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Citation: Xiaowen Li, Xueying Li, Bing Xu. Phylogeography of Highly Pathogenic H5 Avian Influenza Viruses in China [J].VIROLOGICA SINICA, 2020, 35(5) : 548-555.  http://dx.doi.org/10.1007/s12250-020-00193-7

Phylogeography of Highly Pathogenic H5 Avian Influenza Viruses in China

  • Corresponding author: Bing Xu, bingxu@tsinghua.edu.cn, ORCID: http://orcid.org/0000-0001-9159-2512
  • Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12250-020-00193-7) contains supplementary material, which is available to authorized users.
  • Received Date: 26 May 2019
    Accepted Date: 17 December 2020
    Published Date: 08 May 2020
  • The spread of H5 highly pathogenic avian influenza viruses poses serious threats to the poultry industry, wild bird ecology and human health. Circulation of H5 viruses between poultry and wild birds is a significant public health threat in China. Thus, viral migration networks in this region need to be urgently studied. Here, we conducted molecular genetic analyses of the hemagglutinin genes of H5 highly pathogenic avian influenza viruses in multiple hosts from 2000 to 2018 in China. Our aim was to clarify the roles of different hosts in the evolution of H5 viruses. We used a flexible Bayesian statistical framework to simulate viral space diffusion and continuous-time Markov chains to infer the dynamic evolutionary process of spatiotemporal dissemination. Bayesian phylogeographic analysis of H5 viruses showed for the first time that H5 viruses in poultry and wild birds were present in Guangdong Province. Furthermore, Guangdong, Jiangsu, Shanghai and Hunan acted as the epicenters for the spread of various H5 subtypes viruses in poultry, and Henan, Shanghai, Hong Kong and Inner Mongolia acted as epicenters for the spread of various H5 subtypes viruses in wild birds. Thus, H5 viruses exhibited distinct evolutionary dynamics in poultry and wild birds. Our findings extend our understanding of the transmission and spread of highly pathogenic H5 avian influenza viruses in China.

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    1. Bertran K, Dolz R, Majó N (2014) Pathobiology of avian influenza virus infection in minor gallinaceous species: a review. Avian Pathol 43:9–25
        doi: 10.1080/03079457.2013.876529

    2. Bi Y, Zhang Z, Liu W, Yin Y, Lei F (2015) Highly pathogenic avian influenza A(H5N1) virus struck migratory birds in China in 2015. Sci Rep 5:12986
        doi: 10.1038/srep12986

    3. Bi Y, Chen J, Zhang Z, Li M, Cai T, Sharshov K, Susloparov I, Shestopalov A, Wong G, He Y (2016a) Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds, 2014–2015. Virol Sin 31:300–305
        doi: 10.1007/s12250-016-3750-4

    4. Bi Y, Chen Q, Wang Q, Chen J, Jin T, Wong G, Quan C, Liu J, Wu J, Yin R (2016b) Genesis, evolution and prevalence of H5N6 avian influenza viruses in China. Cell Host Microbe 20:810–821
        doi: 10.1016/j.chom.2016.10.022

    5. Bielejec F, Baele G, Vrancken B, Suchard MA, Rambaut A, Lemey P (2016) Sprea D3: interactive visualization of spatiotemporal history and trait evolutionary processes. Mol Biol Evol 33:2167–2169
        doi: 10.1093/molbev/msw082

    6. Brown JD, Stallknecht DE, Swayne DE (2008) Experimental infection of swans and geese with highly pathogenic avian influenza virus (H5N1) of Asian lineage. Emerg Infect Dis 14:136–142
        doi: 10.3201/eid1401.070740

    7. Chen H, Smith GJD, Zhang SY, Qin K, Wang J, Li KS, Webster RG, Peiris JSM, Guan Y (2005) Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature 436:191–192
        doi: 10.1038/nature03974

    8. Chen H, Smith GJD, Li KS, Wang J, Fan XH, Rayner JM, Vijaykrishna D, Zhang JX, Zhang LJ, Guo CT et al (2006) Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control. Proc Natl Acad Sci USA 103:2845–2850
        doi: 10.1073/pnas.0511120103

    9. Claes F, Kuznetsov D, Liechti R, Dobschuetz SV, Bao DT, Gleizes A, Conversa D, Colonna A, Demaio E, Ramazzotto S, Larfaoui F, Pinto J, Le Mercier P, Xenarios I, Dauphin G (2014) The EMPRES-i genetic module: a novel tool linking epidemiological outbreak information and genetic characteristics of influenza viruses. Database (Oxford). 2014: bau008

