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

Juan 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. Jude Children’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 influenza evolution of H9N2 viruses mainly focusing on farm chickens and virus in China raises a pandemic concern. Chicken H9N2 viruses their role in the genesis of the novel H7N9 viruses. provided all six of the novel reassortant’s internal genes. How- ever, it is not fully understood how the prevalence and evolution Results of these H9N2 chicken viruses facilitated the genesis of the novel Increased Isolation of H9N2 Viruses from Farm Chickens Preceded the H7N9 viruses. Here we show that over more than 10 y of cocircu- Outbreak of H7N9 Infection in Humans. We retrospectively in- lation of multiple H9N2 genotypes, a genotype (G57) emerged vestigated the infection of farm chickens with H9N2-subtype in- that had changed antigenicity and improved adaptability in chick- fluenza viruses, mainly in northeast China, from January 2010 to ens. It became predominant in vaccinated farm chickens in China, December 2013 (Fig. 1 and Table S1). Tracheal swabs or lung caused widespread outbreaks in 2010–2013 before the H7N9 samples were taken for virus isolation and identification from 1975

viruses emerged in humans, and finally provided all of their in- chicken farms reporting illness in 19 provinces during 2010–2013. MICROBIOLOGY ternal genes to the novel H7N9 viruses. The prevalence and vari- All of these chickens had been vaccinated against H9N2 virus but ation of H9N2 influenza virus in farmed poultry could provide an showed respiratory disease signs and/or a 5–20% reduction in egg important early warning of the emergence of novel reassortants production. Of the 1975 farms, 637 were positive for H9N2 in- with pandemic potential. fection, with a virus isolation rate as high as 32.25% (Table S1). Mean H9N2 isolation rates increased annually, from 22.08% in H9N2 | H7N9 | chicken influenza virus | genotype | infectivity 2010 to 47.08% in 2013. Further, these H9N2 infections occurred even during the warm season (July through September), when uman infection with a novel avian-origin H7N9 influenza A influenza infection is usually reported less often (Fig. 1). Despite Hvirus causing severe respiratory symptoms and mortality was vaccination, H9N2 virus infection in the investigated regions in- first reported in eastern China in March 2013 (1). To date, the creased dramatically after 2010, indicating an antigenic variant novel virus has caused two outbreaks of human infection, in- cluding 375 known cases and 115 deaths as of 11 March 2014 (2). Significance Phylogenetic analysis suggests that the virus is a triple reassortant of H7, N9, and H9N2 avian influenza viruses (3, 4). The H7 and The emergence of human infection with a novel H7N9 avian N9 genes may have been transferred from migratory birds to influenza reassortant in China raises a pandemic concern. domestic ducks and then to chickens in the live poultry markets However, it is not fully understood how these H9N2 chicken (3–5), after which reassortment with enzootic H9N2 viruses – viruses facilitated the genesis of the novel H7N9 viruses. formed the H7N9 viruses identified in humans (3 5). Here we show that a “fittest” genotype (G57) emerged with H9N2 influenza virus has low pathogenicity for avians, repli- changed antigenicity and improved adaptability in chickens. It cating mainly in the upper respiratory tract and causing mild or became predominant in vaccinated farm chickens and caused no overt signs of illness in specific pathogen-free (SPF) chickens widespread outbreaks before the H7N9 virus emergence, in- (6). In 1994, the H9N2 subtype was first identified in chicken creasing reassortment between H9N2 and other subtype viru- farms in the Guangdong province of south China (7); it has since ses and finally providing all of their internal genes to the novel become widespread in chickens and has caused great economic H7N9 viruses. The prevalence and variation of H9N2 in- loss from reduced egg production and highly lethal coinfections fluenza virus in farmed poultry could provide an important (8–11). To reduce the impact of H9N2 infection in chickens, the early warning of the emergence of novel reassortants with flocks have been vaccinated since 1998 with commercial inacti- pandemic potential. 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/ Author contributions: J.P., G.Z., J.Z., and J.L. designed research; J.P., Y.Y., G.Z., J.W., G.X., Shanghai/F/1998 (Ck/SH/F/98) (8, 12, 13). These H9N2 vaccines 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; and initially limited the outbreaks and virus spread. However, despite J.P., S.D., G.W., J.L., and R.G.W. wrote the paper. multiple doses, the H9N2 vaccines became less effective, espe- The authors declare no conflict of interest. cially after 2007, and H9N2 influenza virus continues to circulate Data deposition: The sequences generated in this study have been deposited in the Gen- in vaccinated chicken flocks and has caused sporadic disease bank database (accession nos. are listed in Table S3). – outbreaks (8, 10, 12 20). However, the recent prevalence and 1J.P., S.W., and Y.Y. contributed equally to this work. molecular evolution of the H9N2 viruses in chickens especially 2 To whom correspondence may be addressed. Email: [email protected] or robert.webster@ in the flocks receiving large-scale vaccination, and their role in stjude.org. the emergence of human H7N9 virus, are not fully understood. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. In this study, we systematically investigated the prevalence and 1073/pnas.1422456112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1422456112 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 provinces in 2007, and its prevalence increased sharply during 60 2009. Since 2010, G57 has been the predominant genotype in Apr Aug Jan Feb Mar May Jun Jul Sep Oct Nov Dec circulation 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). The 40 G57 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- 20 duced in a chicken virus, A/chicken/Shanghai/F/1998 (SH/98), which caused a severe outbreak in chickens in eastern China Isolation rate (%) (26). After 1998, the SH/98-like virus reassorted with the A/quail/Hong Kong/G1/1997-like and A/chicken/Jiangsu/1/2000- 0 like viruses, introducing matrix (M) and the HA genes to gen- 2010 2011 2012 2013 erate the A/chicken/Hunan/5260/2005-like virus in 2005. The Fig. 1. Isolation rate (%) of H9N2 influenza viruses during 2010–2013 on virus had seven of the eight gene segments of the G57 genotype. chicken farms reporting illness in our investigated regions. Histogram indicates Then the final segment, PB2, was transmitted from ducks monthly isolation rates; red horizontal line (connected with dots) indicates (A/duck/Shantou/163/2004-like) to chickens, where it formed the mean annual isolation rates; and gray horizontal dashed line indicates 95% CI. predominant G57 genotype in 2007. Since 2007, G57 viruses have spread to domestic aquatic birds, wild birds, and swine (Dataset S1); in 2014, this genotype was isolated from a human patient in Hong caused an outbreak in farm chickens that was maximal before and Kong (Dataset S1), indicating a wide range of infected hosts. during the 2013 emergence of the novel H7N9 virus in humans. Clearly, the widespread outbreak in 2010–2013 established the single dominant G57 genotype in chickens, ending more than – Reduced Genetic Diversity of H9N2 Viruses in Chickens During 2010 10 y of cocirculation of multiple genotypes and producing nu- 2013 Indicates Widespread Outbreak. Widespread influenza out- merous H9N2 viruses carrying the same gene constellation. breaks caused by antigenic drift variants are usually thought to be characterized by reduced genetic diversity of the viruses, either Improved Infectivity of G57 H9N2 Viruses in Chickens. Reassortment – genome-wide or in the hemagglutinin (HA) segment (21 23). seems advantageous because it can rapidly create high fitness Thus, a widespread outbreak can be reasonably inferred by high genotypes (27). To determine the effect of reassortment on the sequence similarity of viruses isolated during a specified time span infectivity of G57, we selected nine representative H9N2 chicken across a broad region. We wondered whether other regions be- viruses from G57 or other genotypes to test their replication in yond our investigation were also affected. To address this question, inoculated and contact chickens (Fig. 4, Top, A–C). Specifically, we analyzed the temporal dynamics of genome-wide sequence four 2010–2011 viruses were from G57, one virus was from G68 similarities of H9N2 viruses by collecting all available H9N2 (only with different HA segment compared with G57) isolated in sequences from chicken viruses in China during 1994 through 2011, and four viruses were from other major genotypes circu- 2013. Pairwise identity comparison showed a decrease in diversity lating before 2010. After inoculation, although all of the tested in all segments of these viruses, starting mainly in 2009–2010 and viruses had a 100% isolation rate in tracheas (Fig. 4A), most of reaching a peak during 2010–2013 (Fig. 2 and Fig. S1). Somewhat the G57 viruses replicated to significantly higher titers than did less similarity was found in the HA genes of 2011 viruses and the the earlier viruses of other genotypes (titers 10–100 times as neuraminidase (NA) genes of 2013 viruses. The decreased se- high; P < 0.05; Fig. 4B). The G57 viruses also showed clearly quence diversity in almost all segments at approximately the same enhanced replication in the cloaca, to titers 10–1,000 times as high time point suggests that a virus with the greatest fitness was se- as those of earlier isolates (Fig. 4B), explaining the 100% de- lected, gained dominance, and caused a widespread outbreak in tection rate of all inoculated G57 viruses but only 20–60% of in- chickens throughout China. A genetic “bottleneck” is proposed in oculated earlier viruses (Fig. 4A). In addition, most of the G57 influenza virus evolution (22). This is the first observation, to our viruses were shed from the cloaca for a longer period [until 7 days knowledge, of such a bottleneck in the H9N2 viruses (Fig. 2 and postinfection (dpi); Fig. 4C]. The replication ability of G68 virus Fig. S1), despite their prevalence in China for the last 20 y. was moderate between G57 viruses and other genotypes of earlier viruses (Fig. 4 A–C). Similar to its replication in inoculated Genotype G57 of H9N2 Influenza Virus Became Predominant in chickens, G57 exhibited improved infection with higher viral titers Chickens Since 2010. To understand the phylogenetic evolution also in contact chickens followed by the G68 virus, although there of the prevalent H9N2 viruses, we constructed phylogenetic trees was a 100% isolation rate among different genotypes except for for all of the eight segments. Phylogenetic trees were automati- G02 in the contact chickens. (Fig. 4 A–C). These results suggest cally partitioned into clusters (24) using a 20th percentile cutoff. this improved adaptation in chickens may be one reason for the Clades were then manually merged, if necessary, based on the widespread outbreak of G57 H9N2 viruses during 2010–2013. branching posterior and reported classification (19, 20, 25). In each tree of eight segments, most of the 2010–2013 viruses Antigenic Drift of G57 H9N2 Viruses Isolated Since 2010. Antigenic belonged to the same clade (Fig. 3A and Fig. S2). Within each drift, induced mainly in the HA glycoprotein, can decrease a prevalent clade, the majority of 2010–2013 viruses further formed vaccine’s efficacy and cause outbreaks of viral infection (21–23, a major group with few early viruses (Fig. 3A and Fig. S2), in- 28). Phylogenetic analysis revealed that the 2010–2013 H9N2 dicating they shared the common ancestor surviving the bottle- viruses were mainly in clade 6 and clade 9, whereas the vaccine neck event. In the polymerase basic 2 (PB2), NP (nucleoprotein), strains were in clade 2 (Fig. S2). To determine whether antigenic HA, NA, and nonstructural (NS) genes, a minor population of the drift had been induced in the prevalent G57 H9N2 viruses, we prevalent viruses formed a different clade (HA) or minor group investigated this possibility by hemagglutinin inhibition (HI) test- (HA and other segments). On the basis of clade classification, 69 ing of 26 representative H9N2 viruses. As the outbreak of H9N2 genotypes were found in H9N2 chicken viruses in China from influenza in chickens occurred mainly after 2010 and the H7N9 1994 through 2013 (Dataset S1). Most of the 2010–2013 viruses viruses are estimated to have emerged in 2011–2012 (4, 12), we werefromasinglegenotypeclassifiedasgenotype57(G57).G57 selected viruses from 2010 to 2011 for HI testing, on the basis of virus was first detected in chickens in Jiangxi and Jiangsu their HA phylogenic topology. Some early representative viruses

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1422456112 Pu et al. Downloaded by guest on September 28, 2021 1994 1994 HA NA 100 chickens. The vaccinated chickens were divided into groups with low antibody levels (HI titer, 6–9) and high antibody levels (HI titer, 10–12). Nine viruses used in the above unvaccinated ≤90 chicken experiment (Fig. 4 A–C, Upper) were also selected in the vaccinated chicken study because they are also the representa- tives based on the HA lineage (Fig. S2) and HI reaction (Table S2). In the inoculated vaccinated chickens, the G57 viruses from 2010/2011 consistently maintained their growth in trachea, with A 2013 2013 a 100% isolation rate regardless of antibody level (Fig. 4 ). However, the replication of early viruses from other genotypes was clearly inhibited, as shown by a 0–70% isolation rate (except 1994 2013 1994 2013 for A/chicken/Shandong/ZB/2007, ZB/07) in chickens with low 1994 1994 antibody levels (Fig. 4, Middle) and a 0–30% rate in those with PB2 PB1 high antibody levels (Fig. 4A, Bottom). ZB/07 was an antigenic variant identified previously (17). After placing vaccinated chickens in 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 low rate (40%), in chickens with high antibody levels (Fig. 4A). Al- though all of the G57 viruses maintained 100% replication and 2013 2013 transmission, their titers in the trachea and cloaca decreased but remained significantly higher (P < 0.05 or P < 0.01, ANOVA) 1994 2013 1994 2013 than those of the early viruses (Fig. 4B). By comparison of their 1994 1994 shedding time (Fig. 4C), G57 viruses had longer shedding time PA NP until 7 dpi in contact vaccinated chickens with high antibody titers (10–12). Although the shedding time of ZB/07 was similar

with those of G57 viruses, the numbers of H9N2-positive chickens MICROBIOLOGY was 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 was detected (Fig. 4, Middle and Bottom), similar to that observed in unvaccinated chickens (Fig. 4, Top). One G68 virus showed a 2013 2013 moderate level in replication and transmission between G57 and other earlier genotype viruses (Fig. 4). These results suggest that 1994 2013 1994 2013 recent antigenic variants of G57 viruses possessed the fitness to 1994 1994 escape immunization pressure and cause the widespread out- M NS break of H9N2 infection in chickens in 2010–2013.

G57 H9N2 Chicken Virus Provided All Six of Their Internal Genes to the Novel H7N9 Viruses. Previous studies demonstrate that all six of the internal genes of the novel H7N9 viruses are from H9N2 chicken viruses circulating in China (3–5). To determine whether these internal genes were all from the clades of G57 genotype 2013 2013 viruses, we constructed phylogenetic trees of all internal genes of the available 1994–2013 H9N2 chicken viruses and of the H7N9 human viruses isolated during the first 2 wk of the 2013 H7N9 1994 2010 2013 1994 2010 2013 outbreak. Later H7N9 isolates were eliminated to better identify Fig. 2. Reduced genetic diversity of the eight segments of H9N2 influenza the cause of the initial outbreaks because these viruses un- viruses isolated from chickens during the widespread outbreaks in China (2010– derwent further gene exchange with local H9N2 viruses during 2013). Pairwise comparison of nucleotide sequence of all H9N2 chicken viruses is viral transmission after that time point (29). The six internal plotted as a heatmap, if a given pair of sequences shares more than 50% of the genes of the H7N9 human isolates grouped closely with those of full length. Viruses isolated from 1994 through 2013 are ordered by isolation the recent 2010–2013 H9N2 chicken viruses as shown in previous time from left to right on the x axis and from top to bottom on the y axis. Ticks studies (3–5), and all belonged to the clades of the G57 genotype on the axes indicate the isolation years. Red dotted line indicates the year 2010. (Fig. 3A and Fig. S2). Specifically, the internal genes of the initial Color indicates identity levels from ≤90% (red) to 100% (blue) or identity levels unavailable due to the shared sequence length less than 50% (white). H7N9 isolates were the most similar to those of the major groups of H9N2 G57 viruses, with the exception of the NS gene. The NS genes of H7N9 human isolates belonged to a minor group that from different antigenic groups were included (20), as were two included H9N2 G57 viruses isolated mainly in the Jiangsu and representative vaccine strains as controls (Ck/SD/6/96 and Ck/SH/ Zhejiang provinces nearby Shanghai, where the first outbreaks of F/98; Table S2). The G57 viruses prevalent in 2010–2011, as well human H7N9 infection occurred. Eleven novel reassortants of as some earlier viruses (mainly 2009), had lower HI titers eight or subtypes other than H7N9 (e.g., H5N2, H7N7, and H10N8 viru- more times the HI titers of the vaccine strains in reactions with ses) isolated in avian, swine, or humans, mainly after 2010, also vaccine antisera. These results demonstrated that the recent possessed G57-like internal genes (Dataset S1). H9N2 viruses were antigenically distinct from the vaccine strains. To determine whether antigenic drift had reduced the pro- Discussion tective effect of vaccines in the field, we compared the infective The segmented genome feature of influenza viruses allows reas- efficiency of H9N2 viruses in vaccinated inoculated and contact sortment of segments from different viruses, generating novel

