Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus

Evolution of the H9N2 influenza genotype thatfacilitated the genesis of the novel H7N9 virusJuan Pua,b,1, Shuoguo Wangc,1, Yanbo Yind,1, Guozhong Zhanga, Robert A. Carterc, Jinliang Wanga, Guanlong Xua,Honglei Suna, Min Wanga, Chu Wena, Yandi Weia, Dongdong Wangd, Baoli Zhue, Gordon Lemmonc, Yuannian Jiaoc,Susu Duanb, Qian Wanga, Qian Dua, Meng Suna, Jinnan Baoa, Yipeng Suna, Jixun Zhaoa, Hui Zhangf, Gang Wuc,Jinhua Liua,2, and Robert G. Websterb,2

aKey Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193,China; bDepartment of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105; cDepartment of Computational Biology, St. JudeChildren’s Research Hospital, Memphis, TN 38105; dCollege of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109,China; eChinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences,Beijing 100101, China; and fDepartment of Biostatistics, St. Jude Children’s Research Hospital, Memphis, TN 38105

Contributed by Robert G. Webster, November 26, 2014 (sent for review October 22, 2014)

The emergence of human infection with a novel H7N9 influenzavirus in China raises a pandemic concern. Chicken H9N2 virusesprovided all six of the novel reassortant’s internal genes. How-ever, it is not fully understood how the prevalence and evolutionof these H9N2 chicken viruses facilitated the genesis of the novelH7N9 viruses. Here we show that over more than 10 y of cocircu-lation of multiple H9N2 genotypes, a genotype (G57) emergedthat had changed antigenicity and improved adaptability in chick-ens. It became predominant in vaccinated farm chickens in China,caused widespread outbreaks in 2010–2013 before the H7N9viruses emerged in humans, and finally provided all of their in-ternal genes to the novel H7N9 viruses. The prevalence and vari-ation of H9N2 influenza virus in farmed poultry could provide animportant early warning of the emergence of novel reassortantswith pandemic potential.

H9N2 | H7N9 | chicken influenza virus | genotype | infectivity

Human infection with a novel avian-origin H7N9 influenza Avirus causing severe respiratory symptoms and mortality was

first reported in eastern China in March 2013 (1). To date, thenovel virus has caused two outbreaks of human infection, in-cluding 375 known cases and 115 deaths as of 11 March 2014 (2).Phylogenetic analysis suggests that the virus is a triple reassortantof H7, N9, and H9N2 avian influenza viruses (3, 4). The H7 andN9 genes may have been transferred from migratory birds todomestic ducks and then to chickens in the live poultry markets(3–5), after which reassortment with enzootic H9N2 virusesformed the H7N9 viruses identified in humans (3–5).H9N2 influenza virus has low pathogenicity for avians, repli-

cating mainly in the upper respiratory tract and causing mild orno overt signs of illness in specific pathogen-free (SPF) chickens(6). In 1994, the H9N2 subtype was first identified in chickenfarms in the Guangdong province of south China (7); it has sincebecome widespread in chickens and has caused great economicloss from reduced egg production and highly lethal coinfections(8–11). To reduce the impact of H9N2 infection in chickens, theflocks have been vaccinated since 1998 with commercial inacti-vated vaccines, such as A/chicken/Guangdong/SS/1994 (Ck/GD/SS/94), A/chicken/Shandong/6/1996 (Ck/SD/6/96), and A/chicken/Shanghai/F/1998 (Ck/SH/F/98) (8, 12, 13). These H9N2 vaccinesinitially limited the outbreaks and virus spread. However, despitemultiple doses, the H9N2 vaccines became less effective, espe-cially after 2007, and H9N2 influenza virus continues to circulatein vaccinated chicken flocks and has caused sporadic diseaseoutbreaks (8, 10, 12–20). However, the recent prevalence andmolecular evolution of the H9N2 viruses in chickens especiallyin the flocks receiving large-scale vaccination, and their role inthe emergence of human H7N9 virus, are not fully understood.In this study, we systematically investigated the prevalence and

evolution of H9N2 viruses mainly focusing on farm chickens andtheir role in the genesis of the novel H7N9 viruses.

ResultsIncreased Isolation of H9N2 Viruses from Farm Chickens Preceded theOutbreak of H7N9 Infection in Humans. We retrospectively in-vestigated the infection of farm chickens with H9N2-subtype in-fluenza viruses, mainly in northeast China, from January 2010 toDecember 2013 (Fig. 1 and Table S1). Tracheal swabs or lungsamples were taken for virus isolation and identification from 1975chicken farms reporting illness in 19 provinces during 2010–2013.All of these chickens had been vaccinated against H9N2 virus butshowed respiratory disease signs and/or a 5–20% reduction in eggproduction. Of the 1975 farms, 637 were positive for H9N2 in-fection, with a virus isolation rate as high as 32.25% (Table S1).Mean H9N2 isolation rates increased annually, from 22.08% in2010 to 47.08% in 2013. Further, these H9N2 infections occurredeven during the warm season (July through September), wheninfluenza infection is usually reported less often (Fig. 1). Despitevaccination, H9N2 virus infection in the investigated regions in-creased dramatically after 2010, indicating an antigenic variant

Significance

The emergence of human infection with a novel H7N9 avianinfluenza reassortant in China raises a pandemic concern.However, it is not fully understood how these H9N2 chickenviruses facilitated the genesis of the novel H7N9 viruses.Here we show that a “fittest” genotype (G57) emerged withchanged antigenicity and improved adaptability in chickens. Itbecame predominant in vaccinated farm chickens and causedwidespread outbreaks before the H7N9 virus emergence, in-creasing reassortment between H9N2 and other subtype viru-ses and finally providing all of their internal genes to the novelH7N9 viruses. The prevalence and variation of H9N2 in-fluenza virus in farmed poultry could provide an importantearly warning of the emergence of novel reassortants withpandemic potential.

Author contributions: J.P., G.Z., J.Z., and J.L. designed research; J.P., Y.Y., G.Z., J.W., G.X.,H.S., M.W., C.W., Y.W., D.W., B.Z., Y.J., Q.W., Q.D., M.S., and J.B. performed research; J.P.,S.W., G.Z., R.A.C., J.W., M.W., G.L., Y.S., J.Z., H.Z., G.W., J.L., and R.G.W. analyzed data; andJ.P., S.D., G.W., J.L., and R.G.W. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The sequences generated in this study have been deposited in the Gen-bank database (accession nos. are listed in Table S3).1J.P., S.W., and Y.Y. contributed equally to this work.2To whom correspondence may be addressed. Email: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1422456112/-/DCSupplemental.

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caused an outbreak in farm chickens that was maximal before andduring the 2013 emergence of the novel H7N9 virus in humans.

Reduced Genetic Diversity of H9N2 Viruses in Chickens During 2010–2013 Indicates Widespread Outbreak. Widespread influenza out-breaks caused by antigenic drift variants are usually thought to becharacterized by reduced genetic diversity of the viruses, eithergenome-wide or in the hemagglutinin (HA) segment (21–23).Thus, a widespread outbreak can be reasonably inferred by highsequence similarity of viruses isolated during a specified time spanacross a broad region. We wondered whether other regions be-yond our investigation were also affected. To address this question,we analyzed the temporal dynamics of genome-wide sequencesimilarities of H9N2 viruses by collecting all available H9N2sequences from chicken viruses in China during 1994 through2013. Pairwise identity comparison showed a decrease in diversityin all segments of these viruses, starting mainly in 2009–2010 andreaching a peak during 2010–2013 (Fig. 2 and Fig. S1). Somewhatless similarity was found in the HA genes of 2011 viruses and theneuraminidase (NA) genes of 2013 viruses. The decreased se-quence diversity in almost all segments at approximately the sametime point suggests that a virus with the greatest fitness was se-lected, gained dominance, and caused a widespread outbreak inchickens throughout China. A genetic “bottleneck” is proposed ininfluenza virus evolution (22). This is the first observation, to ourknowledge, of such a bottleneck in the H9N2 viruses (Fig. 2 andFig. S1), despite their prevalence in China for the last 20 y.