    10. Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol 29:1969–1973
        doi: 10.1093/molbev/mss075

    11. Duan L, Bahl J, Smith GJD, Wang J, Vijaykrishna D, Zhang LJ, Zhang JX, Li KS, Fan XH, Cheung CL, Huang K, Poon LL, Shortridge KF, Webster RG, Peiris JS, Chen H, Guan Y (2008) The development and genetic diversity of H5N1 influenza virus in China, 1996–2006. Virology 380:243–254
        doi: 10.1016/j.virol.2008.07.038

    12. El-Shesheny R, Barman S, Feeroz MM, Hasan MK, Jones-Engel L, Franks J, Turner J, Seiler P, Walker D, Friedman K, Kercher L, Begum S, Akhtar S, Datta AK, Krauss S, Kayali G, McKenzie P, Webby RJ, Webster RG (2017) Genesis of influenza A (H5N8) viruses. Emerg Infect Dis 23:1368–1371
        doi: 10.3201/eid2308.170143

    13. Flyway EAA (2011) Implementation strategy for the East Asian—Australasian flyway partnership: 2007–2011. Department of the Environment 3:1-8

    14. Flyway EAA (2015) East Asian–Australasian flyway. New Zealand

    15. Gu M, Liu W, Cao Y, Peng D, Wang X, Wan H, Zhao G, Xu Q, Zhang W, Song Q (2011) Novel reassortant highly pathogenic avian influenza (H5N5) viruses in domestic ducks, China. Emerg Infect Dis 17:1060–1063
        doi: 10.3201/eid/1706.101406

    16. Guan Y, Smith GJD, Webby R, Webster RG (2009) Molecular epidemiology of H5N1 avian influenza. Rev Sci Tech 28:39–47
        doi: 10.20506/rst.28.1.1868

    17. Huang PY, Lee CCD, Yip CH, Cheung CL, Yu G, Lam TTY, Smith DK, Zhu H, Guan Y (2016) Genetic characterization of highly pathogenic H5 influenza viruses from poultry in Taiwan, 2015. Infect Genet Evol 38:96–100
        doi: 10.1016/j.meegid.2015.12.006

    18. Keawcharoen J, Van Riel D, Van Amerongen G, Bestebroer T, Beyer WE, van Lavieren R, Osterhaus AD, Fouchier RA, Kuiken T (2008) Wild ducks as long-distance vectors of highly pathogenic avian influenza virus (H5N1). Emerg Infect Dis 14:600–607
        doi: 10.3201/eid1404.071016

    19. Kilpatrick AM, Chmura AA, Gibbons DW, Fleischer RC, Marra PP, Daszak P (2006) Predicting the global spread of H5N1 avian influenza. Proc Natl Acad Sci USA 103:19368–19373
        doi: 10.1073/pnas.0609227103

    20. Kim YI, Park SJ, Kwon HI, Kim EH, Si YJ, Jeong JH, Lee IW, Nguyen HD, Kwon JJ, Choi WS, Song MS, Kim CJ, Choi YK (2017) Genetic and phylogenetic characterizations of a novel genotype of highly pathogenic avian influenza (HPAI) H5N8 viruses in 2016/2017 in South Korea. Infect Genet Evol 53:56–67
        doi: 10.1016/j.meegid.2017.05.001

    21. Lam TT, Wang J, Shen Y, Zhou B, Duan L, Cheung CL, Ma C, Lycett SJ, Leung CY, Chen X, Li L, Hong W, Chai Y, Zhou L, Liang H, Ou Z, Liu Y, Farooqui A, Kelvin DJ, Poon LL, Smith DK, Pybus OG, Leung GM, Shu Y, Webster RG, Webby RJ, Peiris JS, Rambaut A, Zhu H, Guan Y (2013) The genesis and source of the H7N9 influenza viruses causing human infections in China. Nature 502:241–244
        doi: 10.1038/nature12515

    22. Lee YJ, Kang HM, Lee EK, Song BM, Jeong J, Kwon YK, Kim HR, Lee KJ, Hong MS, Jang I, Choi KS, Kim JY, Lee HJ, Kang MS, Jeong OM, Baek JH, Joo YS, Park YH, Lee HS (2014) Novel reassortant influenza A(H5N8) viruses, South Korea, 2014. Emerg Infect Dis 20:1086–1089
        doi: 10.3201/eid2006.140233

    23. Lee DH, Torchetti MK, Winker K, Ip HS, Song CS, Swayne DE (2015) Intercontinental spread of asian-origin H5N8 to North America through Beringia by migratory birds. J Virol 89:6521–6524
        doi: 10.1128/JVI.00728-15