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A/chicken/Anhui/G20/2008A/chicken/Guangdong/V13/2009A/chicken/Jilin/63/2009A/chicken/Guangdong/B87/2009A/chicken/Guangdong/P27/2009A/chicken/Guangdong/R24/2009A/chicken/Guangdong/N4/2009A/chicken/Guangdong/R11/2009A/chicken/Guangdong/Y12/2009 A/chicken/Jilin/P23/2009A/chicken/Jilin/8/2009A/chicken/Hubei/B23/2009A/chicken/Hubei/N14/2009A/chicken/Anhui/10/2009A/chicken/Tianjin/F2/2009A/chicken/Hubei/J4/2009A/chicken/Guangdong/B106/2009A/chicken/Hunan/U6/2010 A/Chicken/Henan/DY/2009A/chicken/Jilin/BHZ/2010A/chicken/Jilin/XJJZ/2010A/chicken/Henan/F17/2010A/chicken/Jilin/CBS/2010A/chicken/Henan/A16/2010A/chicken/Jilin/BHJ/2010A/chicken/Henan/JZ/2011A/chicken/Henan/LH/2012 A/chicken/Henan/E2/2010A/chicken/Henan/F20/2010A/chicken/Henan/F14/2010A/chicken/Henan/F21/2010A/chicken/Henan/F18/2010A/chicken/Henan/E1/2010A/chicken/Ningxia/153/2011A/chicken/Ningxia/04/2011A/chicken/Ningxia/31/2011 A/chicken/Hunan/HA17/2007A/chicken/Hunan/HB5/2007A/chicken/Hunan/HB25/2007A/chicken/Anhui/AI16/2008 A/chicken/Ningxia/99/2011A/chicken/Ningxia/113/2011A/chicken/Ningxia/47/2011A/chicken/Ningxia/112/2011A/chicken/Ningxia/102/2011 A/chicken/Hunan/HB29/2007A/chicken/Anhui/AK25/2008A/chicken/Anhui/AH16/2008A/chicken/Hubei/J9/2009A/chicken/Hubei/N11/2009A/chicken/Ningxia/A1/2011A/chicken/Ningxia/29/2011A/chicken/Ningxia/A2/2011A/chicken/Shaanxi/11/2012 A/chicken/Hubei/D21/2010A/chicken/Hubei/E17/2010A/chicken/Hubei/G21/2010A/chicken/Hubei/E16/2010A/chicken/Hubei/D2/2010A/chicken/Hubei/GYC/2011A/chicken/Shanxi/D24/2011A/chicken/Ningxia/I24/2011A/chicken/Ningxia/A15/2011 A/chicken/Hubei/E9/2010A/chicken/Hubei/E13/2010A/chicken/Hubei/G5/2010A/chicken/Hubei/A4/2010A/chicken/Hubei/G4/2010A/chicken/Hubei/G27/2010A/chicken/Hubei/G19/2010A/chicken/Hubei/E18/2010A/chicken/Hubei/E8/2010 A/chicken/Hubei/A30/2010A/Chicken/Shandong/11/2010A/chicken/Hubei/E20/2010A/chicken/Fujian/SL4/2011A/chicken/Fujian/SN1/2011A/Chicken/Shandong/06/2011A/Chicken/Sichuan/02/2011A/chicken/Fujian/SN2/2012A/chicken/GuangXi/GLLY/2012 A/chicken/Hubei/A25/2010A/chicken/Hubei/G9/2010A/chicken/Hubei/G2/2010A/chicken/Hubei/A27/2010A/chicken/Hubei/A28/2010A/chicken/Hubei/A1/2010A/Chicken/Shandong/07/2011A/Chicken/Shandong/11/2011A/Chicken/Shandong/09/2011 A/chicken/Hubei/G10/2010A/chicken/Hubei/A23/2010A/chicken/Hubei/G6/2010A/chicken/Hubei/580/2011A/chicken/Hubei/ZWC/2011A/chicken/Hubei/HWC/2011A/chicken/Hubei/558/2011A/chicken/Hubei/LLM03/2011A/chicken/Hubei/661/2011 G37 A/chicken/Shandong/lc1124/2007A/chicken/Hebei/C6/2007A/chicken/Hebei/0822/2007A/chicken/Hebei/LC/2008 A/chicken/Hubei/581/2011 G05 A/chicken/Hebei/0617/2007A/chicken/Ningxia/A18/2009A/chicken/Ningxia/N22/2009 A/chicken/Shandong/JN/1999 A/chicken/Jilin/47/2009A/chicken/Ningxia/F15/2009A/chicken/Jilin/209/2009A/chicken/Guizhou/Y6/2009A/chicken/Guizhou/Y76/2009A/chicken/Jilin/M19/2009A/chicken/Jilin/2/2009A/chicken/Jilin/G12/2009 A/chicken/Guangdong/A5/2003A/chicken/Guangdong/A4/2003A/chicken/Guangdong/B2/2006A/chicken/Guangdong/A10/2007A/chicken/Jiangsu/E26/2007A/chicken/Sichuan/B1/2007A/chicken/Hubei/HE27/2008A/chicken/Sichuan/G2/2009A/chicken/Sichuan/G4/2009 A/chicken/Jiangxi/12/2007A/chicken/Shanghai/Q0712/2007A/chicken/Shandong/lx316/2008A/chicken/Jiangsu/DT/2009A/chicken/Shandong/382/2009A/chicken/Shandong/377/2009A/chicken/Guangxi/G8/2009A/chicken/Shandong/N/2010A/chicken/Shandong/384/2011 A/chicken/Shandong/1231/2008A/chicken/Shandong/363/2009A/Chicken/Shandong/zc12/2009A/chicken/Shandong/PD/2009A/chicken/Shandong/SG2/2009A/chicken/Shandong/SG1/2009A/chicken/Shandong/L/2010A/chicken/Shandong/K/2010A/chicken/Chongqing/K7/2011 A/chicken/Tibet/S4/2009A/chicken/Guizhou/Q6/2009A/chicken/Tibet/S1/2009A/chicken/Shandong/02/2009A/chicken/Ningxia/R13/2009A/Chicken/Shandong/zc11/2009A/chicken/Ningxia/S17/2009A/chicken/Guizhou/Q16/2009A/chicken/Shandong/YT2/2010 A/chicken/Shandong/01/2009A/chicken/Shandong/YT15/2011A/chicken/Shandong/YT1/2010A/chicken/Shandong/01/2010A/chicken/Shandong/YT4/2010A/chicken/Shandong/YT4/2011A/chicken/Shandong/YT21/2011A/chicken/Shandong/YT2/2011A/chicken/Shandong/YT3/2011 HA A/chicken/Shandong/02/2008A/chicken/Shandong/12/2008A/chicken/Tibet/L7/2009A/chicken/Guangdong/K25/2009A/chicken/Tibet/L18/2009A/chicken/Guangdong/P26/2009A/Chicken/Shandong/zc2/2009A/chicken/Shandong/TH9/2010A/chicken/Shanxi/xy1/2012 A/chicken/Guangdong/B15/2009A/chicken/Guangdong/K3/2009A/chicken/Guangdong/J22/2009A/chicken/Guangdong/J23/2009A/chicken/Guangdong/G2/2009A/chicken/Chongqing/G21/2011A/chicken/Chongqing/A53/2011A/chicken/Chongqing/K23/2011 A/chicken/Jiangsu/Q7/2010A/chicken/Jiangsu/Q3/2010A/chicken/Jiangsu/U3/2010A/chicken/Shangdong/SDWD/2011A/chicken/Qianzhou/12/2010A/chicken/Chongqing/K4/2011A/chicken/Chongqing/K9/2011A/chicken/Chongqing/K10/2011A/chicken/Chongqing/K3/2011 A/chicken/Jiangsu/H3/2011A/chicken/Jiangsu/LWC/2011A/chicken/Jiangsu/B6/2011A/chicken/Jiangsu/FQD03/2011A/chicken/Jiangsu/B16/2011A/chicken/Jiangsu/H10/2011A/chicken/JiIangsu/I9/2011A/chicken/Jiangsu/B5/2011A/chicken/Jiangsu/I8/2011 A/chicken/Jiangsu/H9/2010A/chicken/Jiangsu/H25/2010A/chicken/Jiangsu/A20/2010A/chicken/Jiangsu/A13/2010A/chicken/Jiangsu/H26/2010A/chicken/Jiangsu/H4/2010A/chicken/Jiangsu/B12/2011A/Chicken/Shandong/05/2011A/chicken/Jiangsu/D8/2011 A/Chicken/Shandong/10/2010A/Chicken/Shandong/06/2010A/chicken/Jiangsu/XZPZ/2011A/chicken/Hubei/TS1/2011A/chicken/Tianjin/DCWD/2011A/chicken/Jiangsu/D10/2011A/chicken/Jiangsu/I16/2011A/chicken/Tongshan/1/2011A/chicken/Shandong/zc4/2012 A/chicken/Henan/G15/2011A/chicken/Henan/D21/2011A/chicken/Jiangsu/ZJDT/2011A/chicken/Hubei/TS3/2011A/chicken/Henan/B6/2011A/chicken/Henan/J14/2011A/chicken/Henan/J22/2011A/chicken/Henan/G20/2011A/chicken/Henan/LY-36/2013 A/Chicken/Jiangsu/TS/2010A/chicken/Jiangsu/A9/2010A/chicken/Shandong/J9/2011A/chicken/Jiangsu/YZ0611/2011A/chicken/Henan/G27/2011A/chicken/Zhejiang/611/2011A/chicken/Anhui/ZYX05/2011A/chicken/Jiangsu/YZ0511/2011A/chicken/Henan/D24/2011 A/chicken/Anhui/B6/2010A/chicken/Anhui/HF/2010A/chicken/Henan/G19/2010A/chicken/Anhui/1/2010A/chicken/Anhui/G15/2010A/chicken/Jiangsu/G17/2010A/chicken/Anhui/G18/2010A/chicken/Anhui/G10/2010A/chicken/Henan/F9/2010 A/chicken/Anhui/B2/2010A/chicken/Guizhou/D26/2011A/chicken/Chongqing/J24/2011A/chicken/Chongqing/G20/2011A/chicken/Guizhou/J22/2011A/chicken/Chongqing/G25/2011A/chicken/Chongqing/G14/2011A/chicken/Chongqing/G8/2011A/chicken/Chongqing/G12/2011 A/chicken/Guangdong/D8/2010A/chicken/Guangdong/D9/2010A/chicken/Guangdong/D7/2010A/chicken/Guangdong/H28/2010A/chicken/Anhui/E13/2011A/chicken/Anhui/E9/2011A/chicken/Anhui/I24/2011A/chicken/Anhui/E7/2011A/chicken/Anhui/E8/2011 G41 A/chicken/Taixing/10/2010A/chicken/Jiangsu/H11/2010A/chicken/China/AH-10-01/2010A/chicken/Guangdong/H29/2010A/chicken/Guangdong/H11/2010A/chicken/Guangdong/H12/2010A/chicken/Yangzhou/11/2010 G06 A/chicken/Anhui/D21/2010A/chicken/Jiangsu/H27/2011A/chicken/Xigou/1/2011A/chicken/Dawang/1/2011A/chicken/Jiangsu/H13/2011A/chicken/Jiangsu/B4/2011A/chicken/Anhui/ZTL/2011A/chicken/Shuanggou/1/2011A/chicken/Jiangsu/CZYWP/2012 A/chicken/Henan/F21/2011A/chicken/Henan/F5/2011A/chicken/Ningxia/A13/2011A/chicken/Henan/F12/2011A/chicken/Henan/F16/2011A/chicken/Henan/F13/2011A/chicken/Jiangsu/B25/2011A/chicken/Ningxia/189/2011A/chicken/Henan/D17/2011 A/chicken/Anhui/K9/2011A/chicken/Anhui/K13/2011A/chicken/Anhui/I4/2011A/chicken/Anhui/E22/2011A/chicken/Anhui/E19/2011A/chicken/Anhui/E16/2011A/chicken/Jiangsu/H2/2011A/chicken/Anhui/I8/2011A/chicken/Wujin/19/2011 A/chicken/Guangdong/Y7/2009A/chicken/Shandong/KD/2009A/Chicken/Shandong/05/2010A/chicken/Shandong/YT7/2010A/chicken/Shandong/03/2010A/Chicken/Shandong/sd02/2010A/Chicken/Shandong/sd01/2010A/chicken/Henan/E2/2011A/chicken/Jiangsu/I2/2011 A/chicken/Guangdong/69/2009A/chicken/Shandong/WE26/2011A/chicken/Henan/E24/2011A/chicken/Henan/I27/2011A/chicken/Henan/E11/2011A/chicken/Henan/E18/2011A/chicken/Henan/I23/2011A/chicken/Henan/E21/2011A/chicken/Henan/E10/2011 A/Chicken/Hebei/ZR/2010A/chicken/Shanxi/I5/2011A/chicken/Jiangsu/I20/2011A/chicken/Shandong/WE23/2011A/chicken/Shanxi/I15/2011A/chicken/Shanxi/D30/2011A/chicken/Shanxi/I13/2011A/chicken/Shanxi/B3/2011A/chicken/Shandong/J6/2011 A/chicken/Shanxi/K1/2011A/chicken/Shanxi/I21/2011A/chicken/Jiangsu/B27/2011A/chicken/Shanxi/B29/2011A/chicken/Shanxi/D25/2011A/chicken/Shanxi/D4/2011A/chicken/Shandong/WE12/2011A/chicken/Shandong/WE9/2011A/Chicken/Hebei/FL/2011 A/chicken/Shanxi/K10/2011A/chicken/Shanxi/E30/2011A/chicken/Henan/H15/2011A/chicken/Shanxi/K16/2011A/chicken/Henan/H13/2011A/chicken/Henan/H11/2011A/chicken/Henan/H22/2011A/chicken/Henan/H29/2011A/chicken/Henan/H24/2011 A/chicken/Shanxi/39/2011A/chicken/Henan/H26/2011A/chicken/Henan/K8/2011A/chicken/Hunan/JYZ11/2011A/chicken/Henan/K1/2011A/chicken/Henan/H5/2011A/chicken/Henan/H6/2011A/chicken/Shanxi/33/2011A/chicken/Shanxi/43/2011 A/chicken/Zhejiang/HF4/2009A/chicken/Zhejiang/HF5/2009A/chicken/Zhejiang/HE7/2009A/chicken/Zhejiang/Q1D4/2010A/chicken/Henan/G15/2010A/chicken/Hubei/E23/2010A/chicken/Henan/B19/2011A/chicken/Henan/H19/2011A/chicken/Henan/B7/2011 A/chicken/Henan/G4/2010A/chicken/Anhui/G14/2010A/chicken/Henan/G1/2010A/chicken/Anhui/B12/2010A/chicken/Anhui/B7/2010A/chicken/Henan/G11/2010A/chicken/Anhui/B1/2010A/chicken/Anhui/G11/2010A/chicken/Henan/G14/2010 A/chicken/Guangdong/J3/2009A/chicken/Guangdong/K23/2009A/chicken/Jilin/181/2009A/chicken/Guangdong/L4/2009A/chicken/Guangdong/K22/2009A/chicken/Guangdong/J5/2009A/chicken/Anhui/G12/2010A/chicken/Henan/B8/2010A/chicken/Anhui/B20/2010 A/chicken/Guangdong/D2/2010A/chicken/Hainan/Q4/2010A/chicken/Guizhou/E25/2010A/chicken/Guizhou/E15/2010A/chicken/Guangdong/D4/2010A/chicken/Guizhou/E9/2010A/chicken/Guangxi/LRW04/2011A/chicken/Guangxi/MFX/2011A/chicken/Guizhou/XK04/2011 A/chicken/Guangdong/Y8/2009A/chicken/Guangdong/Y4/2009A/chicken/Jiande/01/2009A/chicken/Guizhou/E23/2010A/chicken/Guizhou/A4/2010A/chicken/Guizhou/E3/2010A/chicken/Guizhou/E7/2010A/chicken/Guizhou/D15/2010A/Chicken/Hebei/FX05/2010 A/Chicken/Shandong/HL/2010A/Chicken/Hebei/YT/2010A/chicken/Shandong/BD/2010A/chicken/Shandong/397/2010A/Chicken/Beijing/HD/2010A/chicken/Yongcheng/1/2011A/chicken/Jiawang/2/2011A/chicken/Jiangsu/DTNSZ/2011A/chicken/Jilin/I12/2011 A/chicken/Anhui/A20/2010A/chicken/Jiangsu/U2/2010A/chicken/Anhui/A19/2010A/chicken/Hainan/E17/2010A/chicken/Anhui/B17-2/2010A/chicken/Anhui/D25/2010A/chicken/Shandong/P4/2011A/chicken/Shandong/M1/2011A/chicken/Shandong/YS/2012 A/chicken/Zhejiang/HE2/2009A/chicken/Zhejiang/HF3/2009A/chicken/Sichuan/G6/2009A/chicken/Zhejiang/HF2/2009A/chicken/Zhejiang/E5/2009A/chicken/Zhejiang/SJL/2009A/chicken/Zhejiang/E4/2009A/chicken/Anhui/A25/2010A/chicken/Henan/KF/2011 G44 A/chicken/Zhejiang/HE3/2009A/chicken/Jiangsu/H10/2010A/chicken/Wuxi/7/2010A/chicken/Jiangsu/A7/2010A/chicken/Anhui/M25/2011A/ChiCken/Shandong/03/2011A/chicken/Jiangsu/WXY/2011A/chicken/Jiangsu/ZHM/2011A/Chicken/Shandong/02/2011 G07 A/chicken/Anhui/B24/2011A/chicken/Anhui/M2/2011A/chicken/Anhui/M8/2011A/chicken/Anhui/F2/2011A/chicken/Anhui/J19/2011 A/chicken/Anhui/D3/2011A/chicken/Anhui/K17/2011A/chicken/Anhui/D21/2011A/chicken/Anhui/M18/2011A/chicken/Anhui/B6/2011A/chicken/Anhui/M5/2011A/chicken/Anhui/E23-2/2011A/chicken/Anhui/E15/2011A/chicken/Anhui/M26/2011 A/chicken/Shandong/H/2009A/chicken/Shandong/A1/2009A/chicken/Shandong/B/2009A/chicken/Shandong/C/2009A/chicken/Shandong/E/2010A/chicken/Anhui/M29/2011A/chicken/Anhui/K3-2/2011A/chicken/Anhui/M13/2011A/chicken/Anhui/B19/2011 40 A/chicken/Jiangsu/U1/2010A/chicken/Anhui/G11/2011A/chicken/Anhui/G6/2011A/chicken/Anhui/K2/2011A/chicken/Anhui/G18/2011A/chicken/Xiangshui/1/2011A/chicken/Zhejiang/HGH12/2011A/chicken/Jiangsu/DT0112/2012 A/chicken/Zhejiang/WXQ12/2011A/chicken/Zhejiang/607/2011A/chicken/Shandong/wf0202/2012A/chicken/Shandong/qd0920/2012A/chicken/Shandong/FXWH13A/2012A/chicken/Zhejiang/HQD/2012A/chicken/Shandong/qd0516/2012A/chicken/Jiangsu/XZ6/2012A/chicken/Shandong/qd1013/2012 A/chicken/Zhejiang/FXK12/2011A/chicken/Jiangsu/QF/2011A/chicken/Zhejiang/SYP12/2011A/chicken/Zhejiang/DJR12/2011A/chicken/Zhejiang/FYQ12/2011A/chicken/Zhejiang/KHZ12/2011A/chicken/Zhejiang/XBF12/2011A/chicken/Zhejiang/ZGPL12/2011A/chicken/Jiangsu/WJYBF/2012 A/chicken/Liaoning/1116/2012A/chicken/Beijing/325/2013A/chicken/Shandong/LC2/2013A/chicken/Shandong/lc0523/2013A/chicken/Beijing/16/2013A/chicken/Beijing/11/2013A/chicken/Shandong/lc0903/2013A/chicken/Beijing/0512/2013A/chicken/Shandong/qd0312/2013 A/chicken/Anhui/LJT/2010A/chicken/Zhejiang/ZLB/2011A/chicken/Shandong/513/2011A/chicken/Shandong/M4/2011A/chicken/Shandong/M6/2011A/chicken/Shandong/WG10/2011A/chicken/Zhejiang/LAC/2011A/chicken/Anhui/XMS/2011A/chicken/Qingdao/04/2012 A/chicken/Shandong/YT1/2011A/chicken/Anhui/WBQ/2011A/chicken/Gansu/419/2012A/chicken/Liaoning/PJXM15/2012A/chicken/Henan/YX/2012A/chicken/Shandong/P2/2012A/chicken/Shandong/WF4/2012A/chicken/ShanXi/WSDX/2012A/chicken/Shandong/P6/2012 Fig. 3. Phylogenetic analysis of H9N2 influenza A/chicken/Anhui/A12/2011A/chicken/Anhui/D14/2011A/chicken/Anhui/A9/2011A/chicken/Anhui/GQY/2011A/chicken/Anhui/D30/2011A/chicken/Anhui/A3/2011A/chicken/Anhui/A17/2011A/chicken/Hunan/1/2012 A/chicken/Anhui/A13/2011A/chicken/Anhui/A8/2011A/chicken/Jilin/1031/2012A/chicken/Shandong/qd1115/2012A/chicken/Zhejiang/NH5/2013A/chicken/Shandong/qd0307/2013A/chicken/Hebei/0109/2013A/chicken/Shandong/qd0107/2013A/chicken/Shandong/qd0225/2013 A/chicken/Hunan/12/2011A/chicken/Shanxi/YC/2012A/chicken/Shandong/qd1224/2012A/chicken/Hebei/1024/2012A/chicken/Jiangsu/HA/2012A/chicken/Shandong/LC1/2013A/chicken/Liaoning/0517/2013A/chicken/Shaanxi/xa0414/2013A/chicken/Shandong/qd0808/2013 5.0 A/chicken/Henan/ZK2/2012A/Chicken/Nanjing/023/2013A/Chicken/Nantong/031-2/2013A/chicken/Rizhao/249/2013A/chicken/Beijing/0331/2013A/chicken/Beijing/0311/2013A/chicken/Rizhao/85/2013A/Chicken/Suzhou/097-2/2013A/chicken/Rizhao/811/2013 A/chicken/Rizhao/15/2013A/chicken/Rizhao/437/2013A/chicken/Rizhao/81/2013A/chicken/Rizhao/651/2013 A/chicken/Zhejiang/SYX/2010A/chicken/Guangdong/WZD11/2011A/chicken/Hebei/QHDzd/2012A/chicken/Guangdong/ZW/2012A/chicken/Shandong/yt0106/2012A/chicken/Rizhao/723/2013A/chicken/Rizhao/719/2013A/chicken/Rizhao/1313/2013A/chicken/Rizhao/267/2013 A/chicken/Zhejiang/FSQ/2011A/chicken/Zhejiang/329/2011A/chicken/Beijing/HD06/2012A/chicken/Tianjin/61/2012A/chicken/Beijing/L2/2012A/chicken/Tianjin/614/2012A/chicken/Shandong/HK1/2013A/chicken/Shandong/LC3/2013A/chicken/Shandong/wf0514/2013 A/chicken/Shandong/zc0606/2012A/chicken/Jiangsu/XZ38/2012A/chicken/Shanghai/C3/2012A/chicken/Shanxi/0703/2012A/chicken/Liaoning/0704/2012A/chicken/Shanghai/C1/2012A/chicken/Shandong/lc0830/2012A/chicken/Shanghai/C2/2012A/chicken/Shandong/qd0427/2012 A/chicken/Jiangsu/WJHRG/2012A/chicken/Jiangsu/CZJJF/2012A/chicken/Shandong/wf12010/2012A/chicken/Jiangsu/CZZWQ/2012A/chicken/Jiangsu/CZLJG/2012A/chicken/Shandong/wf1206/2012A/chicken/Guangdong/LG1/2013A/chicken/Tianjin/120/2013A/chicken/Shandong/qd0917/2013 G49 A/chicken/Jiangsu/XZMG/2012A/chicken/Shandong/SDWF17/2012 A/chicken/Jiangsu/XZLZG/2012A/chicken/Jiangsu/XZWHL/2012A/chicken/Shangdong/SDWF29/2012A/chicken/Jiangsu/XZZSL/2012A/chicken/Shangdong/SDWF14/2012A/chicken/Jiangsu/WJHDL/2012A/chicken/Jiangsu/MYJMF/2012A/chicken/Beijing/F1/2012A/chicken/Beijing/1115/2013 G09 A/chicken/Jiangsu/CZ1/2012A/chicken/Shandong/qd01417/2013A/chicken/Hebei/0721/2013 A/chicken/Shantou/22116/2005 A/chicken/Henan/HF/2012A/chicken/Jiangsu/ZJ4/2013A/chicken/Shandong/yt0711/2013A/chicken/Rizhao/1339/2013A/chicken/Jiangsu/NTTZ/2013A/chicken/Jiangsu/CZJT2/2013A/chicken/Shanghai/020/2013A/chicken/Wenzhou/630/2013A/chicken/Shandong/wf0712/2013 A/chicken/Beijing/1/1994A/Chicken/Beijing/1/94 A/chicken/Beijing/1/00A/chicken/Jiangsu/2/2001 A/chicken/Jiangsu/ZJDT1/2013A/chicken/Rizhao/1436/2013A/chicken/Rizhao/853/2013A/chicken/Rizhao/1363/2013A/chicken/Rizhao/1207/2013A/chicken/Jiangsu/HA2/2013A/Chicken/Nanjing/503/2013A/chicken/Rizhao/836/2013A/chicken/Hangzhou/50-2/2013 A/Chicken/Beijing/1/95A/Chicken/Hebei/1/96A/chicken/Shandong/7/96A/Chicken/Shandong/6/96A/Chicken/Heilongjiang/10/97 A/chicken/Wenzhou/606/2013A/chicken/Rizhao/55/2013A/Chicken/Nanjing/106-2/2013A/chicken/Wenzhou/628b/2013A/chicken/Wenzhou/598/2013A/chicken/Wenzhou/253/2013A/chicken/Wenzhou/692b/2013A/chicken/Beijing/324/2013A/Chicken/Beijing/0309/2013 A/Chicken/Guangdong/11/97A/chicken/Shandong/B1/1998A/chicken/Shandong/WF/1998A/Chicken/Beijing/3/99 A/chicken/Wenzhou/620/2013A/chicken/Wenzhou/614/2013A/chicken/Wenzhou/632b/2013A/chicken/Wenzhou/594/2013A/chicken/Wenzhou/642/2013A/chicken/Wenzhou/592b/2013A/chicken/Wenzhou/618/2013A/chicken/Wenzhou/648/2013A/chicken/Wenzhou/622/2013 A/chicken/Guangdong/SS/94A/Chicken/Guangdong/SS/94 A/Chicken/Guangdong/97A/chicken/Guangdong/5/97 A/chicken/Guangdong/TS/2004 A/chicken/Wenzhou/688b/2013A/chicken/Jiangsu/CZWJ5/2013A/Chicken/Huzhou/C143-8/2013A/Chicken/Huzhou/C143-2/2013A/chicken/Hangzhou/48-2/2013 