135A, 164M, 174S, 186V, 214V, 226L,
276N, 541N, 542G
#1
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
#2
with a probability > 95%.
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528
Figures
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.
529
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H7N9 surveillance & evolution
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531
532
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Figure 2. Pictures of several typical LBMs in China. These pictures show that pigeons were prevalent and caged closely with other birds on LBMs.
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535
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H7N9 surveillance & evolution
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Figure 3. Phylogenetic relationships among 127 A/China/2013(H7N9) viruses and 268 H9N2 subtype AIVs based on
540
the sequences of their six internal genes. The clades of each internal gene of the viruses are designated
541
alphabetically and shown in deep red, navy blue, violet, cyan, red, light green, blue, purple red, light blue, olive,
542
light red and deep green, respectively, from the top down. The same clade (e.g., Clade A) with different genes could
543
include different viruses. Genotype 1 harbors AIVs where all of the internal genes belong to clade A, including most
544
of the H7N9 AIVs and some H9N2 subtype AIVs, e.g., A/pigeon/Jiangsu/K77/2013(H9N2), which is represented by
545
the triangles in the figure.
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547
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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Figure 5. Phylogenetic relationships among 445 H7 AIVs isolated in Asia during 2008–2013. The A/China/2013(H7N9)
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viruses and other H7 viruses selected for calculating the  ratios are marked with triangles and circles, respectively.
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H7N9 surveillance & evolution
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Figure 6. A possible pathway toward the origin and development of
A/China/2013(H7N9) in China.
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Statements
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Ethics
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The authors declare that they have not conducted plagiarism, falsification or dual submission with respect to
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this article, and that they have been aware of and complied with the ethical requirements of Newpubli regarding
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authorship, human rights, animal welfare, biosecurity and dual use of research.
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The authors also declare that this study was conducted in strict accordance with the recommendations in the
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Guide for the Care and Use of Laboratory Animals of China Animal Health and Epidemiology Center. The feces
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samples, drinking-water samples and swab samples from poultry farms, backyard flocks and live bird markets were
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all collected with permission given by various relevant parties, including the Ministry of Agriculture of China, China
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Animal Health and Epidemiology Center, the relevant veterinary section in the provincial and county or city
566
government, and the owners of the relevant birds.
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Competing Interests
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The authors declare that no competing interests exist with respect to this article, except that some authors are
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editors of Newpubli. Newpubli has established a mechanism using software to ensure that the rating of each peer
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reviewer regarding the value of each article is blind to everyone and cannot be changed by anyone.
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Data Sharing
572
The authors declare that all of the data underlying the findings or conclusions of this article and its preprint are
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fully available without restriction.
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Funding
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This study was supported by the Avian Influenza Surveillance Program of the Ministry of Agriculture and the
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Sci-tech Basic Work Project of the Ministry of Science and Technology (SQ2012FY3260033) in China. The funders
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had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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