Genotype G57 of H9N2 Influenza Virus Became Predominant inChickens Since 2010. To understand the phylogenetic evolutionof the prevalent H9N2 viruses, we constructed phylogenetic treesfor all of the eight segments. Phylogenetic trees were automati-cally partitioned into clusters (24) using a 20th percentile cutoff.Clades were then manually merged, if necessary, based on thebranching posterior and reported classification (19, 20, 25). Ineach tree of eight segments, most of the 2010–2013 virusesbelonged to the same clade (Fig. 3A and Fig. S2). Within eachprevalent clade, the majority of 2010–2013 viruses further formeda major group with few early viruses (Fig. 3A and Fig. S2), in-dicating they shared the common ancestor surviving the bottle-neck event. In the polymerase basic 2 (PB2), NP (nucleoprotein),HA, NA, and nonstructural (NS) genes, a minor population of theprevalent viruses formed a different clade (HA) or minor group(HA and other segments). On the basis of clade classification, 69genotypes were found in H9N2 chicken viruses in China from1994 through 2013 (Dataset S1). Most of the 2010–2013 viruseswere from a single genotype classified as genotype 57 (G57). G57virus was first detected in chickens in Jiangxi and Jiangsu

provinces in 2007, and its prevalence increased sharply during2009. Since 2010, G57 has been the predominant genotype incirculation throughout China (Dataset S1 and Fig. 3B).This predominant genotype was generated by continued re-

assortment that can be traced to the year 1994 (Fig. 3C). TheG57 NA gene was derived from the A/chicken/Beijing/1/1994-like lineage. In 1998, four new lineages of the polymerase basic(PB1), polymerase acidic (PA), NP, and NS genes were pro-duced in a chicken virus, A/chicken/Shanghai/F/1998 (SH/98),which caused a severe outbreak in chickens in eastern China(26). After 1998, the SH/98-like virus reassorted with theA/quail/Hong Kong/G1/1997-like and A/chicken/Jiangsu/1/2000-like viruses, introducing matrix (M) and the HA genes to gen-erate the A/chicken/Hunan/5260/2005-like virus in 2005. Thevirus had seven of the eight gene segments of the G57 genotype.Then the final segment, PB2, was transmitted from ducks(A/duck/Shantou/163/2004-like) to chickens, where it formed thepredominant G57 genotype in 2007. Since 2007, G57 viruses havespread to domestic aquatic birds, wild birds, and swine (Dataset S1);in 2014, this genotype was isolated from a human patient in HongKong (Dataset S1), indicating a wide range of infected hosts.Clearly, the widespread outbreak in 2010–2013 established the

single dominant G57 genotype in chickens, ending more than10 y of cocirculation of multiple genotypes and producing nu-merous H9N2 viruses carrying the same gene constellation.

Improved Infectivity of G57 H9N2 Viruses in Chickens. Reassortmentseems advantageous because it can rapidly create high fitnessgenotypes (27). To determine the effect of reassortment on theinfectivity of G57, we selected nine representative H9N2 chickenviruses from G57 or other genotypes to test their replication ininoculated and contact chickens (Fig. 4, Top, A–C). Specifically,four 2010–2011 viruses were from G57, one virus was from G68(only with different HA segment compared with G57) isolated in2011, and four viruses were from other major genotypes circu-lating before 2010. After inoculation, although all of the testedviruses had a 100% isolation rate in tracheas (Fig. 4A), most ofthe G57 viruses replicated to significantly higher titers than didthe earlier viruses of other genotypes (titers 10–100 times ashigh; P < 0.05; Fig. 4B). The G57 viruses also showed clearlyenhanced replication in the cloaca, to titers 10–1,000 times as highas those of earlier isolates (Fig. 4B), explaining the 100% de-tection rate of all inoculated G57 viruses but only 20–60% of in-oculated earlier viruses (Fig. 4A). In addition, most of the G57viruses were shed from the cloaca for a longer period [until 7 dayspostinfection (dpi); Fig. 4C]. The replication ability of G68 viruswas moderate between G57 viruses and other genotypes of earlierviruses (Fig. 4 A–C). Similar to its replication in inoculatedchickens, G57 exhibited improved infection with higher viral titersalso in contact chickens followed by the G68 virus, although therewas a 100% isolation rate among different genotypes except forG02 in the contact chickens. (Fig. 4 A–C). These results suggestthis improved adaptation in chickens may be one reason for thewidespread outbreak of G57 H9N2 viruses during 2010–2013.

Antigenic Drift of G57 H9N2 Viruses Isolated Since 2010. Antigenicdrift, induced mainly in the HA glycoprotein, can decrease avaccine’s efficacy and cause outbreaks of viral infection (21–23,28). Phylogenetic analysis revealed that the 2010–2013 H9N2viruses were mainly in clade 6 and clade 9, whereas the vaccinestrains were in clade 2 (Fig. S2). To determine whether antigenicdrift had been induced in the prevalent G57 H9N2 viruses, weinvestigated this possibility by hemagglutinin inhibition (HI) test-ing of 26 representative H9N2 viruses. As the outbreak of H9N2influenza in chickens occurred mainly after 2010 and the H7N9viruses are estimated to have emerged in 2011–2012 (4, 12), weselected viruses from 2010 to 2011 for HI testing, on the basis oftheir HA phylogenic topology. Some early representative viruses

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Fig. 1. Isolation rate (%) of H9N2 influenza viruses during 2010–2013 onchicken farms reporting illness in our investigated regions. Histogram indicatesmonthly isolation rates; red horizontal line (connected with dots) indicatesmean annual isolation rates; and gray horizontal dashed line indicates 95% CI.

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from different antigenic groups were included (20), as were tworepresentative vaccine strains as controls (Ck/SD/6/96 and Ck/SH/F/98; Table S2). The G57 viruses prevalent in 2010–2011, as wellas some earlier viruses (mainly 2009), had lower HI titers eight ormore times the HI titers of the vaccine strains in reactions withvaccine antisera. These results demonstrated that the recentH9N2 viruses were antigenically distinct from the vaccine strains.To determine whether antigenic drift had reduced the pro-

tective effect of vaccines in the field, we compared the infectiveefficiency of H9N2 viruses in vaccinated inoculated and contact

chickens. The vaccinated chickens were divided into groups withlow antibody levels (HI titer, 6–9) and high antibody levels (HItiter, 10–12). Nine viruses used in the above unvaccinatedchicken experiment (Fig. 4 A–C, Upper) were also selected in thevaccinated chicken study because they are also the representa-tives based on the HA lineage (Fig. S2) and HI reaction (TableS2). In the inoculated vaccinated chickens, the G57 viruses from2010/2011 consistently maintained their growth in trachea, witha 100% isolation rate regardless of antibody level (Fig. 4A).However, the replication of early viruses from other genotypeswas clearly inhibited, as shown by a 0–70% isolation rate (exceptfor A/chicken/Shandong/ZB/2007, ZB/07) in chickens with lowantibody levels (Fig. 4, Middle) and a 0–30% rate in those withhigh antibody levels (Fig. 4A, Bottom). ZB/07 was an antigenicvariant identified previously (17). After placing vaccinated chickensin contact with inoculated birds, all of the G57 viruses main-tained 100% transmission in trachea regardless of antibody level(Fig. 4A), whereas the transmission rate of early viruses de-creased markedly, and only one (ZB/07) was isolated, at a lowrate (40%), in chickens with high antibody levels (Fig. 4A). Al-though all of the G57 viruses maintained 100% replication andtransmission, their titers in the trachea and cloaca decreased butremained significantly higher (P < 0.05 or P < 0.01, ANOVA)than those of the early viruses (Fig. 4B). By comparison of theirshedding time (Fig. 4C), G57 viruses had longer shedding timeuntil 7 dpi in contact vaccinated chickens with high antibodytiters (10–12). Although the shedding time of ZB/07 was similarwith those of G57 viruses, the numbers of H9N2-positive chickenswas less (Fig. 4A). Notably, G57 viruses showed improved rep-lication and transmission in the cloaca of vaccinated chickens,whereas none of the other genotypes of early viruses wasdetected (Fig. 4, Middle and Bottom), similar to that observed inunvaccinated chickens (Fig. 4, Top). One G68 virus showed amoderate level in replication and transmission between G57 andother earlier genotype viruses (Fig. 4). These results suggest thatrecent antigenic variants of G57 viruses possessed the fitness toescape immunization pressure and cause the widespread out-break of H9N2 infection in chickens in 2010–2013.