A/chicken/Shandong/1/1999A/chicken/Anhui/1/1998A/chicken/Shandong/1/2000A/chicken/Shandong/244/2002A/chicken/Henan/L1/2002 A/chicken/Shijiazhuang/2/98A/Chicken/Shijiazhuang/2/99A/chicken/Henan/ni/2001A/chicken/Yunnan/nh/2001A/chicken/Hebei/nj/2002 A/chicken/Henan/nd/1998A/chicken/Neimonggu/nk/2002A/chicken/Beijing/nl/2002A/chicken/Jiangsu/7/2002A/chicken/Jiangsu/wa/2002 A/chicken/Shanghai/2/1999 A/chicken/Jiangsu/ng/2001A/chicken/Beijing/ne/1999 A/chicken/Shanghai/Y1/2006A/chicken/Hubei/C1/2007 – A/chicken/Shanghai/10/01A/chicken/Zhuhai/A1/2001 A/chicken/Shanghai/3/2000A/chicken/Shanghai/7/2001A/chicken/Shanghai/1/2001 A/chicken/Jiangsu/L2/2005A/chicken/Shanghai/2/2001 A/Chicken/Shanghai/F/98A/chicken/Jiangsu/1/1999 A/chicken/Jiangsu/YZ4/2012A/chicken/Jiangsu/1/2004A/chicken/Shanghai/14/2001 A/chicken/Shanghai/16/2001A/chicken/Shanghai/12/2001A/chicken/Qixian/A1/2001 A/chicken/Jiangsu/L1/2004A/chicken/Guangxi/2389/2005 viruses isolated from chickens during 1994 2013 and A/chicken/Shanghai/Y1/2002 A/chicken/Shantou/2686/2003A/chicken/Guangxi/4745/2005A/chicken/Guangxi/1428/2005A/chicken/Beijing/7/2005A/chicken/Shandong/1/2006 A/chicken/Guangxi/55/2005A/chicken/Hebei/0822/2007A/chicken/Hebei/0617/2007A/chicken/Hebei/C6/2007A/chicken/Hebei/LC/2008 G51 A/chicken/Guangxi/21/2006 A/chicken/Hunan/5700/2005A/chicken/Hunan/2903/2005A/chicken/Beijing/1/2006A/chicken/Guangxi/44/2006A/chicken/Beijing/20/2006 G10 A/chicken/Guangdong/4/00A/Chicken/Shanghai/1/02 A/chicken/Guangxi/1032/2005A/chicken/Hunan/3369/2005A/chicken/Hunan/2536/2005 A/Chicken/Beijing/2/97A/Chicken/Beijing/8/98A/Chicken/Nanchang/4-010/2000A/Chicken/Nanchang/1-0016/2000A/chicken/Nanchang/1-0016/2000 A/chicken/Beijing/8/98A/chicken/Shandong/1/1998A/chicken/Henan/5/98 A/chicken/Hebei/L1/2006A/chicken/Shandong/LY-1/2008 A/Chicken/Ningxia/5/99A/chicken/Fujian/25/00 A/chicken/Beijing/L1/2005A/chicken/Beijing/243/2005 A/chicken/Beijing/243/2010 A/Chicken/Hebei/3/98A/Chicken/Liaoning/99A/chicken/Gansu/2/99A/chicken/Ningxia/4/99 A/chicken/Shandong/1/2003 A/Chicken/Henan/98A/Chicken/Hebei/4/98A/Chicken/Hebei/2/98A/chicken/Liaoning/99 A/chicken/Hebei/1/01 A/chicken/Hubei/01-MA01/1999A/chicken/Hubei/01/1999A/chicken/Hebei/31/00A/chicken/Shandong/241/2001A/chicken/Hebei/B1/2001 A/Chicken/Shaoguan/13/98A/chicken/Henan/26/00A/Chicken/Henan/62/00 A/chicken/Tianjin/B1/2004 A/chicken/Hebei/274/2009 A/chicken/Guangxi/9/99A/chicken/Guangxi/KMIII/1999A/chicken/Yunnan/Xie-1/1999A/chicken/Heilongjiang/48/01 A/chicken/Hebei/274/2004 A/chicken/Henan/C4/2002A/chicken/Hunan/774/2002A/chicken/Henan/2/2004A/chicken/Guangxi/37/2005A/chicken/Guangxi/101/2006 A/chicken/Guangxi/1857/2004A/chicken/Guangxi/2441/2004A/chicken/Guangxi/187/2005A/chicken/Guangxi/521/2005A/chicken/Guangxi/39/2007 A/chicken/Shantou/859/2000A/chicken/Shantou/1690/2000A/chicken/Nanchang/4-301/2001A/Chicken/Nanchang/4-361/2001 A/chicken/Guangxi/17/2007 A/chicken/Shantou/1579/2000A/chicken/Shantou/1322/2000A/chicken/Shantou/212/2000A/chicken/Shantou/94/2000A/chicken/Shantou/4342/2002 A/chicken/Jiangsu/1/1998A/Chicken/Shandong/98 A/chicken/Henan/1/2002A/chicken/Henan/1/2004A/chicken/Henan/1/2005 G53 A/chicken/Anhui/2/2002A/chicken/Jiangsu/1/2002 A/chicken/Shantou/2994/2004A/chicken/Shantou/2692/2004A/chicken/Shantou/1926/2004 A/chicken/Shandong/1/2008A/chicken/Shandong/SD0014/2008 G13 A/chicken/Shangqiu/A1/2000 A/chicken/Henan/1/2003A/chicken/Qixian/A1/2004 A/chicken/Shanghai/1/2002A/chicken/Shanghai/2/2002A/chicken/Huangdong/A1/2003A/chicken/Shandong/1/2004A/chicken/Kaifeng/A1/2005 H7N9 viruses isolated from humans in China during A/chicken/Hebei/A/2007A/chicken/Henan/2.3/2008A/chicken/Shandong/22/2008A/chicken/Henan/1.2/2008 A/chicken/Shandong/B4/2007A/chicken/Hebei/4/2008A/chicken/Hebei/C4/2008A/chicken/Hebei/7/2008A/chicken/Hebei/Y2/2009 A/chicken/Shandong/251/2007A/chicken/Anhui/10/2009A/Chicken/Hebei/ZR/2010A/Chicken/Hebei/FL/2011A/chicken/Beijing/HD06/2012 A/chicken/Shandong/LY/2006A/chicken/Hebei/0329/2007A/chicken/Shanghai/Q0801-2/2008 A/chicken/Tianjin/614/2012A/chicken/Shandong/wf0514/2013 A/chicken/Yunnan/Chuxiong1/2007A/chicken/Kunming/123/2007A/chicken/Puer/711/2008A/chicken/Shanghai/Q0808-1/2008A/chicken/Dehong/411/2009 A/chicken/Hunan/5260/2005A/chicken/Hunan/4444/2005A/chicken/Hunan/6108/2005 A/chicken/Lijiang/711/2008A/chicken/Guangdong/G7/2009 A/chicken/Guangdong/SYX12/2011A/chicken/Guangdong/LJT12/2011A/chicken/Guangdong/YBS10/2011A/chicken/Wenzhou/253/2013A/chicken/Guangdong/LG1/2013 A/chicken/Guangdong/LYH12/2011A/chicken/Shandong/qd0920/2012A/chicken/Beijing/0512/2013A/chicken/Shandong/lc0903/2013A/chicken/Shandong/lc0523/2013 A/chicken/Hangzhou/50-2/2013A/chicken/Hangzhou/48-2/2013A/chicken/Wenzhou/614/2013A/chicken/Wenzhou/632b/2013A/chicken/Wenzhou/618/2013 A/chicken/Shaanxi/11/2012A/chicken/Wenzhou/620/2013A/chicken/Wenzhou/598/2013A/chicken/Wenzhou/630/2013A/chicken/Wenzhou/622/2013 30 A/chicken/Wenzhou/692b/2013A/Chicken/Nanjing/023/2013A/chicken/Beijing/0311/2013 A/chicken/Beijing/0331/2013A/chicken/Rizhao/15/2013A/chicken/Rizhao/1313/2013A/chicken/Rizhao/811/2013A/Chicken/Nantong/031-2/2013 A/chicken/Rizhao/719/2013A/chicken/Rizhao/81/2013A/chicken/Rizhao/723/2013A/chicken/Rizhao/249/2013A/chicken/Rizhao/85/2013 G57 A/chicken/Rizhao/437/2013A/chicken/Rizhao/651/2013 A/chicken/Rizhao/267/2013A/chicken/Rizhao/1436/2013A/Chicken/Nanjing/106-2/2013A/chicken/Rizhao/853/2013A/chicken/Rizhao/1363/2013 G14 A/chicken/Rizhao/836/2013A/chicken/Rizhao/1207/2013A/chicken/Rizhao/1339/2013 A/chicken/Jiangsu/CZ1/2012A/chicken/Beijing/324/2013A/chicken/Wenzhou/606/2013A/Chicken/Beijing/0309/2013A/chicken/Rizhao/55/2013 A/chicken/Shandong/wf0712/2013A/chicken/Beijing/1115/2013A/chicken/Jiangsu/HA2/2013A/chicken/Jiangsu/ZJDT1/2013A/chicken/Shandong/yt0711/2013 A/chicken/Shanghai/020/2013A/chicken/Wenzhou/642/2013A/chicken/Jiangsu/NTTZ/2013A/chicken/Wenzhou/688b/2013A/chicken/Jiangsu/CZJT2/2013 NA A/chicken/Jiangsu/245/2004 A/chicken/Shandong/382/2009A/chicken/Shandong/PD/2009A/chicken/Shandong/384/2011 A/chicken/Jiangsu/ZJ4/2013 A/chicken/Shandong/SG1/2009A/chicken/Shandong/363/2009A/Chicken/Shandong/zc11/2009A/chicken/Shandong/L/2010A/chicken/Shandong/K/2010 A/chicken/Shanghai/Y2/2006A/chicken/Shanghai/Q0704/2007A/chicken/Shanghai/Q0702/2007A/chicken/Henan/323/2009A/chicken/Shandong/N/2010 the first 2 wk of the 2013 outbreak. (A) Phylogenetic A/chicken/Shandong/317/2007 A/chicken/Shandong/B2/2007A/chicken/Shandong/ZB/2007A/chicken/Shandong/L1/2007A/chicken/Shandong/237/2007A/chicken/Shandong/B3/2007 A/chicken/Jiangxi/12/2007A/chicken/Zhejiang/HJ/2007A/Chicken/Shandong/JL/2008A/chicken/Shanghai/Q0808-2/2008A/chicken/Shanghai/Q0801-1/2008 A/Chicken/Shandong/LY/2008A/chicken/Shandong/BD/2008A/Chicken/Beijing/HD/2008A/chicken/Tibet/S4/2009A/chicken/Tibet/S1/2009 A/chicken/Shanghai/Q0712/2007A/chicken/Shandong/02/2008A/chicken/Zibo/L2/2008A/chicken/EasternA/Chicken/Shandong/zc2/2009 China/SD88/2008 A/chicken/Shanghai/Q0707/2007A/chicken/Shanghai/Y1/2007A/chicken/Shanghai/Y2/2007A/chicken/Shanghai/Q0812-1/2008A/chicken/Guangxi/G8/2009 A/chicken/Shandong/12/2008A/chicken/Shanghai/Q0812-2/2008A/chicken/Shandong/01/2009A/chicken/Hubei/10/2009A/chicken/Shandong/01/2010 A/chicken/Shandong/1231/2008A/chicken/Shanghai/Y1/2008A/Chicken/Shandong/zc12/2009A/chicken/Shandong/02/2009A/chicken/Shandong/SG2/2009 G58 A/chicken/Shanghai/Q0709/2007A/chicken/Shandong/KD/2009A/Chicken/Hebei/FX05/2010 A/chicken/Jiangsu/DT/2009A/chicken/Shandong/C/2009A/chicken/Shandong/377/2009A/chicken/Shandong/H/2009A/Chicken/Shandong/02/2011 G15 A/chicken/Yangzhou/11/2010A/chicken/Taixing/10/2010A/chicken/Dawang/1/2011 A/chicken/Shanghai/Y2/2008A/chicken/China/AH-10-01/2010A/chicken/Xigou/1/2011A/Chicken/Shandong/05/2011A/chicken/Shuanggou/1/2011 A/chicken/Anhui/HF/2010A/chicken/Wuxi/7/2010A/chicken/Zhejiang/SYP12/2011A/chicken/Jiangsu/ZJDT/2011A/chicken/Shandong/zc4/2012 A/Chicken/Jiangsu/TS/2010A/chicken/Jiangsu/Q3/2010A/Chicken/Shandong/10/2010A/chicken/Hunan/12/2011A/chicken/Hunan/1/2012 A/chicken/Shandong/513/2011A/chicken/Jiawang/2/2011A/chicken/Jiangsu/XZPZ/2011A/chicken/Yongcheng/1/2011A/chicken/Tongshan/1/2011 A/chicken/Guangdong/LWZ11/2011A/chicken/Guangdong/SQL10/2011A/chicken/Guangxi/LYY12/2011A/chicken/Guangdong/ZCY/2011A/Chicken/Guangdong/01/2011 A/chicken/Guangdong/CYH12/2011A/chicken/Guangdong/ZHJ/2011A/chicken/Guangdong/ZW12/2011A/chicken/Jilin/0519/2012A/chicken/Shandong/qd0917/2013 A/chicken/Shandong/B/2009A/chicken/Shandong/A1/2009A/chicken/Shandong/397/2010A/chicken/Guangdong/LFQ12/2011A/chicken/Guangdong/SJS12/2011 A/chicken/Shandong/E/2010A/Chicken/Henan/DY/2009A/chicken/Shandong/02/2010A/chicken/Shandong/BD/2010A/chicken/Xiangshui/1/2011 A/chicken/Jiande/01/2009A/chicken/Shandong/03/2010A/Chicken/Shandong/05/2010A/Chicken/Shandong/sd01/2010A/Chicken/Shandong/HL/2010 A/chicken/Zhejiang/Q1D4/2010A/Chicken/Shandong/sd02/2010A/Chicken/Shandong/11/2010A/Chicken/Shandong/09/2011A/Chicken/Sichuan/02/2011 A/chicken/Guangxi/067C4/2010A/Chicken/Shandong/07/2011A/Chicken/Shandong/06/2011A/Chicken/Shandong/11/2011A/chicken/Jiangsu/DTNSZ/2011 trees of surface genes with the H9N2 chicken viruses A/chicken/Qianzhou/12/2010A/chicken/Shangdong/SDWD/2011A/chicken/Jiangsu/YZ0611/2011A/chicken/Zhejiang/611/2011A/chicken/Jiangsu/YZ0511/2011 A/Chicken/Hebei/YT/2010A/chicken/Zhejiang/329/2011A/chicken/Jiangsu/XZ38/2012A/chicken/Jiangsu/DT0112/2012A/chicken/Jiangsu/TZXH/2013 G60 A/Chicken/Hebei/DF/2008A/Chicken/Shandong/06/2010A/Chicken/Beijing/HD/2010A/Chicken/Hebei/TS/2008A/chicken/Jiangsu/YCDF/2012 A/chicken/Zhejiang/HGH12/2011A/Chicken/Shandong/03/2011A/chicken/Zhejiang/607/2011A/chicken/Zhejiang/WXQ12/2011A/chicken/Shanghai/C1/2012 G17 A/chicken/Jiangsu/XZ6/2012 A/chicken/Shandong/qd0516/2012A/chicken/Jiangsu/WJYBF/2012A/chicken/Shandong/qd1013/2012A/chicken/Shandong/wf0202/2012A/chicken/Shanghai/C3/2012 A/chicken/Zhejiang/ZGPL12/2011A/chicken/Zhejiang/DJR12/2011A/chicken/Shandong/qd0427/2012A/chicken/Jiangsu/CZLJG/2012A/Chicken/Suzhou/097-2/2013 A/chicken/Zhejiang/XBF12/2011A/chicken/Zhejiang/FYQ12/2011A/chicken/Zhejiang/FXK12/2011A/chicken/Zhejiang/KHZ12/2011A/chicken/Shanghai/C2/2012 A/chicken/Shandong/SDWF17/2012A/chicken/Shangdong/SDWF29/2012A/chicken/Shangdong/SDWF14/2012A/chicken/Jiangsu/XZZSL/2012A/chicken/Beijing/F1/2012 A/chicken/Guangdong/WSL11/2011A/chicken/Gansu/419/2012A/chicken/Jiangsu/XZMG/2012A/chicken/Jiangsu/XZLZG/2012A/chicken/Jiangsu/XZWHL/2012 A/chicken/Hunan/JYZ11/2011A/chicken/Beijing/L2/2012A/chicken/Liaoning/0517/2013A/chicken/Beijing/11/2013A/chicken/Beijing/325/2013 A/chicken/Liaoning/0704/2012A/chicken/Shandong/qd1224/2012A/chicken/Shandong/lc0830/2012A/chicken/Shanxi/0703/2012A/chicken/Tianjin/120/2013 A/chicken/Jilin/1031/2012A/chicken/Shandong/zc0606/2012A/chicken/Shandong/yt0106/2012A/chicken/Shaanxi/xa0414/2013A/chicken/Hebei/0721/2013 A/chicken/Jiangsu/CZJJF/2012A/chicken/Shandong/wf1206/2012A/chicken/Shandong/wf12010/2012A/chicken/Jiangsu/CZZWQ/2012A/chicken/Shandong/qd01417/2013 A/chicken/Tianjin/61/2012A/chicken/Jiangsu/WJHRG/2012A/chicken/Liaoning/1116/2012A/chicken/Beijing/16/2013A/chicken/Shandong/qd0312/2013 A/chicken/Jiangsu/CZYWP/2012A/chicken/Hebei/1024/2012A/chicken/Jiangsu/MYJMF/2012A/chicken/Jiangsu/WJHDL/2012A/chicken/Shandong/qd0107/2013 5.0 A/chicken/Shandong/qd1115/2012A/chicken/Hebei/0109/2013A/chicken/Shandong/qd0808/2013A/chicken/Shandong/qd0307/2013A/chicken/Shandong/qd0225/2013 G18 G61 and those of internal genes with the chicken H9N2 20 G67 G29 and the novel H7N9 human viruses. Internal gene A/Chicken/Shandong/zc12/2009 A/chicken/Zhejiang/HJ/2007A/chicken/Jiangxi/12/2007 A/chicken/Shandong/513/2011A/chicken/Shaanxi/11/2012 A/chicken/Shanghai/Q0801-2/2008A/chicken/Shanghai/Q0801-1/2008A/chicken/Shanghai/Q0808-2/2008A/chicken/Fujian/G9/2009 A/chicken/Shandong/1231/2008A/chicken/Shandong/SG2/2009A/chicken/Hubei/10/2009A/chicken/Tibet/S1/2009 A/chicken/Shandong/02/2009A/chicken/Shandong/01/2009A/chicken/Tibet/S4/2009A/chicken/Shandong/01/2010 G68 A/chicken/Anhui/10/2009 A/Chicken/Henan/DY/2009A/Chicken/Hebei/FL/2011A/Chicken/Shandong/05/2011A/chicken/Xiangshui/1/2011 A/chicken/Taixing/10/2010A/chicken/Yangzhou/11/2010A/Chicken/Hebei/ZR/2010A/chicken/Hunan/1/2012 G30 A/chicken/China/AH-10-01/2010A/chicken/Shandong/qd0917/2013 A/chicken/Zhejiang/611/2011A/chicken/Xigou/1/2011A/chicken/Dawang/1/2011A/chicken/Shuanggou/1/2011 A/chicken/Jiangsu/ZJDT/2011A/chicken/Jiangsu/DT0112/2012A/chicken/Jiangsu/CZLJG/2012A/chicken/Hebei/1024/2012 A/chicken/Jilin/1031/2012A/chicken/Shandong/qd1224/2012A/chicken/Liaoning/0704/2012A/chicken/Liaoning/0517/2013 A/chicken/Shanghai/Q0702/2007 A/chicken/Shandong/zc0606/2012A/chicken/Shandong/lc0830/2012A/chicken/Shanxi/0703/2012 A/chicken/Shanghai/Q0704/2007A/chicken/Shanghai/Q0812-1/2008A/chicken/Shanghai/Y1/2008A/chicken/Shanghai/Q0812-2/2008 A/Chicken/Hebei/DF/2008A/Chicken/Shandong/HL/2010A/chicken/Shandong/BD/2010A/chicken/Shandong/02/2010 A/chicken/Shandong/H/2009A/chicken/Qianzhou/12/2010A/chicken/Shandong/03/2010A/chicken/Shangdong/SDWD/2011 A/Chicken/Shandong/10/2010A/Chicken/Hebei/TS/2008A/Chicken/Hebei/YT/2010A/chicken/Jiangsu/YCDT/2013 A/Chicken/Shandong/06/2010A/Chicken/Jiangsu/TS/2010A/chicken/Yongcheng/1/2011A/chicken/Shandong/zc4/2012 A/chicken/Jiawang/2/2011A/chicken/Tongshan/1/2011A/chicken/Jiangsu/XZPZ/2011A/chicken/Shandong/SDWF17/2012 trees with only H9N2 viruses are not shown here due A/chicken/Jiangsu/XZMG/2012A/chicken/Shangdong/SDWF14/2012A/chicken/Shangdong/SDWF29/2012A/chicken/Jiangsu/XZZSL/2012 A/chicken/Jiangsu/Q3/2010A/chicken/Shanghai/C2/2012A/chicken/Jiangsu/XZLZG/2012A/chicken/Jiangsu/XZWHL/2012 A/chicken/Wuxi/7/2010A/chicken/Anhui/HF/2010A/chicken/Hunan/12/2011A/chicken/Shanghai/C3/2012 A/chicken/Zhejiang/607/2011A/chicken/Shandong/wf0202/2012A/chicken/Shandong/qd0516/2012A/chicken/Gansu/419/2012 A/chicken/Beijing/HD06/2012A/chicken/Tianjin/614/2012A/chicken/Shandong/qd0920/2012A/chicken/Shandong/qd1013/2012 A/chicken/Jiangsu/CZJJF/2012A/chicken/Jiangsu/CZZWQ/2012A/chicken/Jiangsu/WJHRG/2012A/chicken/Shandong/qd0427/2012 PB2 A/chicken/Shandong/wf12010/2012A/chicken/Shandong/wf1206/2012A/Chicken/Nantong/031-2/2013A/Chicken/Nanjing/023/2013 A/chicken/Rizhao/723/2013A/chicken/Rizhao/85/2013A/chicken/Rizhao/811/2013A/chicken/Rizhao/15/2013 A/chicken/Rizhao/437/2013A/chicken/Rizhao/1313/2013A/chicken/Rizhao/249/2013A/chicken/Rizhao/651/2013 A/Chicken/Shandong/sd02/2010A/Chicken/Shandong/05/2010A/Chicken/Shandong/sd01/2010A/Chicken/Hebei/FX05/2010 A/chicken/Zhejiang/Q1D4/2010A/Chicken/Beijing/HD/2010A/chicken/Jiangsu/NTHA1/2012A/chicken/Jiangsu/XZ6/2012 A/chicken/Jiangsu/TZXH/2013A/chicken/Jiangsu/ZJDT1/2013A/chicken/Jiangsu/NTTZ/2013A/chicken/Jiangsu/CZJT2/2013 A/chicken/Jiangsu/GY64/2012A/chicken/Jiangsu/YZ4/2012A/chicken/Jiangsu/CZWJ5/2013A/chicken/Jiangsu/CZ1/2012 A/chicken/Guangdong/ZHJ/2011A/chicken/Guangdong/ZCY/2011A/Chicken/Guangdong/01/2011A/chicken/Jilin/0519/2012 A/Chicken/Shandong/11/2011A/Chicken/Shandong/06/2011A/Chicken/Shandong/09/2011A/Chicken/Sichuan/02/2011 A/Chicken/Shandong/11/2010A/Chicken/Shandong/07/2011A/chicken/Shandong/yt0106/2012A/chicken/Shandong/wf0514/2013 – A/chicken/Zhejiang/329/2011A/chicken/Shaanxi/xa0414/2013 A/chicken/Jiangsu/YZ0511/2011A/chicken/Jiangsu/XZ38/2012A/chicken/Shandong/qd01417/2013A/chicken/Hebei/0721/2013 A/chicken/Tianjin/120/2013 Genotype and counts 10 A/chicken/Shanghai/C1/2012A/chicken/Hangzhou/48-2/2013 A/chicken/Hangzhou/50-2/2013A/Shanghai/1/2013A/Shanghai/4/2013A/Shanghai/2/2013 to the same topology. Clades with most of 2010 A/chicken/Liaoning/1116/2012A/Shanghai/4664T/2013A/Shanghai/3/2013A/Shanghai/02/2013 A/chicken/Beijing/0331/2013A/chicken/Shandong/qd0312/2013A/chicken/Shandong/lc0903/2013A/chicken/Beijing/0311/2013 A/chicken/Jiangsu/YZ0611/2011A/chicken/Shandong/lc0523/2013A/chicken/Beijing/0512/2013A/chicken/Beijing/11/2013 A/chicken/Jiangsu/DTNSZ/2011A/ChiCken/Shandong/03/2011A/Chicken/Shandong/02/2011A/chicken/Jiangsu/WJYBF/2012 A/chicken/Jiangsu/WJHDL/2012A/chicken/Jiangsu/MYJMF/2012A/chicken/Jiangsu/CZYWP/2012A/chicken/Guangdong/LG1/2013 A/chicken/Shandong/qd0107/2013A/chicken/Shandong/qd1115/2012A/chicken/Shandong/qd0808/2013A/chicken/Shandong/qd0307/2013 A/chicken/Shandong/wf0712/2013A/chicken/Shandong/qd0225/2013A/chicken/Hebei/0109/2013A/chicken/Shandong/yt0711/2013 A/chicken/Shanghai/020/2013A/chicken/Wenzhou/614/2013A/chicken/Wenzhou/606/2013A/chicken/Beijing/1115/2013 A/chicken/Wenzhou/598/2013A/chicken/Rizhao/1339/2013A/chicken/Rizhao/853/2013A/chicken/Wenzhou/642/2013 A/chicken/Wenzhou/253/2013A/chicken/Jiangsu/ZJ4/2013A/chicken/Rizhao/1436/2013A/chicken/Rizhao/1363/2013 A/chicken/Jiangsu/HA2/2013A/Chicken/Nanjing/106-2/2013A/Chicken/Beijing/0309/2013A/chicken/Rizhao/55/2013 5.0 A/Chicken/Suzhou/097-2/2013A/Chicken/Nanjing/503/2013A/chicken/Beijing/16/2013A/chicken/Beijing/324/2013 2013 viruses were fully shown, and others were