    24. Lee DH, Bahl J, Torchetti MK, Killian ML, Ip HS, DeLiberto TJ, Swayne DE (2016) Highly pathogenic avian influenza viruses and generation of novel reassortants, United States, 2014–2015. Emerg Infect Dis 22:1283–1285
        doi: 10.3201/eid2207.160048

    25. Lemey P, Rambaut A, Drummond AJ, Suchard MA (2009) Bayesian phylogeography finds its roots. PLoS Comput Biol 5:e1000520
        doi: 10.1371/journal.pcbi.1000520

    26. Leung YHC, Lau EHY, Juan ZL, Yi G, Cowling BJ, Peiris JSM (2012) Avian influenza and ban on overnight poultry storage in live poultry markets, Hong Kong. Emerg Infect Dis 18:1339–1341
        doi: 10.3201/eid1808.111879

    27. Li Y, Liu L, Zhang Y, Duan Z, Tian G, Zeng X, Shi J, Zhang L, Chen H (2011) New avian influenza virus (H5N1) in wild birds, Qinghai, China. Emerg Infect Dis 17:265–267
        doi: 10.3201/eid1702.100732

    28. Li M, Liu H, Bi Y, Sun J, Wong G, Liu D, Li L, Liu J, Chen Q, Wang H, He Y, Shi W, Gao GF, Chen J (2017) Highly pathogenic avian influenza A(H5N8) virus in wild migratory birds, Qinghai Lake, China. Emerg Infect Dis 23:637–641
        doi: 10.3201/eid2304.161866

    29. Li R, Zhang T, Bai Y, Li H, Wang Y, Bi Y, Chang J, Xu B (2018) Live poultry trading drives China's H7N9 viral evolution and geographical network propagation. Front Public Health 6:210
        doi: 10.3389/fpubh.2018.00210

    30. Minin VN, Bloomquist EW, Suchard MA (2008) Smooth skyride through a rough skyline: Bayesian coalescent-based inference of population dynamics. Mol Biol Evol 25:1459–1471
        doi: 10.1093/molbev/msn090

    31. OIE (2019) OIE situation report for avian influenza. https://www.oie.int/en/animal-health-in-the-world/update-on-avian-influenza/2019/. Accessed 26 May 2019

    32. Olsen B, Munster V, Wallensten A, Jonas W, Osterhaus A, Fouchier R (2006) Global patterns of influenza a virus in wild birds. Science 312:384–388
        doi: 10.1126/science.1122438

    33. Pan M, Gao R, Lu Q, Huang S, Zhou Z, Yang L, Li X, Zhao X, Zou X, Tong W et al (2015) Human infection with a novel highly pathogenic avian influenza A (H5N6) virus: virological and clinical findings. J Infect 72:52–59

    34. Rambaut A, Drummond AJ, Dong X, Baele G, Suchard MA (2018) Posterior summarization in bayesian phylogenetics using tracer 1.7. Syst Biol 67:901–904
        doi: 10.1093/sysbio/syy032

    35. Reperant LA, Fuckar NS, Osterhaus AD, Dobson AP, Kuiken T (2010) Spatial and temporal association of outbreaks of H5N1 influenza virus infection in wild birds with the 0 degrees C isotherm. PLoS Pathog 6:e1000854
        doi: 10.1371/journal.ppat.1000854

    36. Shapiro B, Rambaut A, Drummond AJ (2006) Choosing appropriate substitution models for the phylogenetic analysis of protein-coding sequences. Mol Biol Evol 23:7–9
        doi: 10.1093/molbev/msj021

    37. Smith GJ, Fan XH, Wang J, Li KS, Qin K, Zhang JX, Vijaykrishna D, Cheung CL, Huang K, Rayner JM, Peiris JS, Chen H, Webster RG, Guan Y (2006) Emergence and predominance of an H5N1 influenza variant in China. Proc Natl Acad Sci USA 103:16936–16941
        doi: 10.1073/pnas.0608157103

    38. Smith GJ, Vijaykrishna D, Ellis TM, Dyrting KC, Leung YH, Bahl J, Wong CW, Kai H, Chow MK, Duan L, Chan AS, Zhang LJ, Chen H, Luk GS, Peiris JS, Guan Y (2009) Characterization of avian influenza viruses A (H5N1) from wild birds, Hong Kong, 2004–2008. Emerg Infect Dis 15:402–407
        doi: 10.3201/eid1503.081190