G57 H9N2 Chicken Virus Provided All Six of Their Internal Genes to theNovel H7N9 Viruses. Previous studies demonstrate that all six ofthe internal genes of the novel H7N9 viruses are from H9N2chicken viruses circulating in China (3–5). To determine whetherthese internal genes were all from the clades of G57 genotypeviruses, we constructed phylogenetic trees of all internal genes ofthe available 1994–2013 H9N2 chicken viruses and of the H7N9human viruses isolated during the first 2 wk of the 2013 H7N9outbreak. Later H7N9 isolates were eliminated to better identifythe cause of the initial outbreaks because these viruses un-derwent further gene exchange with local H9N2 viruses duringviral transmission after that time point (29). The six internalgenes of the H7N9 human isolates grouped closely with those ofthe recent 2010–2013 H9N2 chicken viruses as shown in previousstudies (3–5), and all belonged to the clades of the G57 genotype(Fig. 3A and Fig. S2). Specifically, the internal genes of the initialH7N9 isolates were the most similar to those of the major groupsof H9N2 G57 viruses, with the exception of the NS gene. The NSgenes of H7N9 human isolates belonged to a minor group thatincluded H9N2 G57 viruses isolated mainly in the Jiangsu andZhejiang provinces nearby Shanghai, where the first outbreaks ofhuman H7N9 infection occurred. Eleven novel reassortants ofsubtypes other than H7N9 (e.g., H5N2, H7N7, and H10N8 viru-ses) isolated in avian, swine, or humans, mainly after 2010, alsopossessed G57-like internal genes (Dataset S1).

DiscussionThe segmented genome feature of influenza viruses allows reas-sortment of segments from different viruses, generating novel

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Fig. 2. Reduced genetic diversity of the eight segments of H9N2 influenzaviruses isolated from chickens during the widespread outbreaks in China (2010–2013). Pairwise comparison of nucleotide sequence of all H9N2 chicken viruses isplotted as a heatmap, if a given pair of sequences shares more than 50% of thefull length. Viruses isolated from 1994 through 2013 are ordered by isolationtime from left to right on the x axis and from top to bottom on the y axis. Tickson the axes indicate the isolation years. Red dotted line indicates the year 2010.Color indicates identity levels from ≤90% (red) to 100% (blue) or identity levelsunavailable due to the shared sequence length less than 50% (white).

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influenza viruses with pandemic potential (28, 30, 31). In thepresent study, we investigated the roles of the evolution of H9N2viruses in the genesis of the reassortant H7N9 viruses. Our resultsrevealed that H9N2 influenza viruses evolved by reassortment andmutation over their 20 y of prevalence in chickens, and the “fittest”genotype, G57, was selected recently. The G57 viruses with changedantigenicity and improved fitness resulted in viral escape from im-munization and caused widespread outbreaks in 2010–2013 andfinally became the single predominant H9N2 genotype in farmchicken population. This genotype contributed its six internal seg-ments to the novel H7N9 viruses by reassortment.The widespread use of vaccines against H9N2 viruses in chickens

has exerted continuous mutation pressure on the viral HA gene,resulting in antigenic changes. Our study showed a weakened effectof vaccine antisera on the recent H9N2 chicken isolates andheightened virus replication and transmission in vaccinated chick-ens. These results, combined with the reduced diversity of the 2010–2013 viruses and the close phylogenetic grouping of their HA genes,suggest that the observed antigenic changes are common in therecent H9N2 chicken viruses. In addition to the antigenic charac-teristics of HA, the genomic context and compatibility of certainsegment combinations may be an important contributor to viralfitness (22). As shown in the study on unvaccinated chickens, G57viruses exhibited the highest replication ability in inoculated andcontact chickens than other genotypes, indicative of an optimizedsegment combination of G57. In another recent genotype G68,which had the same segments with G57 viruses except for the HAsegment, a representative virus (A/chicken/Guangdong/01/2011)still had higher viral titers in inoculated or contact chickens thanearly viruses of other genotypes, indicating other segments also

contributed to the enhanced infection ability for the viruses pre-vailing after 2010. When testing infection in vaccinated chickens,G57 viruses showed superiority in replication and transmissionability. These results suggest that under immunization pressure andselection of the optimal gene constellation by passing through theimmunological bottleneck the G57 virus eventually became thedominant genotype during the 2010–2013 outbreak. This outbreakwas the first country-wide event of H9N2 viruses in chickens since1998, when extensive agricultural vaccination began in China.The formation of the H9N2 ecological environment, as described

above, was the first step in the genesis of the H7N9 viruses, as itgreatly increased the likelihood of reassortment between H9N2 andother subtypes of influenza viruses. The introduction of H7 and/orN9 viruses into chickens and subsequent reassortment with theH9N2 viruses was the final step. Jiangsu, Shanghai, and Zhejiang,the first provinces that experienced outbreaks of the novel H7N9viruses on humans (1, 3), are ideally situated for the cocirculation ofH9N2, H7, and N9 viruses. These provinces are among the largestchicken consumption markets and are located along the avian mi-gratory route in the Yangtze river delta. Molecular clock dating ofdivergence times estimates that these H7N9 viruses emergedin ∼2011–2012 (4, 13), during the widespread H9N2 outbreaks inchickens. Also since the H9N2 outbreaks, the appearance of morethan 10 non-H7N9 novel reassortants further highlights the sig-nificant contribution of the predominant G57 genotype virusesin reassortment with other subtype viruses and raises concernsfor the generation of potential pandemic viruses.In conclusion, our study provides a comprehensive picture of

how H9N2 influenza viruses infecting chickens contributed togeneration of the H7N9 viruses that have infected humans

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A/chicken/Jiangsu/B5/2011

A/chicken/Yunnanws/102/2008

A/chicken/Wujin/19/2011

A/chicken/Jilin/G12/2009

A/chicken/Zhejiang/SJL/2009

A/chicken/Henan/I23/2011

A/chicken/Rizhao/651/2013A/chicken/Shandong/lc0830/2012

A/chicken/Anhui/B19/2011

A/chicken/Guangdong/Y18/2009

A/chicken/Anhui/M26/2011

A/chicken/Ningxia/102/2011

A/chicken/Hangzhou/48-2/2013

A/chicken/Hubei/A1/2010

A/chicken/Henan/D24/2011

A/chicken/Qianzhou/12/2010

A/chicken/Anhui/WBQ/2011

A/chicken/Hainan/A26/2011A/chicken/Guangdong/L14/2009

A/chicken/Rizhao/811/2013

A/chicken/Chongqing/G12/2011

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A/chicken/Shandong/J6/2011A/chicken/Henan/E10/2011

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A/chicken/Guangxi/1032/2005A/chicken/Liaoning/41/17/2007