A/chicken/Guangxi/2441/2004A/chicken/Guangxi/1857/2004A/chicken/Guangxi/521/2005A/chicken/Guangxi/37/2005 A/chicken/Henan/nd/1998A/chicken/Beijing/ne/1999A/chicken/Jiangsu/wa/2002A/chicken/Guangxi/187/2005 collapsed (indicated by gray triangles). Color of line A/chicken/Jiangsu/7/2002A/chicken/Neimonggu/nk/2002 A/chicken/Hubei/C1/2007 A/chicken/Heilongjiang/48/01A/chicken/Beijing/nl/2002A/chicken/Guangxi/2389/2005 A/chicken/Jiangsu/YZ4/2012 A/chicken/Guangdong/56/01A/chicken/Shanghai/10/01A/chicken/Jiangsu/245/2004 A/Chicken/Shanghai/F/98 A/chicken/Hunan/774/2002A/chicken/Shantou/4435/2003A/chicken/Shantou/4617/2003A/chicken/Shantou/3040/2003 A/chicken/Shantou/4101/2003A/chicken/Shantou/3341/2003A/chicken/Shantou/99/2004A/chicken/Shandong/251/2007 A/chicken/Hunan/4444/2005A/chicken/Shandong/LY/2006A/chicken/Hebei/0329/2007 A/chicken/Shaanxi/11/2012 A/chicken/Hunan/6108/2005A/chicken/Hunan/5260/2005A/chicken/Shandong/22/2008A/chicken/Anhui/10/2009 A/Chicken/Shandong/JL/2008A/chicken/Shandong/397/2010A/chicken/Shandong/ZB/2007A/chicken/Shandong/317/2007 A/chicken/Shandong/B3/2007A/chicken/Shandong/237/2007A/chicken/Hubei/10/2009A/chicken/Shandong/377/2009 A/Chicken/Beijing/HD/2008A/chicken/Shandong/02/2008A/Chicken/Shandong/LY/2008A/chicken/Shandong/12/2008 A/chicken/Zibo/L2/2008A/chicken/Shandong/BD/2008A/Chicken/Shandong/zc2/2009A/Chicken/Shandong/zc12/2009 A/chicken/Shandong/1231/2008A/chicken/Tibet/S4/2009A/chicken/Tibet/S1/2009A/chicken/Shandong/SG2/2009 0 A/chicken/Shandong/01/2009A/chicken/Xiangshui/1/2011 A/chicken/Jiangxi/12/2007A/chicken/Shandong/02/2009A/chicken/Shandong/01/2010A/chicken/Yangzhou/11/2010 A/chicken/Hebei/C4/2008A/chicken/Hebei/4/2008A/chicken/Henan/1.2/2008A/chicken/Taixing/10/2010 A/chicken/Hebei/7/2008A/chicken/Hebei/A/2007A/chicken/Hebei/Y2/2009 A/chicken/Shaanxi/xa0414/2013 A/chicken/Shandong/363/2009A/chicken/Shandong/384/2011A/chicken/Shandong/382/2009A/Chicken/Shandong/zc11/2009 A/chicken/Zhejiang/HJ/2007A/chicken/Shandong/SG1/2009A/chicken/Shandong/KD/2009A/chicken/Jiangsu/DT/2009 A/chicken/Guangxi/G8/2009A/chicken/Shuanggou/1/2011A/chicken/Xigou/1/2011A/chicken/Dawang/1/2011 at right of each leaf node indicates year of isolation A/chicken/Zhejiang/611/2011A/chicken/China/AH-10-01/2010A/chicken/Jiangsu/ZJDT/2011A/chicken/Jiangsu/DT0112/2012 A/Chicken/Hebei/TS/2008A/Chicken/Hebei/ZR/2010A/Chicken/Hebei/FL/2011A/chicken/Jiangsu/CZLJG/2012 A/Chicken/Shandong/05/2010A/chicken/Shandong/03/2010A/Chicken/Shandong/sd01/2010A/Chicken/Shandong/sd02/2010 A/chicken/Shandong/wf0514/2013A/chicken/Wenzhou/606/2013A/chicken/Wenzhou/598/2013A/chicken/Beijing/1115/2013 A/chicken/Beijing/0331/2013A/chicken/Beijing/0311/2013A/Shanghai/1/2013A/chicken/Jiangsu/HA2/2013 A/Shanghai/4664T/2013A/Shanghai/2/2013A/Shanghai/3/2013A/Shanghai/4/2013 A/chicken/Shandong/yt0106/2012A/Shanghai/02/2013A/chicken/Hebei/0721/2013A/chicken/Shandong/qd01417/2013 A/chicken/Shandong/SDWF17/2012A/chicken/Shangdong/SDWF14/2012A/chicken/Shangdong/SDWF29/2012A/chicken/Beijing/L2/2012 A/chicken/Liaoning/1116/2012A/chicken/Tianjin/614/2012A/chicken/Shandong/qd0920/2012A/chicken/Beijing/HD06/2012 A/chicken/Tianjin/61/2012A/chicken/Beijing/11/2013A/chicken/Hangzhou/48-2/2013A/chicken/Shandong/qd0312/2013 A/chicken/Beijing/0512/2013A/chicken/Shandong/lc0903/2013A/chicken/Shandong/lc0523/2013A/chicken/Beijing/325/2013 A/chicken/Shandong/H/2009A/Chicken/Henan/DY/2009A/chicken/Shandong/BD/2010A/Chicken/Hebei/FX05/2010 A/Chicken/Hebei/DF/2008A/Chicken/Shandong/HL/2010A/chicken/Henan/323/2009A/chicken/Shandong/02/2010 A/chicken/Yongcheng/1/2011A/chicken/Jiangsu/XZPZ/2011A/chicken/Tongshan/1/2011A/chicken/Jiawang/2/2011 PB1 A/Chicken/Beijing/HD/2010A/Chicken/Hebei/YT/2010A/chicken/Jiangsu/NTHA1/2012A/chicken/Jiangsu/YCDF/2012 A/chicken/Jiangsu/YCDT/2013 A/chicken/Zhejiang/Q1D4/2010A/Chicken/Sichuan/02/2011A/Chicken/Shandong/06/2011A/chicken/Jiangsu/TZXH/2013 A/Chicken/Shandong/07/2011A/Chicken/Shandong/11/2011A/Chicken/Shandong/11/2010A/Chicken/Shandong/09/2011 A/Chicken/Guangdong/01/2011A/chicken/Guangdong/ZCY/2011A/chicken/Guangdong/ZHJ/2011A/chicken/Shandong/513/2011 (see color bar). Timescale is in years. Vertical black A/chicken/Shanghai/C1/2012A/chicken/Jilin/0519/2012 A/Chicken/Shandong/02/2011A/ChiCken/Shandong/03/2011A/chicken/Zhejiang/607/2011A/chicken/Shanghai/C3/2012 A/chicken/Zhejiang/329/2011A/chicken/Shandong/qd0516/2012A/chicken/Shandong/qd1013/2012A/chicken/Shandong/wf0202/2012 A/chicken/Rizhao/723/2013A/chicken/Rizhao/811/2013A/Chicken/Nanjing/023/2013A/Chicken/Nantong/031-2/2013 A/chicken/Rizhao/15/2013A/chicken/Rizhao/1313/2013A/chicken/Rizhao/85/2013A/chicken/Rizhao/437/2013 A/chicken/Anhui/HF/2010A/chicken/Jiangsu/Q3/2010A/chicken/Rizhao/249/2013A/chicken/Rizhao/651/2013 A/Chicken/Shandong/06/2010A/Chicken/Shandong/10/2010A/Chicken/Shandong/05/2011A/chicken/Jiangsu/XZ6/2012 A/Chicken/Jiangsu/TS/2010A/chicken/Jiangsu/YZ0611/2011A/chicken/Guangdong/LG1/2013A/chicken/Wenzhou/614/2013 A/chicken/Gansu/419/2012A/chicken/Jilin/1031/2012A/chicken/Shandong/qd0917/2013A/chicken/Tianjin/120/2013 A/chicken/Liaoning/0704/2012A/chicken/Shandong/zc0606/2012A/chicken/Shandong/lc0830/2012A/chicken/Shanxi/0703/2012 A/chicken/Qianzhou/12/2010A/chicken/Wuxi/7/2010A/chicken/Shangdong/SDWD/2011A/chicken/Jiangsu/DTNSZ/2011 1994 1997 1998 2001 2002 2003 2004 2005 2006 2012 2013 A/chicken/Hunan/12/2011A/chicken/Shandong/zc4/2012A/chicken/Hebei/0109/2013 1996 1999 2000 2007 2008 2009 2010 2011 A/chicken/Jiangsu/WJYBF/2012A/chicken/Jiangsu/CZZWQ/2012A/chicken/Shanghai/C2/2012A/chicken/Jiangsu/WJHRG/2012 A/chicken/Shandong/qd0427/2012A/chicken/Jiangsu/XZ38/2012A/chicken/Shandong/wf12010/2012A/chicken/Shandong/wf1206/2012 A/chicken/Jiangsu/CZ1/2012A/chicken/Jiangsu/CZJJF/2012A/chicken/Jiangsu/ZJ4/2013A/chicken/Jiangsu/NTTZ/2013 A/chicken/Rizhao/1436/2013A/chicken/Rizhao/853/2013A/chicken/Shandong/yt0711/2013A/chicken/Shandong/wf0712/2013 A/chicken/Jiangsu/YZ0511/2011A/chicken/Jiangsu/WJHDL/2012A/chicken/Rizhao/1363/2013A/chicken/Rizhao/1339/2013 A/chicken/Jiangsu/CZYWP/2012A/chicken/Shandong/qd1115/2012A/chicken/Jiangsu/MYJMF/2012A/chicken/Shandong/qd0307/2013 A/chicken/Shandong/qd1224/2012A/chicken/Hebei/1024/2012A/chicken/Liaoning/0517/2013A/chicken/Shandong/qd0225/2013 lines mark major (long line) and minor (short line) 5.0 A/chicken/Shandong/qd0107/2013 A/chicken/Beijing/F1/2012A/chicken/Jiangsu/XZZSL/2012A/chicken/Shandong/qd0808/2013 A/chicken/Jiangsu/XZMG/2012A/chicken/Jiangsu/XZWHL/2012A/chicken/Jiangsu/XZLZG/2012A/chicken/Rizhao/55/2013 A/chicken/Jiangsu/CZWJ5/2013A/chicken/Hangzhou/50-2/2013A/Chicken/Suzhou/097-2/2013A/chicken/Jiangsu/CZJT2/2013 A/Chicken/Nanjing/106-2/2013A/chicken/Jiangsu/ZJDT1/2013A/chicken/Wenzhou/642/2013A/chicken/Wenzhou/253/2013 A/chicken/Shanghai/020/2013A/chicken/Beijing/16/2013A/Chicken/Beijing/0309/2013A/chicken/Beijing/324/2013 A/Chicken/Nanjing/503/2013 groups of H9N2 viruses, mainly from 2010 through