    39. Su S, Bi Y, Wong G, Gray GC, Gao GF, Li S (2015) Epidemiology, evolution, and recent outbreaks of avian influenza virus in China. J Virol 89:8671–8676
        doi: 10.1128/JVI.01034-15

    40. The Global Consortium for H5N8 and Related Influenza Viruses (2016) Role for migratory wild birds in the global spread of avian influenza H5N8. Science 354:213–217
        doi: 10.1126/science.aaf8852

    41. Tian H, Cui Y, Dong L, Zhou S, Li X, Huang S, Yang R, Xu B (2015a) Spatial, temporal and genetic dynamics of highly pathogenic avian influenza A (H5N1) virus in China. BMC Infect Dis 15:54
        doi: 10.1186/s12879-015-0770-x

    42. Tian H, Zhou S, Dong L, Cui Y, Li X, Wu Y, Cazelles B, Huang S, Yang R, Xu B (2015b) Avian influenza H5N1 viral and bird migration networks in Asia. Proc Natl Acad Sci USA 112:172–177
        doi: 10.1073/pnas.1405216112

    43. Van Kerkhove MD, Vong S, Guitian J, Holl D, Mangtani P, San S, Ghani AC (2009) Poultry movement networks in Cambodia: implications for surveillance and control of highly pathogenic avian influenza (HPAI/H5N1). Vaccine 27:6345–6352
        doi: 10.1016/j.vaccine.2009.05.004

    44. Vijaykrishna D, Bahl J, Riley S, Duan L, Zhang J, Chen H, Peiris J, Smith G, Guan Y (2008) Evolutionary dynamics and emergence of panzootic H5N1 influenza viruses. PLoS Pathog 4:e1000161
        doi: 10.1371/journal.ppat.1000161

    45. WHO/OIE/FAO H5N1 Evolution Working Group (2008) Toward a unified nomenclature system for highly pathogenic avian influenza virus (H5N1). Emerg Infect Dis 14:e1

    46. World Health Organization (WHO) (2015) Evolution of the influenza A(H5) haemagglutinin: WHO/OIE/FAO H5 Working Group reports a new clade designated 2.3.4.4. https://www.who.int/influenza/gisrs_laboratory/h5_nomenclature_clade2344/en/. Accessed 26 May 2019

    47. Wu X, Tian H, Zhou S, Chen L, Xu B (2014) Impact of global change on transmission of human infectious diseases. Sci China Earth Sci 57:189–203
        doi: 10.1007/s11430-013-4635-0

    48. Yang L, Zhu W, Li X, Bo H, Zhang Y, Zou S, Gao R, Dong J, Zhao X, Chen W, Dong L, Zou X, Xing Y, Wang D, Shu Y (2017) Genesis and Dissemination of Highly Pathogenic H5N6 Avian Influenza Viruses. J Virol 91. pii: e02199-16

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    Phylogeography of Highly Pathogenic H5 Avian Influenza Viruses in China

      Corresponding author: Bing Xu, bingxu@tsinghua.edu.cn
    • 1. Department of Earth System Science, Tsinghua University, Beijing 100084, China
    • 2. Center for Healthy Cities, Institute for China Sustainable Urbanization, Tsinghua University, Beijing 100084, China

    Abstract: The spread of H5 highly pathogenic avian influenza viruses poses serious threats to the poultry industry, wild bird ecology and human health. Circulation of H5 viruses between poultry and wild birds is a significant public health threat in China. Thus, viral migration networks in this region need to be urgently studied. Here, we conducted molecular genetic analyses of the hemagglutinin genes of H5 highly pathogenic avian influenza viruses in multiple hosts from 2000 to 2018 in China. Our aim was to clarify the roles of different hosts in the evolution of H5 viruses. We used a flexible Bayesian statistical framework to simulate viral space diffusion and continuous-time Markov chains to infer the dynamic evolutionary process of spatiotemporal dissemination. Bayesian phylogeographic analysis of H5 viruses showed for the first time that H5 viruses in poultry and wild birds were present in Guangdong Province. Furthermore, Guangdong, Jiangsu, Shanghai and Hunan acted as the epicenters for the spread of various H5 subtypes viruses in poultry, and Henan, Shanghai, Hong Kong and Inner Mongolia acted as epicenters for the spread of various H5 subtypes viruses in wild birds. Thus, H5 viruses exhibited distinct evolutionary dynamics in poultry and wild birds. Our findings extend our understanding of the transmission and spread of highly pathogenic H5 avian influenza viruses in China.