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A/chicken/Shanxi/K16/2011




A/chicken/Wenzhou/592b/2013

A/chicken/Guizhou/E3/2010

A/chicken/Jiangsu/CZWJ5/2013

A/chicken/Jiangsu/A13/2010A/chicken/Guangdong/J22/2009


A/chicken/Sichuan/G4/2009

A/chicken/Hunan/U6/2010


A/Chicken/Guangdong/10/00

A/chicken/Jiangsu/WJHDL/2012

A/chicken/Hebei/0109/2013

A/chicken/Jilin/CBS/2010

A/Chicken/Shandong/HL/2010


A/chicken/Guizhou/A4/2010


A/chicken/Shaanxi/11/2012

A/chicken/Tianjin/120/2013


A/chicken/Shandong/TH9/2010

A/chicken/Zhejiang/WXQ12/2011


A/chicken/Baoshan/111/2009

A/chicken/Guangdong/D8/2010



A/chicken/Hubei/N14/2009



A/chicken/Rizhao/1207/2013A/chicken/Shandong/wf0712/2013

A/chicken/Yunnandh/101/2008


A/chicken/Fujian/FG13/2007

A/chicken/Jiangsu/ZHM/2011

A/chicken/Shandong/LY/2006


A/chicken/Guangdong/P26/2009



A/chicken/Guizhou/Y6/2009


A/chicken/Hubei/G27/2010


A/chicken/Xinjiang/XJ75/2007



A/chicken/Shandong/yt0106/2012







A/chicken/Zhejiang/611/2011A/chicken/Shandong/zc4/2012

A/chicken/Guangdong/H11/2010

A/chicken/Guangdong/J23/2009



A/chicken/Fujian/FZ04/2011

A/chicken/Shandong/01/2010

A/chicken/Dawang/1/2011

A/chicken/Hainan/A13/2011A/chicken/Guangdong/HE21/2008

A/chicken/Anhui/AI16/2008

A/chicken/Guangdong/R17/2009

A/chicken/Fujian/FH14/2007




A/chicken/Hubei/J9/2009

A/chicken/Lincang/711/2008



A/chicken/Anhui/GQY/2011






A/chicken/Shandong/WE23/2011

A/chicken/Guizhou/D26/2011

A/chicken/Shandong/L/2010



A/chicken/Shandong/LC2/2013

A/chicken/Jiangsu/YZ0611/2011





A/chicken/Jiangsu/WXY/2011

A/chicken/Liaoning/PJXM15/2012A/chicken/Shandong/M4/2011


A/chicken/Hainan/A4/2011

A/chicken/Henan/YX/2012






A/chicken/Shanxi/E30/2011


A/chicken/Shandong/YB06/2006


A/chicken/Guangxi/MFX/2011


A/chicken/Xinjiang/05/2009

A/chicken/Anhui/K17/2011

A/chicken/Zhejiang/NH5/2013




A/chicken/Shandong/lx316/2008

A/chicken/Zhejiang/DJR12/2011

A/chicken/Jiangsu/CZ1/2012



A/chicken/Shandong/H/2009


A/chicken/Fujian/FB3/2007

A/chicken/Yunnan/Zhaotong/2007A/chicken/Yunnan/Kunming/2007


A/chicken/Gansu/419/2012


A/chicken/Henan/E2/2010


A/chicken/Guangdong/B4/2006A/chicken/Shantou/22054/2005

A/chicken/Shanxi/K10/2011A/chicken/Henan/K8/2011A/chicken/Hunan/JYZ11/2011

A/chicken/Hunan/HB29/2007


A/chicken/Fujian/FB22/2007


A/chicken/Shandong/HK1/2013

A/chicken/Zhejiang/HJ/2007

A/chicken/Hainan/Q4/2010



A/chicken/Shandong/A1/2009

A/chicken/Shandong/J9/2011

A/chicken/Shanxi/I21/2011



A/chicken/Hunan/HE5/2007

A/chicken/Taixing/10/2010

A/chicken/Guangxi/LRW04/2011

A/chicken/Yunnan/Wenshan/2007


A/chicken/Tibet/S4/2009

A/chicken/Guangdong/J3/2009A/chicken/Guangdong/D2/2010




A/chicken/Tianjin/F17/2009




A/chicken/Anhui/G18/2010A/chicken/Guangdong/H28/2010

A/chicken/Hubei/HE27/2008

A/chicken/Guangxi/1428/2005



A/chicken/Hubei/D2/2010

A/chicken/Anhui/F2/2011

A/chicken/Xinjiang/XJ154/2007






A/chicken/Tibet/L18/2009



A/chicken/Fujian/WS/2010A/chicken/Guangxi/21/2006



A/chicken/Shandong/WG10/2011

A/chicken/Shangdong/SDWF14/2012



A/chicken/Anhui/AK25/2008

A/chicken/Jiangsu/XZWHL/2012





A/chicken/Wuxi/7/2010





A/chicken/Anhui/AH16/2008

A/chicken/Guangdong/LG1/2013



A/chicken/Hubei/D21/2010



A/chicken/Anhui/1/2010


A/chicken/Guangdong/A7/2002

A/chicken/Jiangsu/LWC/2011

A/chicken/Zhejiang/ZG1/2007

A/chicken/Zhejiang/HE2/2009


A/chicken/Guangdong/K25/2009

A/chicken/Beijing/16/2013A/chicken/Anhui/G18/2011


A/chicken/Shandong/SDWF17/2012






A/chicken/Jiangsu/XZZSL/2012

A/chicken/Shantou/2686/2003A/chicken/Hubei/E13/2008



A/chicken/Hubei/ZWC/2011

A/chicken/Wenshan/412/2009

A/chicken/Zhejiang/FYQ12/2011


A/chicken/Shanxi/B29/2011


A/chicken/Shanxi/D25/2011

A/chicken/Guizhou/J22/2011




A/chicken/Jiangsu/CZJJF/2012






A/chicken/Shandong/mp1224/2007



A/chicken/Hubei/HWC/2011




A/chicken/Ningxia/A2/2011


A/chicken/Shandong/M1/2011

A/chicken/Jiangsu/A20/2010A/chicken/Chongqing/K4/2011A/chicken/Jiangsu/D8/2011A/chicken/Tianjin/DCWD/2011A/chicken/Jiangsu/H10/2011


A/chicken/Shanxi/33/2011

A/chicken/Jiangsu/U3/2010



A/Chicken/Nantong/031-2/2013

A/chicken/Hainan/E17/2010A/chicken/Henan/KF/2011

A/chicken/Xinjiang/07/2009

A/chicken/Shanghai/020/2013

A/chicken/Guangdong/ZW/2012

A/chicken/Jiangsu/FQD03/2011




A/chicken/Henan/B6/2011A/chicken/Henan/J14/2011






A/Chicken/Huzhou/C143-2/2013


A/chicken/Dehong/411/2009

A/chicken/Guangdong/G2/2009

A/chicken/Yunnan/chuxiong07/07

A/chicken/Shuanggou/1/2011


A/chicken/GuangXi/GLLY/2012

A/chicken/Chongqing/K3/2011

A/chicken/Guangdong/V12/2009



A/chicken/Henan/HF/2012


A/chicken/Shandong/YT4/2010A/chicken/Chongqing/K23/2011





A/chicken/Yangzhou/11/2010


A/chicken/Yunnan/Yuxi2/2007


A/chicken/Jiande/01/2009



A/chicken/Hubei/LLM03/2011





A/chicken/Shandong/wd01/2007


A/chicken/Puer/411/2009


A/Chicken/Huzhou/C143-8/2013










A/chicken/Guangdong/L4/2009



A/chicken/Shandong/K/2010






A/chicken/Jiangsu/G17/2010A/chicken/Anhui/ZYX05/2011


A/chicken/Hubei/E17/2010A/chicken/Hubei/N11/2009

A/chicken/Jiangsu/CZZWQ/2012A/chicken/Shandong/wf12010/2012


A/chicken/Shanghai/Q0712/2007A/chicken/Hebei/C6/2007




A/chicken/Jiangsu/DT0112/2012

A/chicken/Anhui/D30/2011

A/Chicken/Beijing/0309/2013

A/chicken/Shandong/E/2010

A/chicken/Guangxi/YZT10/2011

A/chicken/Jiangsu/CZLJG/2012



A/chicken/Baoshan/711/2008



A/Chicken/Shandong/02/2011A/chicken/Anhui/E23-2/2011




A/chicken/Guangxi/QDX04/2011



A/chicken/Shandong/377/2009A/chicken/Guangxi/G8/2009


A/chicken/Shandong/B/2009




A/chicken/Henan/J22/2011



A/chicken/Shandong/02/2009A/chicken/Shandong/YT2/2011



A/chicken/Jiangsu/MYJMF/2012






A/chicken/Henan/A16/2010

A/chicken/Ningxia/S17/2009




A/chicken/Zhejiang/HQD/2012


A/chicken/Guangdong/N4/2009

A/Chicken/Suzhou/097-2/2013


A/chicken/Tianjin/F2/2009



A/chicken/Guangdong/BL/2010

A/chicken/Zhejiang/LAC/2011





A/chicken/Fujian/FZ11/2011

A/chicken/Guizhou/XK04/2011

A/chicken/Jilin/M19/2009A/chicken/Ningxia/N22/2009


A/Chicken/Hebei/YT/2010




A/chicken/Jiangsu/HA2/2013


A/chicken/Guizhou/Y76/2009

A/chicken/Qingdao/04/2012


A/chicken/Shandong/M/2010



A/chicken/Chongqing/A53/2011



A/chicken/Shandong/SG2/2009A/chicken/Ningxia/R13/2009

A/chicken/JiIangsu/I9/2011


A/chicken/Henan/LY-36/2013



A/chicken/Zhejiang/HGH12/2011

A/chicken/Anhui/ZTL/2011





A/chicken/Zhejiang/SYX/2010




A/chicken/Tongshan/1/2011


A/chicken/Guangxi/TJY04/2011



A/chicken/Wenzhou/622/2013A/chicken/Rizhao/836/2013

A/chicken/ShanXi/WSDX/2012

A/chicken/Anhui/I24/2011

A/chicken/Shangdong/SDWD/2011

A/chicken/Henan/622/2006




A/chicken/Anhui/XMS/2011

A/chicken/Fujian/FG14/2007


A/chicken/Zhejiang/329/2011A/chicken/Rizhao/1313/2013A/chicken/Shandong/qd0107/2013