A/chicken/Henan/ni/2001A/chicken/Beijing/nl/2002 C A/chicken/Henan/nd/1998A/chicken/Jiangsu/ng/2001A/chicken/Jiangsu/wa/2002A/chicken/Hebei/nj/2002 A/chicken/Neimonggu/nk/2002A/chicken/Jiangsu/7/2002A/chicken/Hubei/C1/2007 A/chicken/Jiangsu/YZ4/2012 A/chicken/Heilongjiang/48/01 A/chicken/Shangdong/01/2008A/Chicken/Shanghai/F/98A/chicken/Jiangsu/1/2004 A/chicken/Jiangsu/L1/2004A/chicken/Guangxi/2389/2005A/chicken/Jiangxi/12/2007A/chicken/Zhejiang/HJ/2007 A/chicken/Shanghai/10/01A/chicken/Guangdong/56/01A/chicken/Shantou/1404/2003A/chicken/Jiangsu/L2/2005 A/chicken/Hunan/774/2002A/chicken/Guangxi/1857/2004A/chicken/Guangxi/2441/2004A/chicken/Guangxi/37/2005 A/chicken/Shandong/B1/1998 A/chicken/Guangxi/187/2005A/chicken/Guangxi/521/2005A/chicken/Shandong/317/2007 A/chicken/Shandong/237/2007A/chicken/Shandong/ZB/2007A/chicken/Zibo/B1/2008A/chicken/Henan/323/2009 A/chicken/Shandong/B2/2007A/chicken/Shandong/B3/2007A/Chicken/Shandong/LY/2008A/chicken/Shandong/02/2008 A/chicken/Shandong/LY/2006A/chicken/Shandong/251/2007A/chicken/Hebei/0329/2007A/chicken/Shandong/B4/2007 A/chicken/Hunan/6108/2005A/chicken/Hunan/4444/2005A/chicken/Hunan/5260/2005 A/chicken/Shaanxi/11/2012 A/chicken/Jiangsu/245/2004 A/chicken/Shandong/PD/2009A/chicken/Shandong/382/2009A/chicken/Shandong/384/2011 2013. Black arrows mark the position of H7N9 viru- A/chicken/Shandong/363/2009A/chicken/Shandong/SG1/2009A/Chicken/Shandong/zc11/2009 A/chicken/Hebei/7/2008A/chicken/Henan/1.2/2008A/chicken/Anhui/10/2009A/chicken/Hebei/Y2/2009 A/chicken/Hebei/C4/2008A/chicken/Hebei/A/2007A/chicken/Hebei/4/2008A/Chicken/Henan/DY/2009 A/chicken/Jiangsu/ZJDT/2011A/chicken/Wujin/19/2011A/Chicken/Shandong/05/2011A/chicken/Zhejiang/329/2011 A/chicken/Dawang/1/2011A/chicken/Xigou/1/2011A/chicken/Shuanggou/1/2011A/Chicken/Hebei/FL/2011 A/chicken/Shandong/22/2008A/Chicken/Hebei/TS/2008A/Chicken/Hebei/ZR/2010A/Chicken/Shandong/JL/2008 A/chicken/Shandong/377/2009A/chicken/Taixing/10/2010A/chicken/Yangzhou/11/2010A/chicken/China/AH-10-01/2010 A/Chicken/Beijing/HD/2008A/chicken/Jiangsu/DT0112/2012A/chicken/Hunan/1/2012A/chicken/Jiangsu/CZLJG/2012 A/chicken/Shandong/BD/2008A/chicken/Shandong/12/2008A/Chicken/Shandong/zc2/2009A/chicken/Shandong/SG2/2009 A/chicken/Shandong/1231/2008A/chicken/Tibet/S4/2009A/Chicken/Shandong/zc12/2009A/chicken/Tibet/S1/2009 A/chicken/Shandong/01/2009A/chicken/Shandong/02/2009A/chicken/Hubei/10/2009A/chicken/Shandong/01/2010 A/Chicken/Hebei/DF/2008A/chicken/Shandong/KD/2009A/chicken/Jiangsu/DT/2009A/chicken/Guangxi/G8/2009 A/Chicken/Shandong/11/2010A/Chicken/Shandong/06/2011A/Chicken/Shandong/07/2011A/Chicken/Shandong/09/2011 A/chicken/Shandong/H/2009A/chicken/Shandong/02/2010A/Chicken/Shandong/11/2011A/Chicken/Sichuan/02/2011 A/chicken/Shandong/397/2010A/Chicken/Hebei/FX05/2010A/chicken/Xiangshui/1/2011A/chicken/Jiawang/2/2011 A/Chicken/Shandong/06/2010A/chicken/Yongcheng/1/2011A/chicken/Jiangsu/XZPZ/2011A/chicken/Tongshan/1/2011 A/Chicken/Beijing/HD/2010A/Chicken/Hebei/YT/2010A/chicken/Jiangsu/YCDF/2012A/chicken/Jiangsu/YCDT/2013 A/chicken/Jiangsu/Q3/2010A/chicken/Zhejiang/Q1D4/2010A/chicken/Zhejiang/611/2011A/chicken/Jiangsu/TZXH/2013 A/Chicken/Shandong/10/2010A/chicken/Hunan/12/2011A/chicken/Jiangsu/YZ0611/2011A/chicken/Shandong/zc4/2012 ses. Also see Fig. S2. (B) Diversity of genotypes of A/Chicken/Jiangsu/TS/2010 A/chicken/Wuxi/7/2010A/chicken/Anhui/HF/2010A/chicken/Qingdao/04/2012A/chicken/Gansu/419/2012 A/chicken/Jiangsu/YZ0511/2011A/chicken/Beijing/HD06/2012A/chicken/Shandong/SDWF17/2012A/chicken/Tianjin/614/2012 A/chicken/Jilin/0519/2012A/chicken/Shangdong/SDWF29/2012A/chicken/Shangdong/SDWF14/2012A/Chicken/Nanjing/023/2013 A/chicken/Guangdong/ZCY/2011A/chicken/Shandong/513/2011A/Chicken/Guangdong/01/2011A/Chicken/Nantong/031-2/2013 PA A/chicken/Guangdong/ZHJ/2011A/chicken/Rizhao/651/2013A/chicken/Rizhao/723/2013A/chicken/Rizhao/1313/2013 A/chicken/Rizhao/15/2013 A/chicken/Jiangsu/XZ38/2012A/chicken/Rizhao/249/2013A/chicken/Rizhao/437/2013A/chicken/Rizhao/85/2013 A/chicken/Qianzhou/12/2010A/chicken/Shandong/BD/2010A/Chicken/Shandong/HL/2010A/chicken/Jiangsu/DTNSZ/2011 A/chicken/Shandong/03/2010A/Chicken/Shandong/sd01/2010A/Chicken/Shandong/05/2010A/chicken/Shangdong/SDWD/2011 A/Chicken/Shandong/sd02/2010A/chicken/Zhejiang/607/2011A/chicken/Shanghai/C1/2012A/chicken/Shandong/wf0514/2013 A/chicken/Jiangsu/XZ6/2012A/chicken/Wenzhou/642/2013A/chicken/Wenzhou/598/2013A/chicken/Wenzhou/253/2013 A/chicken/Shandong/qd1013/2012A/chicken/Shandong/qd0516/2012A/chicken/Jilin/1031/2012A/chicken/Shandong/yt0106/2012 A/chicken/Shandong/wf0202/2012A/chicken/Shandong/qd0920/2012A/chicken/Shandong/lc0830/2012A/chicken/Beijing/1115/2013 A/chicken/Liaoning/0704/2012A/chicken/Shandong/zc0606/2012A/chicken/Shanxi/0703/2012A/Shanghai/1/2013 Ck/BJ/94 A/Shanghai/2/2013A/chicken/Rizhao/1339/2013A/Shanghai/4664T/2013A/chicken/Hangzhou/50-2/2013 A/chicken/Liaoning/1116/2012A/Shanghai/02/2013A/Shanghai/4/2013A/Shanghai/3/2013 – A/chicken/Beijing/0311/2013 A/chicken/Beijing/L2/2012A/chicken/Shandong/qd0312/2013A/chicken/Beijing/0331/2013A/chicken/Beijing/325/2013 A/chicken/Tianjin/61/2012A/chicken/Shandong/lc0903/2013A/chicken/Tianjin/120/2013A/chicken/Beijing/11/2013 A/ChiCken/Shandong/03/2011A/Chicken/Shandong/02/2011A/chicken/Shandong/lc0523/2013A/chicken/Beijing/0512/2013 H9N2 viruses isolated from chickens in China, 1994 A/chicken/Shanghai/C3/2012A/chicken/Shanghai/C2/2012 A/chicken/Jiangsu/WJYBF/2012A/chicken/Shandong/qd0427/2012A/chicken/Shaanxi/xa0414/2013A/Chicken/Suzhou/097-2/2013 A/chicken/Rizhao/811/2013A/chicken/Hebei/0721/2013A/chicken/Shandong/qd01417/2013A/chicken/Guangdong/LG1/2013 5.0 A/chicken/Jiangsu/XZLZG/2012A/chicken/Rizhao/1436/2013A/chicken/Rizhao/853/2013A/chicken/Rizhao/1363/2013 A/chicken/Jiangsu/XZMG/2012 A/chicken/Beijing/F1/2012A/chicken/Jiangsu/XZZSL/2012A/chicken/Jiangsu/XZWHL/2012A/chicken/Shandong/qd0917/2013 A/chicken/Jiangsu/CZZWQ/2012A/chicken/Jiangsu/CZJJF/2012A/chicken/Shandong/wf12010/2012A/chicken/Jiangsu/WJHDL/2012 A/chicken/Shandong/wf1206/2012A/chicken/Shandong/qd1115/2012A/chicken/Jiangsu/MYJMF/2012A/chicken/Shandong/qd0307/2013 A/chicken/Shandong/qd0808/2013A/chicken/Shandong/qd0107/2013A/chicken/Hebei/0109/2013A/chicken/Liaoning/0517/2013 A/chicken/Jiangsu/WJHRG/2012A/chicken/Shandong/qd0225/2013A/chicken/Hebei/1024/2012A/chicken/Shandong/qd1224/2012 A/chicken/Jiangsu/CZYWP/2012A/chicken/Jiangsu/ZJDT1/2013A/Chicken/Nanjing/106-2/2013A/chicken/Shanghai/020/2013 A/Chicken/Beijing/0309/2013A/Chicken/Nanjing/503/2013A/chicken/Jiangsu/HA2/2013A/chicken/Beijing/324/2013 A/chicken/Jiangsu/CZ1/2012A/chicken/Jiangsu/NTTZ/2013A/chicken/Beijing/16/2013A/chicken/Shandong/wf0712/2013 A/chicken/Wenzhou/614/2013A/chicken/Hangzhou/48-2/2013A/chicken/Shandong/yt0711/2013A/chicken/Wenzhou/606/2013 A/chicken/Jiangsu/CZJT2/2013A/chicken/Rizhao/55/2013A/chicken/Jiangsu/ZJ4/2013A/chicken/Jiangsu/CZWJ5/2013 2013. Here are shown the recurrent genotypes that