    • Influenza A viruses of the H5 subtype can infect multiple species of poultry, wild birds, and mammals. In recent years, H5 subtype viruses have been continuously circulating worldwide. The spread of H5 highly pathogenic avian influenza (HPAI) viruses is a serious threat to the poultry industry, wild bird ecology and human health. H5 subtype viruses have high genetic diversity and a very high rate of evolution (Duan et al. 2008; Vijaykrishna et al. 2008; Guan et al. 2009). Based on the sequence of the hemagglutinin (HA) gene, H5 subtype viruses can be divided into different clades (WHO et al. 2008). Viruses in different clades have different geographical distributions.

      In China, most clade 2.3.2 viruses clustered in Hong Kong, Henan, Inner Mongolia and Qinghai (Smith et al. 2009; Li et al. 2011; Bi et al. 2015; Bi et al. 2016a), while clade 2.2 viruses have persisted for long periods in Qinghai Lake (Chen et al. 2005). Clade 2.3.4 viruses first emerged in the south and subsequently spread to the north (Tian et al. 2015a). In contrast, clade 7 viruses were first isolated in northern China and spread from north to south into Hunan and Yunnan provinces (Tian et al. 2015a). A study of the spatiotemporal patterns of H5N1 HPAI viruses in China found a significant time lag between poultry or wild bird outbreaks and human cases. Viruses circulating in poultry spread faster, but were more spatially clustered (Tian et al. 2015b). The spread of H5 viruses in wild birds is associated with bird migration networks and the peak of migration (Tian et al. 2015b).

      In 2014, a new clade of H5 viruses (2.3.4.4) emerged. This clade includes H5N1, H5N2, H5N3, H5N5, H5N6 and H5N8 subtype viruses (WHO 2015). In 2014, a novel H5N6 virus was first reported in China (Pan et al. 2015). To date, a total of 18 countries have reported cases of H5N6 infection, including China, Korea, Japan, Vietnam, Laos, Myanmar, the Philippines, Greece, Sweden, Switzerland, Germany, Finland, Denmark, Iran, Ireland, Netherlands, Slovakia and the United Kingdom (OIE 2019). H5N6 viruses have become the predominant avian influenza virus subtype circulating in southern China. This virus was derived from reassortment between the internal genes of H9N2/H7N9 viruses and a clade 2.3.2.1c H5N1 virus (Bi et al. 2016b; Yang et al. 2017). In 2014, a novel H5N8 virus caused outbreaks in domestic ducks and wild birds in South Korea (Lee et al. 2014). As these birds migrated, the H5N8 virus spread to Japan, Siberia, North America and Europe (Lee et al. 2015; The Global Consortium for H5N8 and Related Influenza Viruses 2016). In 2016, a novel reassortant H5N8 virus bearing internal genes from a low pathogenic avian influenza virus circulating in Mongolia was identified in wild birds in Qinghai Lake (Li et al. 2017). In North America, novel reassortant H5N1, H5N2 and H5N8 viruses bearing genes of local low pathogenic avian influenza viruses were identified (Lee et al. 2016). In Chinese Taiwan, novel reassortant H5N2, H5N3 and H5N8 viruses bearing Eurasian influenza genes were identified (Huang et al. 2016). In Chinese mainland, novel reassortant H5N5 viruses bearing internal genes from H5N1 viruses were identified (Gu et al. 2011).The genes of HPAI H5 clade 2.3.4.4 viruses around the world show significant diversity and bear internal genes from multiple viral subtypes (El-Shesheny et al. 2017; Kim et al. 2017).

      The evolutionary patterns of HPAI H5 viruses in China are not well understood. Studies in this area would help us to understand the geographical dispersal of H5 viruses and to identify areas that are significant for viral migration and evolution.

    • Full-length HA sequences of H5 HPAI viruses in China were obtained from the GISAID (http://gisaid.org) and the GenBank (http://www.ncbi.nlm.nih.gov/genomes/FLU/) repositories on 1 January 2019. HA sequences were aligned and neighbor-joining (NJ) phylogenies were estimated using MEGA 7 software (https://www.megasoftware.net/). NJ methods were used to perform system reconstruction and to calculate confidence values to estimate the number of repetitions (1000). Residual analyses of phylogenetic trees were performed using TempEst v1.4 (http://tree.bio.ed.ac.uk/software/tempest/), and anomalous sequences were removed based on the estimated root-to-tip distance. To control sampling biases, we removed sequences with nucleotide similarities greater than 99% within the same year and region to ensure fewer than 50 sequences per year were used. After removing duplicate sequences, we analyzed 398 HA gene sequences from poultry (Supplementary Table S1) and 133 HA gene sequences from wild birds (Supplementary Table S2) isolated between 2000 and 2018. All sequences were used to analyze the dispersal of H5 HPAI viruses in China. We collected data on HPAI H5 virus outbreaks in China from the EMPRES Global Animal Disease Information System (Claes et al. 2014). The outbreak data included information on time, latitude and longitude, and infected hosts.