A/Chicken/Nanjing/503/2013


A/chicken/Shandong/BD/2010


A/chicken/Zhejiang/KHZ12/2011



A/chicken/Jiangsu/E26/2007


A/chicken/Ningxia/I24/2011

A/chicken/Anhui/AK13/2008


A/chicken/Guangdong/B7/2003A/chicken/Fujian/FB21/2007


A/chicken/Anhui/HF/2010

A/chicken/Jilin/BHZ/2010



A/chicken/Anhui/A19/2010A/chicken/Jiawang/2/2011




A/chicken/Jiangsu/1/00


A/chicken/Shandong/P4/2011


A/chicken/Guangxi/55/2005A/chicken/Yunnan/qujing09/07



A/chicken/Guangdongg/G9/2009


A/chicken/Anhui/K9/2011A/chicken/Shandong/WE26/2011

A/chicken/Zhejiang/ZLB/2011

A/chicken/Tibet/L7/2009


A/chicken/Zhejiang/FXK12/2011


A/chicken/Hubei/GYC/2011

A/chicken/Jilin/P23/2009




A/chicken/Fujian/SN1/2011

A/chicken/Anhui/K13/2011



A/chicken/Shanxi/K1/2011



A/chicken/Beijing/HD06/2012



A/chicken/Jiangsu/ZJDT1/2013

A/chicken/Hebei/QHDzd/2012

A/chicken/Henan/JZ/2011

A/chicken/Liaoning/21/7/2007





A/chicken/Jiangsu/ZJ4/2013


A/chicken/Yongcheng/1/2011







A/Chicken/Hebei/ZR/2010

A/chicken/Yunnan/YA114/2007



A/chicken/Jiangsu/XZPZ/2011


A/chicken/Jiangsu/NTTZ/2013




A/chicken/Henan/LH/2012

A/chicken/Sichuan/B1/2007





A/chicken/Guangdong/WZD11/2011



A/chicken/Jilin/XJJZ/2010



A/ChiCken/Shandong/03/2011

A/chicken/Guangdong/DJN03/2011

A/chicken/Guizhou/Q6/2009

A/chicken/Anhui/LJT/2010

A/Chicken/Shandong/10/2010A/chicken/Henan/G19/2010




A/chicken/Shanxi/YC/2012



A/chicken/Hunan/HA17/2007

A/chicken/Fujian/SL4/2011


A/chicken/Zhejiang/SYP12/2011


A/chicken/Jiangsu/QF/2011



A/chicken/Hubei/B23/2009



A/chicken/Shandong/FXWH13A/2012


A/chicken/Shandong/PD/2009

A/chicken/Shandong/WF4/2012A/chicken/Shandong/qd0307/2013

A/chicken/Jiangsu/WJYBF/2012


A/chicken/Shandong/N/2010





A/chicken/Henan/K1/2011

A/chicken/Shandong/YS/2012


A/chicken/Shanxi/xy1/2012

A/chicken/Anhui/K2/2011A/chicken/Anhui/K3-2/2011


A/chicken/Jiangsu/WJHRG/2012




A/chicken/Jiangsu/DT/2009



A/chicken/Hubei/TS3/2011



A/chicken/Fujian/G9/2009


A/chicken/Zhejiang/FSQ/2011

A/chicken/Yunnan/YC12/2007

A/chicken/Xiangshui/1/2011





A/Chicken/Henan/DY/2009

A/chicken/Sichuan/G2/2009A/chicken/Hubei/580/2011


A/chicken/Henan/I27/2011


A/chicken/Yunnan/Simao3/2007A/chicken/Xinjiang/02/2009

A/chicken/Jiangsu/Q3/2010



A/chicken/Anhui/AH23/2008



A/chicken/Anhui/B2/2010A/chicken/Hubei/TS1/2011


A/chicken/China/AH-10-01/2010



A/chicken/Fujian/SN2/2012

A/Chicken/Nanjing/106-2/2013

A/chicken/Ningxia/F15/2009

A/chicken/Jiangsu/XZMG/2012

A/chicken/Shandong/P2/2012A/chicken/Anhui/D14/2011


A/chicken/Jiangsu/ZJDT/2011



A/chicken/Guangdong/Q6/2010



A/chicken/Hebei/LC/2008

A/chicken/Shandong/M6/2011



A/chicken/Jiangsu/DTNSZ/2011

A/chicken/Beijing/L2/2012










A/chicken/Anhui/I4/2011




A/chicken/Chongqing/J24/2011


A/chicken/Jiangsu/CZJT2/2013

A/chicken/Shandong/zc0606/2012


A/chicken/Jiangsu/XZLZG/2012


A/chicken/Zhejiang/Q1D4/2010

A/chicken/Jiangsu/A7/2010





A/Shanghai/02/2013



A/chicken/Rizhao/1339/2013A/chicken/Rizhao/1363/2013



A/chicken/Zibo/B1/2008










A/chicken/Rizhao/853/2013A/chicken/Jiangsu/CZ1/2012A/Chicken/Nanjing/503/2013


A/Shanghai/1/2013



A/chicken/Hebei/A/2007






A/chicken/Hebei/0329/2007A/chicken/Hunan/2536/2005



A/chicken/Qianzhou/12/2010A/Chicken/Beijing/HD/2010

A/chicken/Shandong/yt0106/2012A/chicken/Jiawang/2/2011

A/Chicken/Hebei/DF/2008


A/chicken/Fujian/8341/2005A/chicken/Beijing/1/2006


A/chicken/Tianjin/61/2012A/chicken/Tianjin/120/2013







A/Chicken/Henan/DY/2009A/chicken/Shandong/02/2008

A/chicken/Jiangsu/CZLJG/2012A/chicken/Jiangsu/XZZSL/2012





A/chicken/Henan/L3/2008


A/chicken/Guangxi/G8/2009








A/chicken/Guangdong/ZHJ/2011A/Chicken/Shandong/sd02/2010


A/chicken/Zibo/L2/2008







A/Shanghai/2/2013






A/chicken/Hebei/C4/2008












A/chicken/Hebei/Y2/2009
















A/chicken/Hunan/4444/2005A/chicken/Shandong/513/2011A/chicken/Shandong/384/2011








A/chicken/Guangdong/ZCY/2011



A/Chicken/Shandong/LY/2008



A/Chicken/Shandong/JL/2008 A/chicken/Jiangsu/ZJDT1/2013








A/chicken/Shandong/KD/2009A/chicken/Shandong/BD/2010




A/chicken/Hangzhou/50-2/2013A/chicken/Hunan/12/2011



A/chicken/Shandong/12/2008A/chicken/Shandong/B4/2007 A/chicken/Shaanxi/11/2012





A/chicken/Jiangsu/CZJJF/2012A/chicken/Shandong/wf1206/2012






A/chicken/Jiangsu/CZZWQ/2012A/chicken/Shandong/qd0427/2012





















A/Chicken/Hebei/FX05/2010A/chicken/Xiangshui/1/2011








A/Shanghai/4664T/2013










A/chicken/Henan/1.2/2008








A/Chicken/Hebei/TS/2008A/chicken/Wenzhou/606/2013












A/chicken/Shandong/qd0516/2012A/chicken/Shanghai/C3/2012






















A/chicken/Jiangsu/XZLZG/2012A/chicken/Jiangsu/CZWJ5/2013A/chicken/Shandong/qd1013/2012

A/Chicken/Shanghai/1/02










A/Chicken/Hebei/TS/2008


A/chicken/Guangdong/ZW12/2011

A/chicken/Guangxi/KMIII/1999



A/chicken/Shandong/B/2009

A/chicken/Guangdong/SYX12/2011




A/chicken/Tianjin/B1/2004

A/chicken/Zhejiang/SYP12/2011


A/Chicken/Guangdong/97









A/chicken/Henan/nd/1998





A/chicken/Hebei/L1/2006

A/Chicken/Shanghai/F/98

A/chicken/Guangdong/LJT12/2011A/chicken/Guangdong/LG1/2013


A/Chicken/Hebei/1/96

A/chicken/Jiangsu/CZZWQ/2012



A/Chicken/Nanchang/4-361/2001







A/chicken/Shanghai/Y1/2007



A/chicken/Zhejiang/KHZ12/2011



A/chicken/Shanghai/Q0702/2007









A/chicken/Lijiang/711/2008

A/chicken/Shandong/N/2010


A/chicken/Hebei/0721/2013A/chicken/Shandong/qd01417/2013

A/chicken/Zhejiang/DJR12/2011





A/chicken/Guangdong/ZHJ/2011






A/chicken/Qixian/A1/2004



A/chicken/Shandong/K/2010A/chicken/Shandong/ZB/2007



A/chicken/Shandong/qd0516/2012A/chicken/Shanghai/C2/2012






A/chicken/Beijing/ne/1999

A/chicken/Guangxi/067C4/2010





A/chicken/Shandong/LY-1/2008



A/chicken/Nanchang/1-0016/2000

A/Chicken/Shijiazhuang/2/99

A/Chicken/Shandong/98

A/chicken/Zhejiang/611/2011A/Chicken/Hebei/YT/2010



A/chicken/Shandong/B3/2007




A/chicken/Hubei/C1/2007

A/chicken/Heilongjiang/48/01






A/chicken/Hubei/10/2009A/chicken/Shanghai/Q0812-1/2008A/chicken/Shanghai/Q0707/2007