A/chicken/Shandong/1/2004 A/chicken/Jiangsu/1/2002A/chicken/Anhui/2/2002A/chicken/Guangxi/2389/2005A/chicken/Shanghai/Q0702/2007 A/chicken/Jiangsu/1/1999A/chicken/Shanghai/16/2001A/chicken/Shanghai/12/2001A/chicken/Shanghai/Y1/2002 A/chicken/Beijing/ne/1999A/chicken/Henan/ni/2001A/chicken/Jiangsu/245/2004A/chicken/Jiangsu/L1/2004 A/chicken/Henan/nd/1998A/chicken/Heilongjiang/48/01A/chicken/Hebei/nj/2002A/chicken/Beijing/nl/2002 A/chicken/Jiangsu/ng/2001A/chicken/Yunnan/nh/2001A/chicken/Shanghai/14/2001 A/chicken/Jiangsu/YZ4/2012 A/chicken/Shanghai/2/1999A/chicken/Jiangsu/wa/2002 A/chicken/Jiangsu/1/2004A/Chicken/Shanghai/F/98 A/chicken/Neimonggu/nk/2002A/chicken/Jiangsu/7/2002A/chicken/Shantou/3040/2003 A/chicken/Hubei/C1/2007 A/chicken/Shantou/4617/2003A/chicken/Shantou/4435/2003A/chicken/Shantou/3341/2003A/chicken/Shantou/99/2004 A/chicken/Shantou/5630/2004A/chicken/Shantou/6319/2004A/chicken/Shantou/6911/2004 A/chicken/Henan/321/2008 A/chicken/Henan/L3/2008A/chicken/Henan/L2/2008A/chicken/Henan/L1/2008A/chicken/Henan/323/2008 A/chicken/Shantou/4726/2004A/chicken/Shantou/6781/2005A/chicken/Fujian/5683/2005A/chicken/Fujian/4332/2005 A/chicken/Fujian/7884/2005A/chicken/Fujian/7386/2005A/chicken/Shantou/5269/2005A/chicken/Fujian/4820/2005 were persistent across at least 2 y. All of the geno- A/chicken/Fujian/6960/2005 A/chicken/Shantou/1138/2004A/chicken/Fujian/3080/2005A/chicken/Fujian/5214/2005A/chicken/Fujian/6188/2005 A/chicken/Hunan/5700/2005A/chicken/Beijing/7/2005A/chicken/Beijing/1/2006A/chicken/Beijing/20/2006 A/chicken/Hunan/4444/2005A/chicken/Guangxi/4745/2005A/chicken/Hunan/2536/2005A/chicken/Guangxi/55/2005 A/chicken/Guangxi/1428/2005A/chicken/Hunan/3369/2005A/chicken/Guangxi/1032/2005A/chicken/Hunan/2903/2005 A/chicken/Shandong/1/2006A/chicken/Hebei/0822/2007A/chicken/Hebei/C6/2007A/chicken/Hebei/LC/2008 A/chicken/Shantou/9909/2005A/chicken/Shantou/19465/2005A/chicken/Hebei/0617/2007A/chicken/Shandong/lx929/2007 A/chicken/Fujian/10954/2005A/chicken/Shantou/22054/2005A/chicken/Shantou/22504/2005A/chicken/Shandong/lx1023/2007 A/chicken/Fujian/11302/2005A/chicken/Fujian/12252/2005A/chicken/Fujian/11488/2005A/chicken/Fujian/9104/2005 A/chicken/Fujian/8341/2005A/chicken/Shantou/17139/2005A/chicken/Shantou/14907/2005A/chicken/Shantou/20817/2005 A/chicken/Fujian/10308/2005A/chicken/Fujian/9290/2005A/chicken/Fujian/9752/2005 A/chicken/Fujian/G9/2009 A/chicken/Shanghai/3/2000A/chicken/Shanghai/10/01A/chicken/Shantou/2686/2003A/chicken/Shantou/1404/2003 A/chicken/Shanghai/2/2001A/chicken/Shanghai/1/2001 A/chicken/Jiangsu/L2/2005A/chicken/Shanghai/7/2001 A/chicken/Guangdong/56/01A/chicken/Hunan/774/2002A/chicken/Henan/1/2002A/chicken/Guangxi/37/2005 A/chicken/Guangxi/1857/2004A/chicken/Guangxi/2441/2004A/chicken/Guangxi/187/2005A/chicken/Guangxi/521/2005 A/chicken/Shanghai/1/2002A/chicken/Shanghai/2/2002A/chicken/Jiangsu/2/2001A/chicken/Henan/1/2003 A/chicken/Henan/2/2004A/chicken/Henan/1/2004A/chicken/Henan/1/2005A/chicken/Shanghai/Y2/2007 A/chicken/Shanghai/Y1/2006A/chicken/Shanghai/Q0704/2007A/chicken/Shanghai/Y2/2008A/chicken/Shanghai/Y1/2008 A/chicken/Hunan/5260/2005A/chicken/Hunan/6108/2005A/chicken/Shandong/LY/2006A/chicken/Hebei/0329/2007 A/chicken/Shandong/251/2007A/chicken/Shandong/B4/2007A/chicken/Hebei/4/2008A/chicken/Guangdong/G7/2009 A/chicken/Hebei/C4/2008A/chicken/Hebei/7/2008A/chicken/Henan/1.2/2008A/chicken/Hebei/Y2/2009 A/chicken/Hebei/A/2007 A/chicken/Jilin/1031/2012A/chicken/Shanxi/0703/2012A/chicken/Liaoning/0704/2012 types can be found in Dataset S1.(C) Summary of A/Chicken/Beijing/HD/2008A/Chicken/Shandong/HL/2010A/chicken/Shandong/zc0606/2012A/chicken/Shandong/lc0830/2012 A/chicken/Shandong/BD/2008A/chicken/Zibo/L2/2008A/Chicken/Shandong/LY/2008A/Chicken/Henan/DY/2009 A/chicken/Anhui/10/2009A/chicken/Jiangsu/ZJDT/2011A/chicken/Jiangsu/DT0112/2012A/chicken/Jiangsu/CZLJG/2012 A/chicken/Taixing/10/2010A/chicken/Yangzhou/11/2010A/chicken/China/AH-10-01/2010A/Chicken/Shandong/02/2011 A/Chicken/Shandong/05/2011A/chicken/Hunan/12/2011A/chicken/Hunan/1/2012A/chicken/Shandong/qd0917/2013 A/chicken/Xigou/1/2011A/chicken/Shuanggou/1/2011A/chicken/Dawang/1/2011A/chicken/Shaanxi/xa0414/2013 A/chicken/Rizhao/15/2013A/chicken/Rizhao/651/2013A/Chicken/Nanjing/023/2013A/Chicken/Nantong/031-2/2013 A/chicken/Rizhao/723/2013A/chicken/Rizhao/85/2013A/chicken/Rizhao/249/2013A/chicken/Rizhao/437/2013 A/chicken/Shanghai/Q0712/2007A/chicken/Shanghai/Q0709/2007A/chicken/Shanghai/Q0801-2/2008 A/chicken/Rizhao/1313/2013 A/chicken/Shanghai/Q0801-1/2008A/chicken/Shanghai/Q0808-1/2008A/chicken/Hubei/10/2009A/chicken/Shandong/01/2009 A/chicken/Shandong/1231/2008A/chicken/Shandong/02/2009A/chicken/Shandong/SG2/2009A/chicken/Shandong/01/2010 A/chicken/Shanghai/Y2/2006A/chicken/Shanghai/Y1/2007A/chicken/Shanghai/Q0707/2007A/Chicken/Shandong/zc12/2009 A/chicken/Zhejiang/HJ/2007A/chicken/Jiangxi/12/2007A/chicken/Shandong/22/2008A/chicken/Shandong/12/2008 A/chicken/Shandong/02/2008A/Chicken/Shandong/zc2/2009A/chicken/Tibet/S4/2009A/chicken/Tibet/S1/2009 A/chicken/Shandong/B2/2007A/chicken/Shandong/317/2007A/chicken/Shandong/H/2009A/chicken/Henan/323/2009 NP A/chicken/Shandong/B3/2007A/chicken/Zibo/B1/2008A/chicken/Shandong/ZB/2007A/chicken/Shandong/L1/2007 A/chicken/Shandong/363/2009A/Chicken/Shandong/zc11/2009 A/chicken/Shandong/SG1/2009A/chicken/Shandong/PD/2009A/chicken/Shandong/382/2009A/chicken/Shandong/384/2011 A/chicken/Shandong/KD/2009A/chicken/Shandong/377/2009A/chicken/Jiangsu/DT/2009A/chicken/Shaanxi/11/2012 A/Chicken/Shandong/05/2010A/chicken/Jiangsu/YZ0611/2011A/chicken/Xiangshui/1/2011A/chicken/Zhejiang/607/2011 A/Chicken/Shandong/sd02/2010A/Chicken/Shandong/10/2010A/chicken/Shandong/zc4/2012A/chicken/Jiangsu/XZ6/2012 A/chicken/Shandong/02/2010A/Chicken/Beijing/HD/2010A/chicken/Tongshan/1/2011A/chicken/Jiangsu/XZPZ/2011 the genesis of genotype G57 by reassortment A/Chicken/Shandong/06/2010A/Chicken/Hebei/YT/2010A/chicken/Yongcheng/1/2011 A/chicken/Jiawang/2/2011A/chicken/Jiangsu/NTHA1/2012A/chicken/Jiangsu/YCDF/2012A/chicken/Jiangsu/YCDT/2013 A/Chicken/Hebei/ZR/2010A/Chicken/Hebei/FL/2011A/chicken/Beijing/L2/2012A/chicken/Jiangsu/TZXH/2013 A/chicken/Beijing/HD06/2012A/chicken/Tianjin/614/2012A/chicken/Tianjin/61/2012A/chicken/Shandong/wf0514/2013 A/Chicken/Shandong/sd01/2010A/chicken/Beijing/0512/2013A/chicken/Shandong/lc0903/2013A/chicken/Shandong/lc0523/2013 A/chicken/Shanghai/Q0808-2/2008A/chicken/Shanghai/Q0812-2/2008A/chicken/Shanghai/Q0812-1/2008A/chicken/Guangxi/G8/2009 A/Chicken/Hebei/DF/2008A/Chicken/Shandong/JL/2008A/Chicken/Hebei/TS/2008A/Chicken/Hebei/FX05/2010 A/Chicken/Shandong/06/2011A/Chicken/Sichuan/02/2011A/Chicken/Shandong/09/2011A/Chicken/Shandong/11/2011 A/ChiCken/Shandong/03/2011A/Chicken/Shandong/07/2011A/Chicken/Shandong/11/2010A/chicken/Shanghai/C1/2012 A/chicken/Shandong/397/2010A/Chicken/Jiangsu/TS/2010A/Chicken/Suzhou/097-2/2013A/Shanghai/1/2013 A/chicken/Zhejiang/Q1D4/2010A/chicken/Shandong/03/2010A/chicken/Shandong/513/2011A/chicken/Guangdong/LG1/2013 A/chicken/Shandong/BD/2010A/chicken/Guangdong/ZCY/2011A/chicken/Guangdong/ZHJ/2011A/Chicken/Guangdong/01/2011 A/chicken/Jilin/0519/2012A/chicken/Wenzhou/642/2013A/chicken/Wenzhou/598/2013A/chicken/Wenzhou/253/2013 A/chicken/Wuxi/7/2010A/chicken/Zhejiang/329/2011A/chicken/Gansu/419/2012A/chicken/Beijing/0331/2013 A/chicken/Jiangsu/Q3/2010A/chicken/Shangdong/SDWD/2011A/chicken/Qianzhou/12/2010A/chicken/Jiangsu/DTNSZ/2011 A/chicken/Anhui/HF/2010A/chicken/Shandong/qd0920/2012A/chicken/Shandong/wf0202/2012A/chicken/Shandong/qd0516/2012 A/chicken/Zhejiang/611/2011A/chicken/Shandong/qd1013/2012A/chicken/Shanghai/C2/2012A/chicken/Shanghai/C3/2012 A/chicken/Jiangsu/YZ0511/2011A/chicken/Shandong/yt0106/2012A/chicken/Jiangsu/WJYBF/2012A/chicken/Beijing/0311/2013 A/chicken/Liaoning/1116/2012A/chicken/Shandong/qd0312/2013A/chicken/Beijing/16/2013A/chicken/Beijing/325/2013 A/Shanghai/3/2013A/chicken/Rizhao/1339/2013A/Shanghai/4/2013A/Shanghai/2/2013 5.0 A/chicken/Shangdong/SDWF14/2012A/chicken/Shandong/SDWF17/2012A/Shanghai/4664T/2013A/Shanghai/02/2013 A/chicken/Jiangsu/XZMG/2012A/chicken/Shangdong/SDWF29/2012 events. Virus particles are represented by ovals. The A/chicken/Jiangsu/XZZSL/2012A/chicken/Jiangsu/XZWHL/2012A/chicken/Jiangsu/XZLZG/2012 A/chicken/Jiangsu/XZ38/2012A/chicken/Jiangsu/WJHRG/2012A/chicken/Beijing/F1/2012A/chicken/Jiangsu/CZZWQ/2012 A/chicken/Shandong/qd0427/2012A/chicken/Hangzhou/50-2/2013A/chicken/Jiangsu/CZWJ5/2013A/chicken/Jiangsu/NTTZ/2013 A/chicken/Shandong/wf0712/2013A/chicken/Beijing/11/2013A/chicken/Jiangsu/ZJDT1/2013A/chicken/Tianjin/120/2013 Qa/HK/97 Ck/SH/98 Ck/JS/00 A/chicken/Rizhao/55/2013A/chicken/Shandong/yt0711/2013A/chicken/Rizhao/811/2013 A/chicken/Shandong/wf12010/2012A/chicken/Shandong/wf1206/2012A/chicken/Hebei/0721/2013A/chicken/Beijing/1115/2013 A/chicken/Jiangsu/CZJJF/2012A/chicken/Jiangsu/WJHDL/2012A/chicken/Jiangsu/MYJMF/2012A/chicken/Jiangsu/CZYWP/2012 A/chicken/Hebei/1024/2012A/chicken/Rizhao/853/2013A/chicken/Rizhao/1436/2013A/chicken/Rizhao/1363/2013 A/chicken/Shandong/qd1115/2012A/chicken/Shandong/qd0808/2013A/chicken/Hebei/0109/2013A/chicken/Shandong/qd0107/2013 A/chicken/Jiangsu/ZJ4/2013A/chicken/Jiangsu/CZJT2/2013A/chicken/Shandong/qd0225/2013A/chicken/Shandong/qd0307/2013 A/chicken/Shandong/qd1224/2012A/chicken/Liaoning/0517/2013A/chicken/Shanghai/020/2013A/chicken/Shandong/qd01417/2013 A/chicken/Wenzhou/606/2013A/chicken/Wenzhou/614/2013A/chicken/Jiangsu/HA2/2013A/chicken/Hangzhou/48-2/2013 A/chicken/Beijing/324/2013A/Chicken/Nanjing/106-2/2013A/Chicken/Nanjing/503/2013A/Chicken/Beijing/0309/2013 eight gene segments are horizontal bars (from the

