Evolutionary dynamics and comparative pathogenicity of clade 2.3.4.4b H5 subtype avian influenza viruses, China, 2021-2022

Siru Lin; Junhong Chen; Ke Li; Yang Liu; Siyuan Fu; Shumin Xie; Aimin Zha; Aiguo Xin; Xinyu Han; Yuting Shi; Lingyu Xu; Ming Liao; Weixin Jia

doi:10.1016/j.virs.2024.04.004

June 2024

Siru Lin, Junhong Chen, Ke Li, Yang Liu, Siyuan Fu, Shumin Xie, Aimin Zha, Aiguo Xin, Xinyu Han, Yuting Shi, Lingyu Xu, Ming Liao and Weixin Jia. Evolutionary dynamics and comparative pathogenicity of clade 2.3.4.4b H5 subtype avian influenza viruses, China, 2021-2022[J]. Virologica Sinica, 2024, 39(3): 358-368. doi: 10.1016/j.virs.2024.04.004

Citation: Siru Lin, Junhong Chen, Ke Li, Yang Liu, Siyuan Fu, Shumin Xie, Aimin Zha, Aiguo Xin, Xinyu Han, Yuting Shi, Lingyu Xu, Ming Liao, Weixin Jia. Evolutionary dynamics and comparative pathogenicity of clade 2.3.4.4b H5 subtype avian influenza viruses, China, 2021-2022 .VIROLOGICA SINICA, 2024, 39(3) : 358-368. http://dx.doi.org/10.1016/j.virs.2024.04.004

2021-2022年中国Clade 2.3.4.4b分支H5亚型禽流感病毒的演化动态和致病性差异分析

林思茹 ^a ,
陈俊宏 ^a ,
李珂 ^c ,
柳洋 ^a ,
付思源 ^a ,
谢淑敏 ^a ,
查爱敏 ^a ,
信爱国 ^a ,
韩心雨 ^a ,
史玉婷 ^a ,
徐玲玉 ^a ,
廖明 ^a,b,, ,
贾伟新 ^a,b,,

cstr: 32224.14.j.virs.2024.04.004

a. 华南农业大学兽医学院, 国家禽流感专业实验室(广州), 广东省人兽共患病防控制剂工程实验室, 岭南现代农业科学与技术广东省实验室;
b. 农业农村部人畜共患病重点实验室, 农业农村部兽用疫苗创制重点实验室;
c. 云南省畜牧兽医科学院养禽与禽病研究所

通讯作者： 廖明, mliao@scau.edu.cn; 贾伟新, jiaweixin@scau.edu.cn
收稿日期： 2023-09-26
录用日期： 2024-04-18

摘要

当前, 新型H5N1、H5N6和H5N8禽流感病毒在全球范围广泛传播，并导致了大量鸟类感染和死亡。自 2020 年以来，禽流感病毒在人与动物之间的互动频繁发生。为了深入了解新型 H5 亚型禽流感病毒（即 H5N1、H5N6 和 H5N8），我们利用2021年1月至2022年9月期间从中国分离的41株H5亚型禽流感病毒进行了系统发育分析和生物特性等分析。系统进化分析表明，41株 H5亚型 AIV 属于2.3.4.4b支系, 其中13株 H5N1禽流感病毒，19株 H5N6 禽流感病毒，9 株H5N8 禽流感病毒。基于全球2.3.4.4b支系毒株的溯源分析表明，所有病毒的共同祖先均为H5N8病毒。但结合病毒的种群动态分析来看，新型H5N1和新型H5N6病毒的进化模式并非完全一致。在此基础上，我们进一步计算三种H5亚型毒株的进化速率和选择压力位点，发现H5N1在2021-2022年的进化率较高，在2015-2022年受到正向选择压力的位点较多，因此在传播方面可能具有竞争优势。新型H5N1和H5N6的抗原谱表现出显著差异。血凝抑制试验表明，某些甲型H5N1病毒在抗原性上可能与现有的H5N6和H5N8株不同。哺乳动物试验表明，H5N8病毒（21GD001_H5N8）对小鼠的致病性最高，其次是H5N1病毒（B1557_H5N1），然后是H5N6病毒（220086_H5N6），这表明 H5 禽流感病毒在哺乳动物宿主中的毒力特征各不相同。基于以上结果，我们认为 A型H5N1禽流感病毒在未来流行的风险较高。综上所述，这些发现揭示了 2.3.4.4b支系中新型H5禽流感病毒不同的进化历史和生物学特征，有助于更好地理解新型H5禽流感病毒，从而设计出更有效的防控策略。
- 禽流感病毒
- , H5 亚型禽流感病毒
- , 进化
- , 致病性

Evolutionary dynamics and comparative pathogenicity of clade 2.3.4.4b H5 subtype avian influenza viruses, China, 2021-2022