A/chicken/Shandong/SG2/2009A/chicken/Shandong/377/2009

A/chicken/Jiangsu/XZPZ/2011A/chicken/Guangdong/SJS12/2011


A/chicken/Zhejiang/HGH12/2011




A/chicken/Shandong/C/2009

A/chicken/Shandong/WF/1998

A/chicken/Guangdong/LWZ11/2011

A/chicken/Jiangsu/1/2004A/chicken/Jiangsu/ng/2001






A/chicken/Hunan/JYZ11/2011





A/chicken/Shanghai/Q0808-2/2008




A/chicken/Jiangsu/L1/2004

A/chicken/Zhejiang/Q1D4/2010A/chicken/Jiande/01/2009





A/chicken/Yunnan/Chuxiong1/2007





















A/chicken/Guangxi/LYY12/2011

A/chicken/Zhejiang/FYQ12/2011

A/chicken/Hubei/01-MA01/1999

A/chicken/Shandong/L/2010

A/chicken/Zhejiang/XBF12/2011

A/chicken/Hebei/nj/2002

A/chicken/Zhejiang/ZGPL12/2011



A/chicken/Shangqiu/A1/2000

A/chicken/Henan/C4/2002


A/chicken/Tibet/S4/2009A/chicken/Shandong/02/2008





A/chicken/Hebei/B1/2001



A/chicken/Shandong/A1/2009


A/chicken/Zhejiang/WXQ12/2011




A/chicken/Fujian/25/00A/Chicken/Beijing/2/97

A/chicken/Guangdong/LYH12/2011A/chicken/Wenzhou/622/2013A/Chicken/Nanjing/023/2013


A/chicken/Jiangsu/WJHDL/2012A/chicken/Shandong/qd0225/2013


























A/Chicken/Ningxia/5/99

A/chicken/Henan/ni/2001










A/Chicken/Hebei/DF/2008A/chicken/Jiangsu/DT0112/2012A/chicken/Shandong/wf0202/2012A/chicken/Shandong/qd1013/2012


A/chicken/Zhejiang/607/2011A/chicken/Jiangsu/YCDF/2012



A/chicken/Eastern China/SD88/2008


A/chicken/Yunnan/Xie-1/1999



A/Chicken/Liaoning/99









A/chicken/Huangdong/A1/2003



A/chicken/Shandong/PD/2009A/chicken/Shandong/L1/2007



A/chicken/Guangdong/TS/2004


A/chicken/Guangdong/YBS10/2011

A/Chicken/Shaoguan/13/98

A/chicken/Kaifeng/A1/2005

A/chicken/Jiangsu/wa/2002

A/chicken/Shandong/SD0014/2008



A/chicken/Dehong/411/2009


A/chicken/Kunming/123/2007










A/chicken/Shantou/212/2000A/chicken/Anhui/2/2002

A/chicken/Neimonggu/nk/2002








A/chicken/Zhejiang/FXK12/2011



A/chicken/Zhuhai/A1/2001



A/chicken/Liaoning/99

A/chicken/Puer/711/2008



A/Chicken/Heilongjiang/10/97



A/chicken/Guangxi/55/2005A/chicken/Beijing/1/2006



A/chicken/Jiangsu/1/1999A/chicken/Qixian/A1/2001




A/chicken/Yunnan/nh/2001






A/Chicken/Hebei/FX05/2010A/chicken/Yangzhou/11/2010





A/chicken/Guangdong/G7/2009A/chicken/Shanghai/Q0801-2/2008

A/chicken/Guangdong/CYH12/2011


A/chicken/Shandong/E/2010


A/Chicken/Henan/98



A/chicken/Jiangsu/XZMG/2012A/chicken/Shandong/qd1224/2012


A/chicken/Guangdong/WSL11/2011


A/chicken/Shandong/wf0514/2013A/chicken/Hebei/4/2008






A/Chicken/Shandong/JL/2008





























A/chicken/Nanchang/4-301/2001




A/chicken/Shijiazhuang/2/98A/chicken/Beijing/nl/2002



A/chicken/Jiangsu/TZXH/2013


A/Chicken/Henan/62/00A/chicken/Henan/26/00




A/chicken/Guangdong/SQL10/2011







A/chicken/Guangdong/LFQ12/2011

A/chicken/Hebei/31/00A/chicken/Hebei/274/2004


A/chicken/Jiawang/2/2011







A/chicken/Shandong/qd0307/2013A/chicken/Jiangsu/WJHRG/2012

A/Chicken/Guangdong/SS/94A/chicken/Guangdong/SS/94




A/chicken/Jiangsu/CZYWP/2012A/chicken/Jiangsu/MYJMF/2012

A/Shanghai/4/2013








A/Shanghai/2/2013







A/chicken/Jiangsu/XZLZG/2012A/chicken/Jiangsu/Q3/2010





A/Shanghai/02/2013



A/chicken/Rizhao/853/2013A/chicken/Shandong/yt0711/2013

A/chicken/Shandong/SDWF17/2012A/chicken/Yongcheng/1/2011














A/chicken/Jiangsu/YCDT/2013




A/chicken/Shandong/SG2/2009A/chicken/Shandong/01/2009







A/chicken/Guangdong/ZCY/2011A/Chicken/Shandong/09/2011



A/chicken/Xiangshui/1/2011A/chicken/Anhui/10/2009



A/Shanghai/3/2013


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A/chicken/Hebei/0721/2013A/chicken/Tianjin/614/2012






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A/chicken/China/AH-10-01/2010A/chicken/Jiangsu/CZLJG/2012


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A/Chicken/Nanjing/023/2013A/chicken/Shanghai/C3/2012











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A/chicken/Wenzhou/614/2013A/chicken/Beijing/324/2013

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A/Chicken/Shandong/05/2011A/chicken/Jiangsu/YCDT/2013






A/chicken/Rizhao/1313/2013A/chicken/Rizhao/723/2013







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A/chicken/Hangzhou/50-2/2013A/chicken/Shuanggou/1/2011



A/chicken/Guangxi/1857/2004A/chicken/Kaifeng/A1/2005


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A/chicken/Jiangsu/YZ0611/2011A/chicken/Shandong/qd0427/2012






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A/Chicken/Shijiazhuang/2/99A/chicken/Qixian/A1/2001




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A/chicken/Shandong/317/2007A/chicken/Shandong/WL/2009












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A/chicken/Zhejiang/NH5/2013

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A/chicken/Beijing/1115/20135.0

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5.0 Fig. 3. Phylogenetic analysis of H9N2 influenzaviruses isolated from chickens during 1994–2013 andH7N9 viruses isolated from humans in China duringthe first 2 wk of the 2013 outbreak. (A) Phylogenetictrees of surface genes with the H9N2 chicken virusesand those of internal genes with the chicken H9N2and the novel H7N9 human viruses. Internal genetrees with only H9N2 viruses are not shown here dueto the same topology. Clades with most of 2010–2013 viruses were fully shown, and others werecollapsed (indicated by gray triangles). Color of lineat right of each leaf node indicates year of isolation(see color bar). Timescale is in years. Vertical blacklines mark major (long line) and minor (short line)groups of H9N2 viruses, mainly from 2010 through2013. Black arrows mark the position of H7N9 viru-ses. Also see Fig. S2. (B) Diversity of genotypes ofH9N2 viruses isolated from chickens in China, 1994–2013. Here are shown the recurrent genotypes thatwere persistent across at least 2 y. All of the geno-types can be found in Dataset S1. (C) Summary ofthe genesis of genotype G57 by reassortmentevents. Virus particles are represented by ovals. Theeight gene segments are horizontal bars (from thetop: PB2, PB1, PA, HA, NP, NA, M, and NS). Redsegments are clades of G57; and blue segments areother clades. A broken arrow indicates an inter-species gene transmission. Ck/BJ/94, A/chicken/Beijing/1/1994; Qa/HK/97, A/quail/HongKong/G1/1997; Ck/SH/98, A/chicken/Shanghai/F/1998; Ck/JS/00, A/chicken/Jiangsu/1/2000; Ck/HuN/05, A/chicken/Hunan/5260/2005; Dk/ST/04, A/duck/Shantou/163/2004; Ck/ZJ/07,A/chicken/Zhejiang/HJ/2007 (G57 genotype). AnotherG57 virus (A/chicken/Jiangxi/12/2007) detected in 2007is not shown.