A/chicken/Hunan/4246/2005 A/chicken/Shantou/19465/2005A/chicken/Fujian/11302/2005A/chicken/Shantou/22054/2005 A/chicken/Fujian/G9/2009 A/chicken/Shantou/20817/2005A/chicken/Shantou/17139/2005A/chicken/Fujian/10954/2005A/chicken/Shantou/14907/2005 A/chicken/Fujian/9104/2005A/chicken/Shantou/9909/2005A/chicken/Fujian/9752/2005A/chicken/Fujian/9290/2005 A/chicken/Fujian/8341/2005A/chicken/Fujian/12252/2005A/chicken/Fujian/11488/2005A/chicken/Fujian/10308/2005 A/chicken/Shantou/6786/2004A/chicken/Shantou/2402/2004A/chicken/Beijing/1/2006A/chicken/Beijing/20/2006 A/chicken/Henan/323/2008A/chicken/Zibo/B1/2008A/chicken/Guangdong/G7/2009A/chicken/Shandong/377/2009 A/chicken/Jiangxi/12/2007A/chicken/Henan/L1/2008A/chicken/Henan/L3/2008A/chicken/Henan/L2/2008 A/chicken/Guangxi/55/2005A/chicken/Guangxi/1428/2005A/chicken/Guangxi/4745/2005A/chicken/Guangxi/1032/2005 A/chicken/Jiangsu/245/2004A/chicken/Beijing/7/2005A/chicken/Hebei/C6/2007A/chicken/Shandong/12/2008 A/chicken/Shandong/1/2006A/Chicken/Beijing/HD/2008A/chicken/Shandong/BD/2008A/Chicken/Shandong/LY/2008 A/chicken/Zhejiang/HJ/2007A/chicken/Shandong/B4/2007A/chicken/Zibo/L2/2008 A/chicken/Shaanxi/11/2012 A/chicken/Hebei/0617/2007A/chicken/Hebei/0822/2007A/chicken/Hebei/LC/2008A/chicken/Hebei/4/2008 A/chicken/Hebei/A/2007A/chicken/Hebei/C4/2008A/chicken/Hebei/Y2/2009A/chicken/Henan/1.2/2008 A/chicken/Shandong/02/2008A/chicken/Hebei/7/2008A/chicken/Tibet/S1/2009A/Chicken/Shandong/zc2/2009 A/chicken/Anhui/10/2009A/Chicken/Henan/DY/2009A/chicken/Hubei/10/2009A/chicken/Tibet/S4/2009 A/chicken/Shandong/1231/2008A/chicken/Shandong/SG2/2009A/chicken/Shandong/01/2009A/Chicken/Shandong/zc12/2009 top: PB2, PB1, PA, HA, NP, NA, M, and NS). Red A/chicken/Shandong/02/2009A/chicken/Shandong/SG1/2009A/chicken/Shandong/01/2010 A/chicken/Shandong/382/2009A/chicken/Shandong/PD/2009A/Chicken/Shandong/zc11/2009A/chicken/Shandong/513/2011 A/chicken/Hunan/3369/2005A/chicken/Hunan/5700/2005A/chicken/Hunan/5260/2005 A/chicken/Shandong/384/2011 A/chicken/Hunan/2536/2005A/chicken/Hunan/2903/2005A/chicken/Hunan/4444/2005A/chicken/Hunan/6108/2005 A/chicken/Shandong/LY/2006A/chicken/Hebei/0329/2007A/chicken/Shandong/22/2008A/Chicken/Shandong/sd01/2010 A/chicken/Shandong/KD/2009A/chicken/Qianzhou/12/2010A/Chicken/Hebei/YT/2010A/chicken/Shangdong/SDWD/2011 A/chicken/Guangxi/G8/2009A/chicken/Jiangsu/DT/2009A/chicken/Shandong/H/2009A/Chicken/Hebei/FX05/2010 A/Chicken/Beijing/HD/2010A/chicken/Shandong/02/2010A/chicken/Zhejiang/Q1D4/2010A/chicken/Xiangshui/1/2011 A/Chicken/Shandong/05/2010A/Chicken/Shandong/HL/2010A/chicken/Shandong/BD/2010A/chicken/Shandong/03/2010 A/Chicken/Shandong/sd02/2010A/chicken/Guangdong/ZHJ/2011A/chicken/Guangdong/ZCY/2011A/Chicken/Guangdong/01/2011 A/Chicken/Hebei/DF/2008 A/Chicken/Sichuan/02/2011A/Chicken/Shandong/06/2011A/chicken/Jilin/0519/2012 A/Chicken/Shandong/11/2011A/Chicken/Shandong/07/2011A/Chicken/Shandong/09/2011 A/Chicken/Shandong/11/2010 A/chicken/Zhejiang/611/2011A/chicken/Jiangsu/ZJDT/2011A/chicken/Jiangsu/DT0112/2012A/chicken/Jiangsu/YZ0511/2011 A/Chicken/Jiangsu/TS/2010A/chicken/Jiangsu/Q3/2010A/chicken/Shanghai/C2/2012A/chicken/Wenzhou/598/2013 A/Chicken/Shandong/10/2010A/Chicken/Shandong/06/2010A/chicken/Tongshan/1/2011A/chicken/Yongcheng/1/2011 A/chicken/Jiangsu/XZPZ/2011A/chicken/Jiawang/2/2011A/chicken/Jiangsu/XZ6/2012A/chicken/Gansu/419/2012 A/chicken/Jiangsu/DTNSZ/2011A/Chicken/Shandong/02/2011A/chicken/Zhejiang/607/2011A/chicken/Shandong/zc4/2012 A/Chicken/Shandong/05/2011A/chicken/Jiangsu/YZ0611/2011A/chicken/Jiangsu/XZ38/2012A/chicken/Jilin/1031/2012 A/chicken/Shandong/yt0106/2012A/Shanghai/1/2013A/chicken/Guangdong/LG1/2013A/chicken/Hangzhou/50-2/2013 A/chicken/Dawang/1/2011A/Shanghai/2/2013A/Shanghai/02/2013A/Shanghai/4664T/2013 A/chicken/China/AH-10-01/2010A/chicken/Shuanggou/1/2011A/chicken/Xigou/1/2011A/chicken/Hunan/12/2011 segments are clades of G57; and blue segments are A/chicken/Shanxi/0703/2012A/chicken/Liaoning/0704/2012A/chicken/Hunan/1/2012A/chicken/Shandong/zc0606/2012 A/chicken/Shandong/lc0830/2012A/chicken/Liaoning/0517/2013A/chicken/Shandong/qd0225/2013A/chicken/Hebei/0109/2013 A/chicken/Hebei/1024/2012A/chicken/Shandong/qd1115/2012A/chicken/Shandong/qd1224/2012A/chicken/Shandong/qd0107/2013 M A/Chicken/Shandong/JL/2008 A/chicken/Shandong/qd0307/2013A/chicken/Hebei/0721/2013A/chicken/Shandong/qd0808/2013 A/chicken/Jiangsu/CZJT2/2013A/chicken/Shandong/qd01417/2013 A/Chicken/Nanjing/503/2013A/Chicken/Beijing/0309/2013A/chicken/Jiangsu/ZJDT1/2013A/chicken/Jiangsu/HA2/2013 A/chicken/Beijing/324/2013A/chicken/Shandong/yt0711/2013A/chicken/Shandong/wf0712/2013A/Chicken/Nanjing/106-2/2013 A/chicken/Jiangsu/NTTZ/2013A/chicken/Beijing/1115/2013A/Chicken/Suzhou/097-2/2013A/chicken/Shanghai/020/2013 A/chicken/Rizhao/853/2013A/chicken/Jiangsu/CZ1/2012A/chicken/Rizhao/1339/2013A/chicken/Beijing/0331/2013 A/chicken/Wenzhou/253/2013A/chicken/Rizhao/1363/2013A/chicken/Rizhao/1436/2013A/chicken/Hangzhou/48-2/2013 A/Chicken/Hebei/FL/2011A/chicken/Wenzhou/642/2013A/chicken/Jiangsu/ZJ4/2013A/chicken/Wenzhou/614/2013 A/Chicken/Hebei/TS/2008A/Chicken/Hebei/ZR/2010A/chicken/Shanghai/C1/2012A/chicken/Liaoning/1116/2012 A/chicken/Beijing/0311/2013A/chicken/Wenzhou/606/2013A/chicken/Beijing/16/2013A/chicken/Shandong/qd0312/2013 A/chicken/Rizhao/811/2013A/chicken/Rizhao/55/2013A/chicken/Shaanxi/xa0414/2013A/chicken/Shandong/qd0917/2013 A/chicken/Rizhao/85/2013A/chicken/Rizhao/15/2013A/chicken/Rizhao/651/2013A/chicken/Rizhao/723/2013 A/chicken/Rizhao/437/2013A/chicken/Rizhao/1313/2013A/chicken/Rizhao/249/2013A/Chicken/Nantong/031-2/2013 A/chicken/Taixing/10/2010A/chicken/Wuxi/7/2010A/chicken/Yangzhou/11/2010 A/Chicken/Nanjing/023/2013 A/chicken/Anhui/HF/2010A/chicken/Jiangsu/CZYWP/2012A/chicken/Jiangsu/WJHDL/2012A/chicken/Jiangsu/MYJMF/2012 A/ChiCken/Shandong/03/2011A/chicken/Jiangsu/WJYBF/2012A/chicken/Shandong/qd0427/2012A/chicken/Jiangsu/WJHRG/2012 A/chicken/Shandong/qd1013/2012A/chicken/Shandong/qd0516/2012A/chicken/Shandong/wf0202/2012A/chicken/Jiangsu/CZZWQ/2012 A/chicken/Shanghai/C3/2012A/chicken/Shandong/wf12010/2012A/chicken/Shandong/wf1206/2012A/chicken/Jiangsu/CZJJF/2012 other clades. A broken arrow indicates an inter- A/chicken/Jiangsu/CZWJ5/2013A/chicken/Shandong/SDWF17/2012A/chicken/Jiangsu/CZLJG/2012 A/chicken/Zhejiang/329/2011A/chicken/Shangdong/SDWF29/2012A/chicken/Jiangsu/XZZSL/2012A/chicken/Shangdong/SDWF14/2012 5.0 A/chicken/Jiangsu/XZLZG/2012A/chicken/Jiangsu/XZMG/2012A/chicken/Beijing/F1/2012A/chicken/Jiangsu/XZWHL/2012 A/chicken/Beijing/0512/2013A/chicken/Shandong/lc0903/2013 A/chicken/Tianjin/614/2012A/chicken/Shandong/qd0920/2012A/chicken/Beijing/HD06/2012A/chicken/Shandong/lc0523/2013 A/chicken/Tianjin/61/2012A/chicken/Beijing/L2/2012A/chicken/Beijing/11/2013A/chicken/Beijing/325/2013 A/chicken/Tianjin/120/2013A/chicken/Shandong/wf0514/2013 species gene transmission. Ck/BJ/94, A/chicken/Beijing/ 1/1994; Qa/HK/97, A/quail/HongKong/G1/1997; Ck/SH/ A/chicken/Anhui/1/1998 A/chicken/Shandong/1/1999A/chicken/Shijiazhuang/2/98A/Chicken/Shijiazhuang/2/99A/chicken/Shandong/1/2000 Dk/ST/04 Ck/HuN/05 A/chicken/Qixian/A1/2001A/chicken/Shandong/241/2001 A/chicken/Shanghai/14/2001A/chicken/Shandong/244/2002A/chicken/Henan/L1/2002 A/chicken/Jiangsu/YZ4/2012 A/chicken/Shangqiu/A8/2000A/chicken/Qixian/A1/2002 A/Chicken/Shanghai/F/98A/chicken/Jiangsu/1/2004 A/chicken/Xuchang/A10/2000A/chicken/Henan/1/2002A/chicken/Hunan/774/2002A/chicken/Kaifeng/A1/2005 A/chicken/Guangdong/56/01A/chicken/Henan/1/2003A/chicken/Guangxi/37/2005A/chicken/Guangxi/521/2005 A/chicken/Shanghai/1/2001A/chicken/Guangxi/187/2005A/chicken/Guangxi/1857/2004A/chicken/Guangxi/2441/2004 A/chicken/Zhuhai/A1/2001A/chicken/Shanghai/2/1999A/chicken/Shanghai/7/2001A/chicken/Shantou/1404/2003 A/chicken/Shandong/251/2007A/Chicken/Shandong/LY/2008A/chicken/Shandong/B4/2007A/chicken/Shandong/02/2008 A/chicken/Hebei/4/2008A/chicken/Anhui/10/2009A/Chicken/Henan/DY/2009A/chicken/Hebei/Y2/2009 A/chicken/Hebei/A/2007A/chicken/Henan/1.2/2008A/chicken/Hebei/C4/2008A/chicken/Hebei/7/2008 A/chicken/Guangdong/A1/2003A/chicken/Henan/1/2004A/chicken/Henan/2/2004A/chicken/Henan/1/2005 A/chicken/Hunan/5260/2005A/chicken/Hunan/4444/2005A/chicken/Shandong/LY/2006A/chicken/Hebei/0329/2007 A/chicken/Hunan/6108/2005A/chicken/Zhejiang/HJ/2007A/chicken/Shandong/12/2008A/chicken/Hubei/10/2009 A/chicken/Shandong/1231/2008A/Chicken/Shandong/zc2/2009A/chicken/Shandong/KD/2009A/chicken/Shaanxi/11/2012 A/Chicken/Shandong/zc12/2009A/chicken/Shandong/SG2/2009A/chicken/Shandong/01/2009A/chicken/Shandong/01/2010 A/chicken/Shandong/02/2009A/chicken/Tibet/S4/2009A/chicken/Tibet/S1/2009A/chicken/China/AH-10-01/2010 A/chicken/Taixing/10/2010A/chicken/Yangzhou/11/2010A/chicken/Dawang/1/2011A/chicken/Shuanggou/1/2011 A/chicken/Xigou/1/2011A/chicken/Jiangsu/ZJDT/2011A/chicken/Jiangsu/CZLJG/2012A/chicken/Jiangsu/DT0112/2012 98, A/chicken/Shanghai/F/1998; Ck/JS/00, A/chicken/ A/chicken/Zhejiang/NH5/2013A/Shanghai/4/2013A/Chicken/Suzhou/097-2/2013A/chicken/Hangzhou/48-2/2013 A/Shanghai/2/2013A/Shanghai/1/2013A/chicken/Hangzhou/50-2/2013A/chicken/Jiangsu/HA2/2013 A/Chicken/Hebei/ZR/2010A/Chicken/Hebei/FL/2011A/chicken/Hunan/12/2011A/Shanghai/02/2013 A/chicken/Tianjin/61/2012A/chicken/Beijing/HD06/2012A/chicken/Shandong/qd0920/2012A/chicken/Tianjin/614/2012 A/chicken/Wenzhou/606/2013A/chicken/Shaanxi/xa0414/2013A/chicken/Wenzhou/598/2013A/chicken/Shandong/wf0514/2013 A/chicken/Shangdong/SDWF14/2012A/chicken/Shandong/SDWF17/2012A/chicken/Jiangsu/XZZSL/2012A/chicken/Shangdong/SDWF29/2012 A/chicken/Jiangsu/XZMG/2012A/chicken/Jiangsu/XZLZG/2012A/chicken/Jiangsu/XZWHL/2012A/chicken/Beijing/F1/2012 A/chicken/Shanghai/1/2002A/chicken/Shanghai/2/2002 A/chicken/Shandong/BD/2008A/Chicken/Beijing/HD/2008 A/chicken/Jiangxi/12/2007A/chicken/Zibo/L2/2008A/chicken/Zibo/B1/2008A/chicken/Shandong/363/2009 A/chicken/Henan/323/2008A/chicken/Shandong/382/2009A/chicken/Shandong/SG1/2009A/Chicken/Shandong/zc11/2009 A/chicken/Shandong/22/2008A/chicken/Shandong/PD/2009A/chicken/Shandong/WL/2009A/chicken/Shandong/384/2011 A/chicken/Shandong/317/2007A/chicken/Shandong/237/2007A/chicken/Shandong/B3/2007A/chicken/Henan/323/2009 A/chicken/Shandong/ZB/2007A/chicken/Shandong/L1/2007A/chicken/Shandong/B2/2007A/Chicken/Shandong/JL/2008 NS A/Chicken/Hebei/DF/2008A/chicken/Shandong/377/2009A/chicken/Xiangshui/1/2011A/chicken/Shandong/zc4/2012 A/Chicken/Shandong/06/2011A/Chicken/Shandong/11/2011A/Chicken/Shandong/09/2011A/Chicken/Sichuan/02/2011 A/chicken/Guangxi/G8/2009A/chicken/Qianzhou/12/2010A/Chicken/Shandong/07/2011A/Chicken/Shandong/11/2010 A/Chicken/Shandong/sd01/2010A/chicken/Shangdong/SDWD/2011A/chicken/Zhejiang/Q1D4/2010A/Chicken/Jiangsu/TS/2010 A/Chicken/Shandong/10/2010A/Chicken/Shandong/06/2010A/chicken/Shanghai/C2/2012A/chicken/Shanghai/C3/2012 A/chicken/Wuxi/7/2010A/Chicken/Shandong/02/2011A/Chicken/Shandong/05/2011A/ChiCken/Shandong/03/2011 A/Chicken/Hebei/YT/2010A/chicken/Jiangsu/NTHA1/2012A/chicken/Jiangsu/YCDF/2012A/chicken/Jiangsu/YCDT/2013 A/chicken/Jiangsu/Q3/2010A/chicken/Jiangsu/DTNSZ/2011A/chicken/Shanxi/0703/2012A/chicken/Jiangsu/TZXH/2013 Jiangsu/1/2000; Ck/HuN/05, A/chicken/Hunan/5260/ A/chicken/Jilin/1031/2012 A/chicken/Zhejiang/607/2011A/chicken/Liaoning/0704/2012A/chicken/Shandong/lc0830/2012A/chicken/Shandong/zc0606/2012 A/chicken/Zhejiang/611/2011A/Chicken/Guangdong/01/2011A/chicken/Guangdong/ZCY/2011A/chicken/Guangdong/ZHJ/2011 A/Chicken/Shandong/sd02/2010A/chicken/Shandong/03/2010A/chicken/Jilin/0519/2012A/chicken/Guangdong/LG1/2013 A/Chicken/Shandong/05/2010A/chicken/Jiangsu/XZ38/2012A/chicken/Hunan/1/2012A/chicken/Shandong/yt0106/2012 A/chicken/Zhejiang/329/2011A/chicken/Shandong/513/2011A/chicken/Gansu/419/2012A/chicken/Shandong/qd0917/2013 A/chicken/Shandong/qd0516/2012A/chicken/Shandong/wf0202/2012A/chicken/Shandong/qd1013/2012A/chicken/Jiangsu/XZ6/2012 A/chicken/Shanghai/C1/2012A/Chicken/Nantong/031-2/2013A/Chicken/Nanjing/023/2013A/chicken/Rizhao/15/2013 A/chicken/Rizhao/85/2013A/chicken/Rizhao/1313/2013A/chicken/Rizhao/811/2013A/chicken/Rizhao/55/2013 A/chicken/Rizhao/437/2013A/chicken/Rizhao/723/2013A/chicken/Rizhao/651/2013A/chicken/Rizhao/249/2013 A/chicken/Jiangsu/DT/2009A/chicken/Shandong/H/2009A/Chicken/Hebei/FX05/2010A/Chicken/Hebei/TS/2008 A/Chicken/Beijing/HD/2010A/chicken/Shandong/BD/2010A/Chicken/Shandong/HL/2010A/chicken/Shandong/02/2010 A/chicken/Shandong/397/2010A/chicken/Jiawang/2/2011A/chicken/Tongshan/1/2011A/chicken/Yongcheng/1/2011 A/chicken/Jiangsu/XZPZ/2011A/chicken/Jiangsu/WJHRG/2012A/chicken/Jiangsu/WJYBF/2012A/chicken/Shandong/qd0427/2012 A/chicken/Jiangsu/YZ0511/2011A/chicken/Jiangsu/YZ0611/2011A/chicken/Jiangsu/CZZWQ/2012A/chicken/Jiangsu/CZJT2/2013 A/chicken/Shandong/wf12010/2012A/chicken/Shandong/wf1206/2012A/chicken/Beijing/1115/2013A/chicken/Jiangsu/ZJ4/2013 A/chicken/Beijing/L2/2012A/chicken/Jiangsu/CZWJ5/2013A/chicken/Wenzhou/642/2013A/chicken/Wenzhou/614/2013 A/chicken/Liaoning/1116/2012A/chicken/Shandong/qd0312/2013A/chicken/Beijing/16/2013A/chicken/Shandong/lc0903/2013 A/chicken/Beijing/11/2013A/chicken/Shandong/lc0523/2013A/chicken/Beijing/325/2013A/chicken/Beijing/0512/2013 A/chicken/Shandong/qd1224/2012A/chicken/Shandong/qd1115/2012A/chicken/Shandong/qd0307/2013A/chicken/Shandong/qd0808/2013 A/chicken/Shandong/qd0225/2013A/chicken/Hebei/0109/2013A/chicken/Liaoning/0517/2013A/chicken/Shandong/qd0107/2013 A/chicken/Jiangsu/CZJJF/2012A/chicken/Jiangsu/CZYWP/2012A/chicken/Jiangsu/WJHDL/2012A/chicken/Jiangsu/MYJMF/2012 2005; Dk/ST/04, A/duck/Shantou/163/2004; Ck/ZJ/07, A/chicken/Hebei/0721/2013A/chicken/Shandong/qd01417/2013A/chicken/Beijing/0331/2013A/chicken/Tianjin/120/2013 A/chicken/Jiangsu/CZ1/2012A/chicken/Wenzhou/253/2013A/chicken/Jiangsu/ZJDT1/2013A/chicken/Jiangsu/NTTZ/2013 A/chicken/Rizhao/853/2013A/chicken/Rizhao/1436/2013A/chicken/Rizhao/1363/2013A/chicken/Shandong/wf0712/2013 A/chicken/Shandong/yt0711/2013A/chicken/Beijing/0311/2013A/chicken/Shanghai/020/2013A/chicken/Rizhao/1339/2013 5.0 A/chicken/Beijing/324/2013A/Chicken/Beijing/0309/2013A/Chicken/Nanjing/106-2/2013A/Chicken/Nanjing/503/2013 A/chicken/Zhejiang/HJ/2007 (G57 genotype). Another