    • The study area included twenty-seven provinces, municipalities and autonomous regions in China. The HA sequences from wild birds were distributed across thirteen regions including the Hong Kong Special Administrative Region of China and the provinces of Qinghai, Henan, Heilongjiang, Inner Mongolia, Jilin, Jiangxi, Shanghai, Hubei, Jiangsu, Guangdong, Guangxi and Hunan. The HA sequences from poultry were distributed across twenty-six regions including the Hong Kong Special Administrative Region of China and the provinces of Zhejiang, Yunnan, Xinjiang, Tibet, Qinghai, Sichuan, Shanxi, Shanghai, Shandong, Ningxia, Liaoning, Jilin, Jiangxi, Jiangsu, Hunan, Hubei, Henan, Hebei, Guizhou, Guangxi, Guangdong, Gansu, Fujian, Anhui and Heilongjiang.

    • A continuous time Markov chain Monte Carlo (MCMC) model was constructed to estimate time-resolved phylogenetic trees using BEAST 1.8.2 software (Drummond et al. 2012). For all runs, the SRD06 nucleotide substitution model (Shapiro et al. 2006) was used to estimate the divergence times of viruses, along with the uncorrelated log-normal relaxed clock model and a Gaussian Markov random field (GMRF) Bayesian Skyride coalescent prior (Minin et al. 2008). The MCMC chain was run for 500 million iterations, with a 10% burn-in and sub-sampling every 50, 000 iterations. GMRF Bayesian Skyride analysis was used to elucidate the population dynamics of H5 viruses.

      The discrete-state continuous time Markov chain method was used to estimate the transition rates of H5 viruses between discrete sampling locations. A Bayesian stochastic search variable selection model with asymmetric substitution was used. The effective sample size values of the pairwise migration rate estimates were > 200 using Tracer v1.6 (Rambaut et al. 2018). We visualized and annotated phylogenetic trees in Figtree (http://tree.bio.ed.ac.uk/software/figtree/) and visualized geographic transition routes in SPREA D3 (version 0.9.7.1) (Bielejec et al. 2016). The Bayes factor (BF) provided statistical support for geographic transition routes. BF values > 100 indicated robust statistical support. BF values > 30 and ≤ 100 indicated very strong statistical support. BF values > 10 and ≤ 30 indicated strong statistical support. BF values > 6 and ≤ 10 indicated substantial statistical support. BF values > 3 and ≤ 6 indicated poor statistical support (Lemey et al. 2009).

    • We constructed a Bayesian genealogical tree to display the time scale of H5 virus HA gene evolution in poultry in China. Twenty-six geographical regions were assigned different colors in the phylogenetic tree (Fig. 1). H5 viruses originated in various provinces in China, and showed complex cladistics and rapid evolution. The genealogical tree showed that H5 viruses isolated from poultry belonged to clades 0, 1, 3, 4, 5, 6, 7, 7.2, 8, 9, 2.4, 2.5, 2.3.1, 2.3.2, 2.3.2.1, 2.3.4 and 2.3.4.4. H5 HPAI viruses were first isolated in Guangdong Province, and then rapidly spread northward, eastward and westward. These viruses evolved quickly, with clade 2.3.2 giving rise to clades 2.3.2.1a, b and c, clade 7 giving rise to clade 7.2, and clade 2.3.4 giving to clades 2.3.4.4.a, b, c and d.

      Figure 1.  Phylogenetic tree of H5Nx HA sequences isolated from poultry. The tree is colored based on the twenty-six geographical regions in which the viruses were sampled. The black points represent nodes, and the horizontal bar indicates the 95% highest posterior density of each node.

      We also constructed a Bayesian genealogical tree to display time scale of H5 virus HA gene evolution in wild birds in China. Thirteen geographical regions were assigned different colors in the phylogenetic tree (Fig. 2). H5 viruses originated in various provinces in China, and again showed complex cladistics and rapid evolution. The genealogical tree showed that H5 viruses isolated from wild birds belonged to clades 1, 3, 4, 6, 7, 9, 2.2, 2.3.2, 2.3.2.1, 2.3.4 and 2.3.4.4. H5 HPAI viruses were first isolated in Guangdong Province in 2002, and subsequently spread rapidly northward and westward. Clade 2.2 viruses were first isolated in Qinghai Province and were not found in other provinces in 2005. The viruses evolved quickly, with clade 2.3.2 giving rise to clades 2.3.2.1b and c and clade 2.3.4 giving rise to clades 2.3.4.4.a, b, c and d.