MICRO

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GY


(Fig. S3). We demonstrated how selection and enrichment of thefittest G57 H9N2 genotype in farm chickens and explained theacquisition of this genotype’s internal genes by the novel H7N9viruses. The prevalence and variation of H9N2 influenza virus infarmed poultry could provide an important early warning of theemergence of novel reassortants with pandemic potential.

Materials and MethodsEthics Statement.All animal research was approved by the Beijing Associationfor Science and Technology (approval ID SYXK, Beijing, 2007–0023) andperformed in compliance with the Beijing Laboratory Animal Welfare andEthics guidelines, as issued by the Beijing Administration Committee ofLaboratory Animals, and in accordance with the China Agricultural Univer-sity (CAU) Institutional Animal Care and Use Committee guidelines (ID:SKLAB-B-2010-003) approved by the Animal Welfare Committee of CAU.

Virus Isolation and Identification. From January 2010 to December 2013,chicken farms in 19 provinces of China (Anhui, Beijing, Guangdong, Guangxi,Fujuan, Hebei, Heilongjiang, Henan, Hubei, Jiangsu, Jiangxi, Jilin, Liaoning,Neimenggu, Shaanxi, Shandong, Sichuan, Tianjin, and Xinjiang) sent chickenswith disease signs or lung tissues from deceased chickens to our laboratoryfor diagnosis. The chicken flocks that were sampled were reported to exhibitovert respiratory illness and/or a 5–20% reduction in egg production, despitehaving received H9N2-subtype influenza vaccination. For virus isolation,tracheal swabs were taken and placed in 1.0 mL transmission medium [50%(vol/vol) glycerol in PBS] containing antibiotics, as previously described (12).Lungs were homogenized in PBS containing antibiotics. Samples werecentrifuged at 1,000 × g for 10 min at 4 °C, and 0.2 mL of the supernatants wasinoculated into the allantoic cavities of 10-d-old SPF embryonated chicken eggsand incubated for 48 h at 37 °C. Allantoic fluid was harvested and tested for HAactivity. HA-positive samples were identified serologically with antisera to eachknown influenza virus subtype, as previously described (20). H9N2-containingallantoic fluid was harvested and stored as stock at−80 °C until use. Usually,

several chickens or samples were sent from one farm to the laboratory at onetime. If one or more than one H9N2 virus was isolated, the farm was noted as anH9N2-positive farm. The virus isolation rate was determined by dividing thenumber of H9N2-positive farms by the total number of farms surveyed.

Genomic Sequencing. H9N2 viruses (n = 76) isolated from 2010 through 2013were selected for sequencing on the basis of isolation time and location. Atleast one strain per province per month was sequenced; if there were mul-tiple isolates in a single month, additional viruses were selected according tolocation. H9N2 viruses (n = 11) isolated during 2008–2009 from diseased,vaccinated farm chickens were also sequenced genomically. Viral RNA wasextracted by using the QIAamp Viral RNA Minikit (Qiagen), and standardRT-PCR was performed with primers specific for H9N2 influenza virus, usingthe One Step RT-PCR Kit (Qiagen). Full genomic DNA was sequenced by ei-ther Sanger sequencing on an ABI 3730 genetic analyzer (Applied Bio-systems) or high-throughput next-generation sequencing (Ion torrent).

Sequence Collection and Alignment.All previously published sequences of ChineseH9N2 influenza A virus (1994–2013) and relative sequences were collated fromInfluenza Research Database (FluDB) (www.FluDB.org), Global Initiative onSharing Avian Influenza Data (www.gisaid.org), and the Influenza Virus Re-source at the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/genomes/FLU). All replicate submissions were removed by identi-fying sets of isolates with identical sequences for any segment, with each isolatecontaining one to eight segments. All sequences of H7N9 influenza A virusespublished before April 2014 were acquired from the above databases andcombined with the sequences generated in this study (Table S3) for furtheranalysis. All of the accession codes of public sequences can be found in Table S4and Dataset S2. The resulting sequences of each gene segment were alignedusing MAFFT v6 (32), manually adjusted to correct frame shift errors, and sub-sequently translated. Downstream phylogenetic analyses were performed onregions of the alignments containing few gaps across sequences. These regionsconsist of the following intervals: PB2, 1,168–2,199; PB1, 61–1,437; PA, 796–2,103; HA, 190–1,563; NP, 67–1,014; NA, 91–1,272; MP, 88–948; and NS, 79–807.

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Fig. 4. Infection of H9N2 chicken viruses in unvaccinated and vaccinated chickens. Nine representative H9N2 chicken viruses were selected from G57 or othergenotypes to test their infection in inoculated and contact chickens. As shown in the figure, from left to right on each x axis, they are A/chicken/Beijing/3/1999(G02); A/chicken/Hebei/0617/2007 (G51); A/chicken/Shandong/22/2008 (G49); A/chicken/Shandong/ZB/2007 (G60); A/chicken/Jiangsu/TS/2010 (G57); A/chicken/Hebei/YT/2010 (G57); A/chicken/Shandong/sd01/2010 (G57); A/chicken/Shandong/06/2011 (G57); and A/chicken/Guangdong/01/2011 (G68). Chickens (n =10 per group) were inoculated intranasally with 1× 106 EID50 of each virus to test viral replication. After 24 h, five chickens were placed in physical contactwith inoculated birds to test viral transmission. Tracheal (red dashed line) and cloacal (green dashed line) swabs were collected at 3, 5, and 7 dpi for virusdetection and/or titration. All of the infection experiments were carried out on unvaccinated (Top) and vaccinated chickens (Middle, with antibody titers of 6–9; Bottom, with antibody titers of 10–12). Antibody titers were expressed as the log2 reciprocal of the end point in HI test. The dashed lines show the dif-ferences of infection among different genotype viruses but have no meanings for the real connection with these genotypes. (A) Isolation rates of H9N2viruses in chickens. (B) Mean titers of H9N2 viruses at 3 dpi. Error bars are SD. The titers of the G57/G68 viruses were significantly higher than those of othergenotypes of the earlier viruses (*P < 0.05, **P < 0.01, one-way ANOVA). (C) The viral shedding time of H9N2 viruses.

552 | www.pnas.org/cgi/doi/10.1073/pnas.1422456112 Pu et al.


http://www.FluDB.org

http://www.gisaid.org

http://www.ncbi.nlm.nih.gov/genomes/FLU

http://www.ncbi.nlm.nih.gov/genomes/FLU





Phylogenetic Analysis and Clade Classification. RAxML was used to constructmaximum likelihood phylogenies for each segment (33). The general-time re-versible model of nucleotide substitution was assumed, and the rates were as-sumed to vary according to the discrete Gamma distribution with four ratecategories. Two sets of trees were constructed: one set including all H9N2 iso-lates from all hosts since 1994 isolated from China and Hong Kong (all segments)and one set additionally including all H7N9 isolates from all hosts from Chinaand Hong Kong (all six internal segments). One hundred bootstrap maximum-likelihood (ML) trees were used to construct a majority rule consensus tree fromeach bootstrap sample using sumtrees (34). Bayesian phylogenetic trees wereconstructed for each segment using Bayesian Evolutionary Analysis SamplingTrees Software (BEAST) (35). Themodel of substitution and rate variation in eachtree was the same as in the corresponding RAxML tree, with the additionalassumption of a log-normally distributed molecular clock. BEAST trees wereinitiated with the RAxML majority rule consensus tree to reduce the computa-tion time required for the MArcov Chain Monte Carlow (MCMC) sampler toreach the stationary distribution. Maximum clade credibility (MCC) trees wereconstructed using 2,000 equidistant samples from the final 30% of the posteriorsample of trees (n = 25 × 106 total samples) using a python script and tree an-notator (35). The resulting MCC trees were partitioned into clades using a me-dian branch length distance threshold of 0.20 (24). Briefly, clades with medianroot-to-tip distances equal to or less than 20% of the median root-to-tip dis-tances of all clades in the tree were selected as clusters. Clades were thenmanually merged, if necessary, based on the branching posterior and reportedclassification (19, 20, 25). Each clade was assigned a unique clade ID.

Genotypic Analysis. Virus genotypes were analyzed in H9N2 viruses isolatedfrom chickens in China during 1994–2013. Isolates were genotyped ifsequences and clade assignments were available for all eight segments. Thegenotypes of the resulting isolates were determined by the combination ofclade assignments of each of the eight segments. Genotype IDs wereassigned according to the isolation time of the initial founder isolate.

HI Assay. The HI assay was used to antigenically characterize the H9N2 virusesisolated in China in this and previous studies. The assay used chicken antiserato two representative H9N2 vaccine strains (A/chicken/Shandong/6/1996 andA/chicken/Shandong/F/1998). The HI assay was performed as previously

described (36), with an initial serum dilution of 1:10. The HI titer wasexpressed as the reciprocal of the highest serum dilution in which hemag-glutination was inhibited.