1994- 2001- 2007- 2010- 2007 2004-2006G57 1997-2003virus 1994-1996 (A/chicken/Jiangxi/12/2007) detected in 2007 2000 2006 2009 2011 2012 2013 Ck/ZJ/07 (G57) is not shown.

influenza viruses with pandemic potential (28, 30, 31). In the contributed to the enhanced infection ability for the viruses pre- present study, we investigated the roles of the evolution of H9N2 vailing after 2010. When testing infection in vaccinated chickens, viruses in the genesis of the reassortant H7N9 viruses. Our results G57 viruses showed superiority in replication and transmission revealed that H9N2 influenza viruses evolved by reassortment and ability. These results suggest that under immunization pressure and mutation over their 20 y of prevalence in chickens, and the “fittest” selection of the optimal gene constellation by passing through the genotype, G57, was selected recently. The G57 viruses with changed immunological bottleneck the G57 virus eventually became the antigenicity and improved fitness resulted in viral escape from im- dominant genotype during the 2010–2013 outbreak. This outbreak munization and caused widespread outbreaks in 2010–2013 and was the first country-wide event of H9N2 viruses in chickens since finally became the single predominant H9N2 genotype in farm 1998, when extensive agricultural vaccination began in China. chicken population. This genotype contributed its six internal seg- The formation of the H9N2 ecological environment, as described ments to the novel H7N9 viruses by reassortment. above, was the first step in the genesis of the H7N9 viruses, as it The widespread use of vaccines against H9N2 viruses in chickens greatly increased the likelihood of reassortment between H9N2 and has exerted continuous mutation pressure on the viral HA gene, other subtypes of influenza viruses. The introduction of H7 and/or resulting in antigenic changes. Our study showed a weakened effect N9 viruses into chickens and subsequent reassortment with the of vaccine antisera on the recent H9N2 chicken isolates and H9N2 viruses was the final step. Jiangsu, Shanghai, and Zhejiang, heightened virus replication and transmission in vaccinated chick- the first provinces that experienced outbreaks of the novel H7N9 ens. These results, combined with the reduced diversity of the 2010– viruses on humans (1, 3), are ideally situated for the cocirculation of 2013 viruses and the close phylogenetic grouping of their HA genes, H9N2, H7, and N9 viruses. These provinces are among the largest suggest that the observed antigenic changes are common in the chicken consumption markets and are located along the avian mi- recent H9N2 chicken viruses. In addition to the antigenic charac- gratory route in the Yangtze river delta. Molecular clock dating of teristics of HA, the genomic context and compatibility of certain divergence times estimates that these H7N9 viruses emerged segment combinations may be an important contributor to viral in ∼2011–2012 (4, 13), during the widespread H9N2 outbreaks in fitness (22). As shown in the study on unvaccinated chickens, G57 chickens. Also since the H9N2 outbreaks, the appearance of more viruses exhibited the highest replication ability in inoculated and than 10 non-H7N9 novel reassortants further highlights the sig- contact chickens than other genotypes, indicative of an optimized nificant contribution of the predominant G57 genotype viruses segment combination of G57. In another recent genotype G68, in reassortment with other subtype viruses and raises concerns which had the same segments with G57 viruses except for the HA for the generation of potential pandemic viruses. segment, a representative virus (A/chicken/Guangdong/01/2011) In conclusion, our study provides a comprehensive picture of still had higher viral titers in inoculated or contact chickens than how H9N2 influenza viruses infecting chickens contributed to early viruses of other genotypes, indicating other segments also generation of the H7N9 viruses that have infected humans

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1422456112 Pu et al. Downloaded by guest on September 28, 2021 ABC 100 8 7 * * **** 80 6 * **** **** * 5 60 **** **** 4 **** 40 **** * *** **** 3 TTrachearache 2 * 20 Virus titers Virus time (day) 1 Unvaccinated CloacaCloaca

Isolation rate (%) 0 0 (log10EID50/ml) Virus shedding Virus

100 8 **** **** 7 80 6 **** * **** **** **** 5 60 4 40 3 2 20 Virus titers Virus time (day) 1 Isolation rate (%) Vaccinated (6-9) Vaccinated 0 0 (log10EID50/ml) Virus shedding Virus

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Isolation rate (%) 0 0 (log10EID50/ml) Virus shedding Virus Vaccinated (10-12) Vaccinated G02 G51 G49 G60 G57 G57 G57 G57 G68 G02 G51 G49 G60 G57 G57 G57 G57 G68 G02 G51 G49 G60 G57 G57 G57 G57 G68 G02 G51 G49 G60 G57 G57 G57 G57 G68 G02 G51 G49 G60 G57 G57 G57 G57 G68 G02 G51 G49 G60 G57 G57 G57 G57 G68 Inoculated Contact Inoculated Contact Inoculated Contact

Fig. 4. Infection of H9N2 chicken viruses in unvaccinated and vaccinated chickens. Nine representative H9N2 chicken viruses were selected from G57 or other genotypes 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/ MICROBIOLOGY 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 = 6 10 per group) were inoculated intranasally with 1× 10 EID50 of each virus to test viral replication. After 24 h, five chickens were placed in physical contact with 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 virus detection 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 H9N2 viruses 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 other genotypes of the earlier viruses (*P < 0.05, **P < 0.01, one-way ANOVA). (C) The viral shedding time of H9N2 viruses.

(Fig. S3). We demonstrated how selection and enrichment of the several chickens or samples were sent from one farm to the laboratory at one fittestG57H9N2genotypeinfarm chickens and explained the time. If one or more than one H9N2 virus was isolated, the farm was noted as an acquisition of this genotype’s internal genes by the novel H7N9 H9N2-positive farm. The virus isolation rate was determined by dividing the viruses. The prevalence and variation of H9N2 influenza virus in number of H9N2-positive farms by the total number of farms surveyed. farmed poultry could provide an important early warning of the Genomic Sequencing. H9N2 viruses (n = 76) isolated from 2010 through 2013 emergence of novel reassortants with pandemic potential. were selected for sequencing on the basis of isolation time and location. At least one strain per province per month was sequenced; if there were mul- Materials and Methods tiple isolates in a single month, additional viruses were selected according to Ethics Statement. All animal research was approved by the Beijing Association location. H9N2 viruses (n = 11) isolated during 2008–2009 from diseased, for Science and Technology (approval ID SYXK, Beijing, 2007–0023) and vaccinated farm chickens were also sequenced genomically. Viral RNA was performed in compliance with the Beijing Laboratory Animal Welfare and extracted by using the QIAamp Viral RNA Minikit (Qiagen), and standard Ethics guidelines, as issued by the Beijing Administration Committee of RT-PCR was performed with primers specific for H9N2 influenza virus, using Laboratory Animals, and in accordance with the China Agricultural Univer- the One Step RT-PCR Kit (Qiagen). Full genomic DNA was sequenced by ei- sity (CAU) Institutional Animal Care and Use Committee guidelines (ID: ther Sanger sequencing on an ABI 3730 genetic analyzer (Applied Bio- SKLAB-B-2010-003) approved by the Animal Welfare Committee of CAU. systems) or high-throughput next-generation sequencing (Ion torrent).

Virus Isolation and Identification. From January 2010 to December 2013, Sequence Collection and Alignment. All previously published sequences of Chinese chicken farms in 19 provinces of China (Anhui, Beijing, Guangdong, Guangxi, H9N2 influenza A virus (1994–2013) and relative sequences were collated from Fujuan, Hebei, Heilongjiang, Henan, Hubei, Jiangsu, Jiangxi, Jilin, Liaoning, Influenza Research Database (FluDB) (www.FluDB.org), Global Initiative on Neimenggu, Shaanxi, Shandong, Sichuan, Tianjin, and Xinjiang) sent chickens Sharing Avian Influenza Data (www.gisaid.org), and the Influenza Virus Re- with disease signs or lung tissues from deceased chickens to our laboratory source at the National Center for Biotechnology Information (NCBI) (www.ncbi. for diagnosis. The chicken flocks that were sampled were reported to exhibit nlm.nih.gov/genomes/FLU). All replicate submissions were removed by identi- overt respiratory illness and/or a 5–20% reduction in egg production, despite fyingsetsofisolateswithidenticalsequences for any segment, with each isolate having received H9N2-subtype influenza vaccination. For virus isolation, containing one to eight segments. All sequences of H7N9 influenza A viruses tracheal swabs were taken and placed in 1.0 mL transmission medium [50% published before April 2014 were acquired from the above databases and (vol/vol) glycerol in PBS] containing antibiotics, as previously described (12). combined with the sequences generated in this study (Table S3) for further Lungs were homogenized in PBS containing antibiotics. Samples were analysis. All of the accession codes of public sequences can be found in Table S4 centrifuged at 1,000 × g for 10 min at 4 °C, and 0.2 mL of the supernatants was and Dataset S2. The resulting sequences of each gene segment were aligned inoculated into the allantoic cavities of 10-d-old SPF embryonated chicken eggs using MAFFT v6 (32), manually adjusted to correct frame shift errors, and sub- and incubated for 48 h at 37 °C. Allantoic fluid was harvested and tested for HA sequently translated. Downstream phylogenetic analyses were performed on activity. HA-positive samples were identified serologically with antisera to each regions of the alignments containing few gaps across sequences. These regions known influenza virus subtype, as previously described (20). H9N2-containing consist of the following intervals: PB2, 1,168–2,199; PB1, 61–1,437; PA, 796– allantoic fluid was harvested and stored as stock at−80 °C until use. Usually, 2,103; HA, 190–1,563; NP, 67–1,014; NA, 91–1,272; MP, 88–948; and NS, 79–807.

Pu et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 Phylogenetic Analysis and Clade Classification. RAxML was used to construct described (36), with an initial serum dilution of 1:10. The HI titer was maximum likelihood phylogenies for each segment (33). The general-time re- expressed as the reciprocal of the highest serum dilution in which hemag- versible model of nucleotide substitution was assumed, and the rates were as- glutination was inhibited. sumed to vary according to the discrete Gamma distribution with four rate categories. Two sets of trees were constructed: one set including all H9N2 iso- Virus Infection in Chickens. These experiments were designed as previously de- lates from all hosts since 1994 isolated from China and Hong Kong (all segments) scribed (19, 20) and performed in unvaccinated and vaccinated chickens. Three- and one set additionally including all H7N9 isolates from all hosts from China week-old SPF white leghorn chickens were vaccinated s.c. once with A/chicken/ and Hong Kong (all six internal segments). One hundred bootstrap maximum- Shandong/6/1996-based commercial vaccine (HA content, 10 log2). HI titers to likelihood (ML) trees were used to construct a majority rule consensus tree from homologous antigen were measured in twofold serum dilutions 3 wk after in- each bootstrap sample using sumtrees (34). Bayesian phylogenetic trees were oculation and expressed as the log reciprocal of the end point. The vaccinated constructed for each segment using Bayesian Evolutionary Analysis Sampling 2 chickens were divided into two groups for infection experiments: those with Trees Software (BEAST) (35). The model of substitution and rate variation in each a low antibody level (HI titer, 6–9) and those with a high antibody level (HI titer, tree was the same as in the corresponding RAxML tree, with the additional – assumption of a log-normally distributed molecular clock. BEAST trees were 10 12). Six-week-old SPF white Leghorn chickens were used as the unvaccinated initiated with the RAxML majority rule consensus tree to reduce the computa- group. Nine H9N2 viruses (Fig. 4) were selected to inoculate chickens. In in- = 6 tion time required for the MArcov Chain Monte Carlow (MCMC) sampler to oculated groups, birds (n 10 per group) were inoculated intranasally with 10 reach the stationary distribution. Maximum clade credibility (MCC) trees were EID50 (egg infective dose at which 50% of inoculated eggs are infected) of each constructed using 2,000 equidistant samples from the final 30% of the posterior virus in a 0.2-mL volume. Tracheal and cloacal swabs were collected at 3, 5, and 6 sample of trees (n = 25 × 10 total samples) using a python script and tree an- 7 dpi. The virus titers of positive samples were measured by determining the EID50; notator (35). The resulting MCC trees were partitioned into clades using a me- the lower limit of detection was 1.0 log10 EID50/mL In contact groups, at 24 h after dian branch length distance threshold of 0.20 (24). Briefly, clades with median infection, five chickens were placed in physical contact (i.e., in the same cage, root-to-tip distances equal to or less than 20% of the median root-to-tip dis- sharing food and water) with inoculated birds. Tracheal and cloacal swabs were tances of all clades in the tree were selected as clusters. Clades were then collected at 3, 5, and 7 dpi for viral detection and titration, as described above. manually merged, if necessary, based on the branching posterior and reported classification (19, 20, 25). Each clade was assigned a unique clade ID. Statistical Analysis. Experimental groups were statistically compared by ANOVA. P < 0.05 was considered to indicate a statistically significant difference. Genotypic Analysis. Virus genotypes were analyzed in H9N2 viruses isolated – from chickens in China during 1994 2013. Isolates were genotyped if ACKNOWLEDGMENTS. We thank Furkat Anwar for assistance with animal sequences and clade assignments were available for all eight segments. The experiments; Robert Huether and Amber Smith for helpful suggestions on genotypes of the resulting isolates were determined by the combination of data analysis; and Sharon Naron, Kin-chow Chang, and Lu Lu for great clade assignments of each of the eight segments. Genotype IDs were editorial assistance. This work was supported by National Natural Science assigned according to the isolation time of the initial founder isolate. Foundation of China Grants 31430086 and 31272535; National Basic Re- search Program (973 Program) Grant 2011CB504702; the China Scholarship Foundation; Key Technologies Research and Development Program of China HI Assay. The HI assay was used to antigenically characterize the H9N2 viruses Grant 2013BAD12B01; the China Agriculture Research System, Poultry- isolated in China in this and previous studies. The assay used chicken antisera Related Science and Technology Innovation Team of Peking; National In- to two representative H9N2 vaccine strains (A/chicken/Shandong/6/1996 and stitute of Allergy and Infectious Disease Contract HHSN266200700005C; and A/chicken/Shandong/F/1998). The HI assay was performed as previously the American Lebanese Syrian Associated Charities.

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