      Figure 2.  Phylogenetic tree of H5Nx HA sequences isolated from wild birds. The tree is colored based on the thirteen geographical regions in which the viruses were sampled. The black points represent nodes, and the horizontal bar indicates the 95% highest posterior density of each node.

    • We reconstructed the spatial dispersal networks of HPAI H5 viruses in poultry and wild birds in China. Overall, we found that the transmission routes of viruses in poultry and wild birds were quite different. Transmission routes with BF values > 10 were selected for analysis. There were 46 significant transmission routes of viruses in poultry (Supplementary Table S3). Guangdong, Hunan, Shanghai and Jiangsu acted as the epicenters for the spread of various H5 subtypes viruses in poultry (Fig. 3). Guangdong was linked with ten locations (Fujian, Guangxi, Hong Kong, Hubei, Hunan, Anhui, Jiangsu, Liaoning, Shanxi, and Heilongjiang), Jiangsu was linked with eleven locations (Guangdong, Guangxi, Jiangxi, Shanghai, Tibet, Hong Kong, Hubei, Hunan, Jilin, Heilongjiang, and Zhejiang), Shanghai was linked with eight locations (Hebei, Hunan, Sichuan, Heilongjiang, Jiangsu, Jiangxi, Ningxia, and Shandong), and Hunan was linked with seven locations (Guangdong, Jilin, Shanghai, Heilongjiang, Jiangsu, Jiangxi, and Anhui). There were 14 major transmission routes of viruses in wild birds (Supplementary Table S4). Henan Province, Shanghai, Hong Kong and Inner Mongolia acted as epicenters for the spread of various H5 subtypes viruses in wild birds (Fig. 4). Henan was linked with three locations (Hunan, Shanghai, and Inner Mongolia), Shanghai was linked with three locations (Jilin, Jiangxi, and Henan), Hong Kong was linked with three locations (Jilin, Jiangsu, and Inner Mongolia), and Inner Mongolia was linked with four locations (Henan, Heilongjiang, Hong Kong, and Jiangsu).

      Figure 3.  Migration routes of H5Nx viruses isolated from poultry with in twenty-six geographic regions in China. The red, green and blue arrows indicate the reconstructed spatial dispersal patterns of H5Nx viruses isolated from poultry in China. The Bayes factor (BF) test provides statistical support for geographic transition routes. BF values > 100 indicate robust statistical support. BF values > 30 and ≤ 100 indicate very strong statistical support. BF values > 10 and ≤ 30 indicate strong statistical support.

      Figure 4.  Migration routes of H5Nx viruses isolated in wild birds within thirteen geographic regions in China. The red, green and blue arrows indicate the reconstructed spatial dispersal patterns of H5Nx viruses isolated from wild birds in China. The Bayes factor (BF) test provides statistical support for geographic transition routes. BF values > 100 indicate robust statistical support. BF values > 30 and ≤ 100 indicate very strong statistical support. BF values > 10 and l ≤ 30 indicate strong statistical support.

    • We inferred H5 virus demographic history using the GMRF Bayesian Skyride model to examine changes in genetic diversity from 2000 to 2018 in China. In poultry, the effective population size fluctuated slightly from 2000 to 2012, and from 2012 to 2014, genetic diversity rapidly increased. After 2014, the effective population size decreased year by year (Fig. 5). In wild birds, the effective population size decreased gradually from 2000 to 2005, and from 2005 to 2013, the effective population size increased slowly. From 2013 to 2015, genetic diversity decreased, and after 2015, the effective population size remained constant (Fig. 6).

      Figure 5.  Gaussian Markov random field (GMRF) Bayesian Skyride plot of H5Nx viruses isolated in poultry in China. The plot was constructed from HA. A GMRF Bayesian Skyride analyses of HA genes to display changes in the effective population size over time. The solid blue line indicates the median value, and the shaded blue area represents the 95% highest posterior density of genetic diversity estimates.

      Figure 6.  Gaussian Markov random field (GMRF) Bayesian Skyride plot of H5Nx viruses isolated in wild birds in China. The plot was constructed from HA. A GMRF Bayesian Skyride analyses of HA genes to display changes in the effective population size over time. The solid blue line indicates the median value, and the shaded blue area represents the 95% highest posterior density of genetic diversity estimates.