Virus Infection in Chickens. These experiments were designed as previously de-scribed (19, 20) and performed in unvaccinated and vaccinated chickens. Three-week-old SPF white leghorn chickens were vaccinated s.c. once with A/chicken/Shandong/6/1996-based commercial vaccine (HA content, 10 log2). HI titers tohomologous antigen were measured in twofold serum dilutions 3 wk after in-oculation and expressed as the log2 reciprocal of the end point. The vaccinatedchickens were divided into two groups for infection experiments: those witha low antibody level (HI titer, 6–9) and those with a high antibody level (HI titer,10–12). Six-week-old SPF white Leghorn chickens were used as the unvaccinatedgroup. Nine H9N2 viruses (Fig. 4) were selected to inoculate chickens. In in-oculated groups, birds (n = 10 per group) were inoculated intranasally with 106

EID50 (egg infective dose at which 50% of inoculated eggs are infected) of eachvirus in a 0.2-mL volume. Tracheal and cloacal swabs were collected at 3, 5, and7dpi. The virus titers of positive samplesweremeasuredbydetermining theEID50;the lower limit of detectionwas 1.0 log10 EID50/mL In contact groups, at 24 h afterinfection, five chickens were placed in physical contact (i.e., in the same cage,sharing food and water) with inoculated birds. Tracheal and cloacal swabs werecollected at 3, 5, and 7 dpi for viral detection and titration, as described above.

Statistical Analysis. Experimental groups were statistically compared byANOVA. P < 0.05 was considered to indicate a statistically significant difference.

ACKNOWLEDGMENTS. We thank Furkat Anwar for assistance with animalexperiments; Robert Huether and Amber Smith for helpful suggestions ondata analysis; and Sharon Naron, Kin-chow Chang, and Lu Lu for greateditorial assistance. This work was supported by National Natural ScienceFoundation of China Grants 31430086 and 31272535; National Basic Re-search Program (973 Program) Grant 2011CB504702; the China ScholarshipFoundation; Key Technologies Research and Development Program of ChinaGrant 2013BAD12B01; the China Agriculture Research System, Poultry-Related Science and Technology Innovation Team of Peking; National In-stitute of Allergy and Infectious Disease Contract HHSN266200700005C; andthe American Lebanese Syrian Associated Charities.

1. Gao R, et al. (2013) Human infection with a novel avian-origin influenza A (H7N9)virus. N Engl J Med 368(20):1888–1897.

2. World Health Organization (2014) Human infections with avian influenza A(H7N9)virus. Available at www.who.int/influenza/human_animal_interface/influenza_h7n9/140225_H7N9RA_for_web_20140306FM.pdf?ua=1. Accessed December 9, 2014.

3. Liu D, et al. (2013) Origin and diversity of novel avian influenza A H7N9 virusescausing human infection: Phylogenetic, structural, and coalescent analyses. Lancet381(9881):1926–1932.

4. Lam TT, et al. (2013) The genesis and source of the H7N9 influenza viruses causinghuman infections in China. Nature 502(7470):241–244.

5. Wu A, et al. (2013) Sequential reassortments underlie diverse influenza H7N9 geno-types in China. Cell Host Microbe 14(4):446–452.

6. Guo YJ, et al. (2000) Characterization of the pathogenicity of members of the newlyestablished H9N2 influenza virus lineages in Asia. Virology 267(2):279–288.

7. Chen B, Zhang ZJ, Chen WB (1994) Isolation and identification of avian influenzavirus. Chin J Vet Med 20(10):3–5.

8. Li C, et al. (2005) Evolution of H9N2 influenza viruses from domestic poultry inMainland China. Virology 340(1):70–83.

9. Dong G, et al. (2011) Reassortant H9N2 influenza viruses containing H5N1-like PB1genes isolated from black-billed magpies in Southern China. PLoS ONE 6(9):e25808.

10. Bi J, et al. (2010) Phylogenetic and molecular characterization of H9N2 influenzaisolates from chickens in Northern China from 2007-2009. PLoS ONE 5(9):e13063.

11. Choi YK, et al. (2004) Continuing evolution of H9N2 influenza viruses in SoutheasternChina. J Virol 78(16):8609–8614.

12. Zhang P, et al. (2008) Characterization of H9N2 influenza viruses isolated from vac-cinated flocks in an integrated broiler chicken operation in eastern China during a5 year period (1998-2002). J Gen Virol 89(Pt 12):3102–3112.

13. Wu ZQ, et al. (2010) Cloning and phylogenetic analysis of hemagglutinin gene ofH9N2 subtype avian influenza virus from different isolates in China during 2002 to2009. Poult Sci 89(6):1136–1143.

14. Chen F, et al. (2012) Phylogenetic analysis of hemagglutinin genes of 40 H9N2 sub-type avian influenza viruses isolated from poultry in China from 2010 to 2011. VirusGenes 45(1):69–75.

15. Huang Y, et al. (2010) Diversified reassortant H9N2 avian influenza viruses in chickenflocks in northern and eastern China. Virus Res 151(1):26–32.

16. Liu H, et al. (2003) Phylogenetic analysis of the hemagglutinin genes of twenty-sixavian influenza viruses of subtype H9N2 isolated from chickens in China during 1996-2001. Avian Dis 47(1):116–127.

17. Sun Y, et al. (2012) Evaluation of the protective efficacy of a commercial vaccineagainst different antigenic groups of H9N2 influenza viruses in chickens. Vet Micro-biol 156(1-2):193–199.

18. Zhang Y, et al. (2012) Molecular and antigenic characterization of H9N2 avian in-fluenza virus isolates from chicken flocks between 1998 and 2007 in China. Vet Mi-crobiol 156(3-4):285–293.

19. Zhang P, et al. (2009) A novel genotype H9N2 influenza virus possessing human H5N1internal genomes has been circulating in poultry in eastern China since 1998. J Virol83(17):8428–8438.

20. Sun Y, et al. (2010) Genotypic evolution and antigenic drift of H9N2 influenza virusesin China from 1994 to 2008. Vet Microbiol 146(3-4):215–225.

21. Rambaut A, et al. (2008) The genomic and epidemiological dynamics of human in-fluenza A virus. Nature 453(7195):615–619.

22. McHardy AC, Adams B (2009) The role of genomics in tracking the evolution of in-fluenza A virus. PLoS Pathog 5(10):e1000566.

23. Boni MF (2008) Vaccination and antigenic drift in influenza. Vaccine 26(Suppl 3):C8–C14.

24. Prosperi MC, et al.; ARCA collaborative group (2011) A novel methodology for large-scale phylogeny partition. Nat Commun 2:321.

25. Xu KM, et al. (2007) The genesis and evolution of H9N2 influenza viruses in poultryfrom southern China, 2000 to 2005. J Virol 81(19):10389–10401.

26. Zhang P, et al. (2008) Characterization of H9N2 influenza viruses isolated from vac-cinated flocks in an integrated broiler chicken operation in eastern China duringa 5 year period (1998-2002). J Gen Virol 89(Pt 12):3102–3112.

27. Moya A, Holmes EC, González-Candelas F (2004) The population genetics and evo-lutionary epidemiology of RNA viruses. Nat Rev Microbiol 2(4):279–288.

28. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y (1992) Evolution andecology of influenza A viruses. Microbiol Rev 56(1):152–179.

29. Cui L, et al. (2014) Dynamic reassortments and genetic heterogeneity of the human-infecting influenza A (H7N9) virus. Nat Commun 5:3142.

30. Nelson MI, Holmes EC (2007) The evolution of epidemic influenza. Nat Rev Genet 8(3):196–205.

31. Cox NJ, Subbarao K (2000) Global epidemiology of influenza: Past and present. AnnuRev Med 51:407–421.

32. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: A novel method for rapid multiplesequence alignment based on fast Fourier transform. Nucleic Acids Res 30(14):3059–3066.

33. Stamatakis A (2014) RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9):1312–1313.

34. Sukumaran J, Holder MT (2010) DendroPy: A Python library for phylogenetic com-puting. Bioinformatics 26(12):1569–1571.

35. Bouckaert R, et al. (2014) BEAST 2: A software platform for Bayesian evolutionaryanalysis. PLOS Comput Biol 10(4):e1003537.

36. Edwards S (2006) OIE laboratory standards for avian influenza. Dev Biol (Basel) 124:159–162.


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http://www.who.int/influenza/human_animal_interface/influenza_h7n9/140225_H7N9RA_for_web_20140306FM.pdf?ua=1

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Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus

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