Siru Lin ^a ,
Junhong Chen ^a ,
Ke Li ^c ,
Yang Liu ^a ,
Siyuan Fu ^a ,
Shumin Xie ^a ,
Aimin Zha ^a ,
Aiguo Xin ^a ,
Xinyu Han ^a ,
Yuting Shi ^a ,
Lingyu Xu ^a ,
Ming Liao ^a,b,, ,
Weixin Jia ^a,b,,

a. National Avian Influenza Para-Reference Laboratory, Guangdong Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Laboratory for Lingnan Modern Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China;
b. Key Laboratory of Zoonoses, Key Laboratory of Animal Vaccine Development, Ministry of Agriculture, Guangzhou, 510642, China;
c. Institute of Poultry Management and Diseases, Yunnan Animal Science and Veterinary Institute, Kunming, 650000, China

Corresponding author: Ming Liao, mliao@scau.edu.cn Weixin Jia, jiaweixin@scau.edu.cn
Received Date: 26 September 2023
Accepted Date: 18 April 2024

Abstract

The recent concurrent emergence of H5N1, H5N6, and H5N8 avian influenza viruses (AIVs) has led to significant avian mortality globally. Since 2020, frequent human-animal interactions have been documented. To gain insight into the novel H5 subtype AIVs (i.e., H5N1, H5N6 and H5N8), we collected 6102 samples from various regions of China between January 2021 and September 2022, and identified 41 H5Nx strains. Comparative analyses on the evolution and biological properties of these isolates were conducted. Phylogenetic analysis revealed that the 41 H5Nx strains belonged to clade 2.3.4.4b, with 13 related to H5N1, 19 to H5N6, and 9 to H5N8. Analysis based on global 2.3.4.4b viruses showed that all the viruses described in this study were likely originated from H5N8, exhibiting a heterogeneous evolutionary history between H5N1 and H5N6 during 2015-2022 worldwide. H5N1 showed a higher rate of evolution in 2021-2022 and more sites under positive selection pressure in 2015-2022. The antigenic profiles of the novel H5N1 and H5N6 exhibited notable variations. Further hemagglutination inhibition assay suggested that some A(H5N1) viruses may be antigenically distinct from the circulating H5N6 and H5N8 strains. Mammalian challenge assays demonstrated that the H5N8 virus (21GD001_H5N8) displayed the highest pathogenicity in mice, followed by the H5N1 virus (B1557_H5N1) and then the H5N6 virus (220086_H5N6), suggesting a heterogeneous virulence profile of H5 AIVs in the mammalian hosts. Based on the above results, we speculate that A(H5N1) viruses have a higher risk of emergence in the future. Collectively, these findings unveil a new landscape of different evolutionary history and biological characteristics of novel H5 AIVs in clade 2.3.4.4b, contributing to a better understanding of designing more effective strategies for the prevention and control of novel H5 AIVs.
- Avian influenza virus (AIV)
- , H5 subtypes AIVs
- , Evolutionary
- , Pathogenicity