    • In this study, we investigated the transmission and evolution of HPAI H5 viruses in poultry and wild birds in China. We found that H5 HPAI viruses exhibited distinct evolutionary dynamics in poultry and wild birds. Bayesian phylogeographic analysis of H5 viruses showed that Guangdong Province was the origin for the spread and migration network of these viruses in poultry and wild birds. Guangdong, Jiangsu, Shanghai and Hunan acted as epicenters for the spread of various H5 subtypes viruses in poultry, and Henan, Shanghai, Hong Kong and Inner Mongolia acted as epicenters for the spread of various H5 subtypes viruses in wild birds.

      HPAI H5 viruses have circulated widely in Asian countries over the past decade (Chen et al. 2006; Smith et al. 2006). China is one of the countries seriously threatened by HPAI H5 viruses (Wu et al. 2014). The viral transmission network in poultry showed long-distance migration links in the north–south and east–west directions as well as short-distance migration links between adjacent geographical regions. Studies have shown the HPAI viruses spread over long distances through live poultry movement and spreads short distances through the poultry market (Li et al. 2018). Guangdong, Jiangsu, Shanghai and Hunan acted as epicenters for the spread of various H5 subtypes viruses in poultry. Poultry trade and transportation are well developed in Guangdong, Jiangsu, Shanghai and Hunan. Backyard poultry farming in southern China is high-density and free-range. Infected poultry in backyard farms could spread the virus through transportation (Van Kerkhove et al. 2009) and live poultry trading (Kilpatrick et al. 2006). Live poultry markets are a high-risk areas for transmission and spread of HPAI viruses (Lam et al. 2013). Many birds are placed in close proximity in these markets, increasing the probability of emergence of novel viruses and of poultry–poultry and poultry–human transmission (Leung et al. 2012; Su et al. 2015). Live poultry markets and backyard farms lack biosafety measures. These highrisk environmental factors can cause the spread of HPAI viruses. Increased biosafety measures in the poultry industry and surveillance of H5 HPAI viruses in these four regions could reduce the risk of a future epidemic.

      Henan, Shanghai, Hong Kong and Inner Mongolia acted as epicenters for the spread of various H5 subtypes viruses in wild birds. Studies have shown that Henan and Inner Mongolia are high-risk areas for viral transmission in wild birds along the flyways (Bi et al. 2015, 2016a). The main migration route of HPAI H5 viruses in Chinese migratory birds are from south to north and from north to south. This may be due to long-distance migration of migratory birds in the East Asia-Australia flyway as well as short-term foraging activities. The East Asia-Australia migration zone is the most significant migration route passing through China. Each year, 50 million waterfowl, including more than 55 migratory species, migrate through this zone (Olsen et al. 2006; Flyway 2011; Flyway 2015). Avian influenza viruses are host-specific and these viruses can persist in aquatic environments. Avian influenza viruses can be transmitted among poultry and wild birds living in the same aquatic environments through the fecal–oral route (Bertran et al. 2014). Wild birds acquire HPAI H5 viruses from poultry and transport the viruses to more distant locations. Studies have shown that migratory birds first gather in the vicinity of the 0 ℃ isotherm (Reperant et al. 2010), and then carry out migratory activities, leading to the geographical spread of the virus. During geographical spread, the virus is constantly evolving to accommodate the long-distance migratory activities of birds. In China, the spread of HPAI H5 viruses in wild birds may be caused by migration of wild birds.

      This study had several limitations. First, sampling bias may have affected the Bayesian phylogenetic reconstruction and inference of transmission networks. Second, because gene sequence data and the road length data for each province were not matched by time, we could not compare our results with poultry transportation data. Third, the spatiotemporal information for HA sequences used in the article may not have accurately represented the times and locations in which wild birds were infected with H5 HPAI viruses. Some wild birds are carriers of these viruses in the absence of clinical symptoms (Brown et al. 2008; Keawcharoen et al. 2008). This could result in increased viral spread by migratory birds on a large scale, a possibility that was not considered in our study.

    • This study was partially supported by the National Research Program of the Ministry of Science and Technology of the People's Republic of China (2016YFA0600104) and by donations from Delos Living LLC and the Cyrus Tang Foundation to Tsinghua University.

    • XWL and BX conceived and designed the research. XYL provided helpful suggestions regarding the study. XWL and BX wrote the manuscript.

    • The authors declare that they have no conflict of interest.

    • This article does not contain any studies with human or animal subjects performed by any of the authors.

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