References
1. Bhat, S., Bhatia, S., Pillai, A.S., Sood, R., Singh, V.K., Shrivas, O.P., Mishra, S.K., Mawale, N., 2015. Genetic and antigenic characterization of H5N1 viruses of clade 2.3.2.1 isolated in India. Microb Pathog 88, 87-93.
2. Bi, F., Jiang, L., Huang, L., Wei, J., Pan, X., Ju, Y., Mo, J., Chen, M., Kang, N., Tan, Y., Li, Y., Wang, J., 2021. Genetic Characterization of Two Human Cases Infected with the Avian Influenza A (H5N6) Viruses-Guangxi Zhuang Autonomous Region, China, 2021. China CDC Wkly 3, 923-928.
3. Bi, Y., Chen, J., Zhang, Z., Li, M., Cai, T., Sharshov, K., Susloparov, I., Shestopalov, A., Wong, G., He, Y., Xing, Z., Sun, J., Liu, D., Liu, Y., Liu, L., Liu, W., Lei, F., Shi, W., Gao, G.F., 2016a. Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds, 2014-2015. Virol Sin 31, 300-305.
4. Bi, Y., Chen, Q., Wang, Q., Chen, J., Jin, T., Wong, G., Quan, C., Liu, J., Wu, J., Yin, R., Zhao, L., Li, M., Ding, Z., Zou, R., Xu, W., Li, H., Wang, H., Tian, K., Fu, G., Huang, Y., Shestopalov, A., Li, S., Xu, B., Yu, H., Luo, T., Lu, L., Xu, X., Luo, Y., Liu, Y., Shi, W., Liu, D., Gao, G.F., 2016b. Genesis, Evolution and Prevalence of H5N6 Avian Influenza Viruses in China. Cell Host Microbe 20, 810-821.
5. Bui, C.H.T., Kuok, D.I.T., Yeung, H.W., Ng, K.C., Chu, D.K.W., Webby, R.J., Nicholls, J.M., Peiris, J.S.M., Hui, K.P.Y., Chan, M.C.W., 2021. Risk assessment for highly pathogenic avian influenza a(H5N6/H5N8) Clade 2.3.4.4 Viruses. Emerg Infect Dis 27, 2619-2627.
6. Caliendo, V., Lewis, N.S., Pohlmann, A., Baillie, S.R., Banyard, A.C., Beer, M., Brown, I.H., Fouchier, R.A.M., Hansen, R.D.E., Lameris, T.K., Lang, A.S., Laurendeau, S., Lung, O., Robertson, G., van der Jeugd, H., Alkie, T.N., Thorup, K., van Toor, M.L., Waldenstrom, J., Yason, C., Kuiken, T., Berhane, Y., 2022. Transatlantic spread of highly pathogenic avian influenza H5N1 by wild birds from Europe to North America in 2021. Sci Rep 12, 11729.
7. Chen, J., Li, X., Xu, L., Xie, S., Jia, W., 2021. Health threats from increased antigenicity changes in H5N6-dominant subtypes, 2020 China. J Infect 83, e9-e11.
8. Chen, J., Xu, L., Liu, T., Xie, S., Li, K., Li, X., Zhang, M., Wu, Y., Wang, X., Wang, J., Shi, K., Niu, B., Liao, M., Jia, W., 2022. Novel Reassortant Avian Influenza A(H5N6) Virus, China, 2021. Emerg Infect Dis 28, 1701-1707.
9. Cui, P., Shi, J., Wang, C., Zhang, Y., Xing, X., Kong, H., Yan, C., Zeng, X., Liu, L., Tian, G., Li, C., Deng, G., Chen, H., 2022. Global dissemination of H5N1 influenza viruses bearing the clade 2.3.4.4b HA gene and biologic analysis of the ones detected in China. Emerg Microbes Infect 11, 1693-1704.
10. Gu, W., Shi, J., Cui, P., Yan, C., Zhang, Yaping, Wang, C., Zhang, Yuancheng, Xing, X., Zeng, X., Liu, L., Tian, G., Suzuki, Y., Li, C., Deng, G., Chen, H., 2022. Novel H5N6 reassortants bearing the clade 2.3.4.4b HA gene of H5N8 virus have been detected in poultry and caused multiple human infections in China. Emerg Microbes Infect 11, 1174-1185.
11. Gunther, A., Krone, O., Svansson, V., Pohlmann, A., King, J., Hallgrimsson, G.T., Skarphedhinsson, K.H., Sigurdhardottir, H., Jonsson, S.R., Beer, M., Brugger, B., Harder, T., 2022. Iceland as Stepping Stone for Spread of Highly Pathogenic Avian Influenza Virus between Europe and North America. Emerg Infect Dis 28, 2383-2388.
12. Hill, V., Baele, G., 2019. Bayesian Estimation of Past Population Dynamics in BEAST 1.10 Using the Skygrid Coalescent Model. Mol Biol Evol 36, 2620-2628.
13. Huang, J., Wu, S., Wu, W., Liang, Y., Zhuang, H., Ye, Z., Qu, X., Liao, M., Jiao, P., 2021. The Biological Characteristics of Novel H5N6 Highly Pathogenic Avian Influenza Virus and Its Pathogenesis in Ducks. Front Microbiol 12, 628545.
14. Isoda, N., Onuma, M., Hiono, T., Sobolev, I., Lim, H.Y., Nabeshima, K., Honjyo, H., Yokoyama, M., Shestopalov, A., Sakoda, Y., 2022. Detection of New H5N1 High Pathogenicity Avian Influenza Viruses in Winter 2021-2022 in the Far East, Which Are Genetically Close to Those in Europe. Viruses 14, 2168.
15. Jiang W, Dong C, Liu S, Peng C, Yin X, Liang S, Zhang L, Li J, Yu X, Li Y, Wang J, Hou G, Zeng Z, Liu H, 2022. Emerging Novel Reassortant Influenza A(H5N6) Viruses in Poultry and Humans, China, 2021. Emerg Infect Dis 28, 1064-1066.
16. Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., Von Haeseler, A., Jermiin, L.S., 2017. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat Methods 14, 587-589.
17. Ke, X., Yao, Z., Tang, Y., Yang, M., Li, Y., Yang, G., Chen, J., Chen, G., Feng, W., Zheng, H., Chen, Q., 2022. Highly Pathogenic Avian Influenza A (H5N1) Virus in Swans, Central China, 2021. Microbiol Spectr 10.
18. Kuiken, T., Cromie, R., 2022. Protect wildlife from livestock diseases. Science 738, 5.
19. Lo, Fatou T., Zecchin, B., Diallo, A.A., Racky, O., Tassoni, L., Diop Aida, Diouf, Moussa, Diouf, Mayekor, Samb, Y.N., Ambra, P., Federica, G., Ellero, F., Diop, M., Lo, M.M., Diouf, M.N., Fall, M., Ndiaye, A.A., Ndumu, D.B., Gaye, A.M., Badiane, M., Lo, M., Youm, B.N., Ndao, I., Niaga, M., Terregino, C., Diop, B., Ndiaye, Y., Angot, A., Seck, I., Niang, M., Soumare, B., Fusaro, A., Monne, I., 2022. Intercontinental Spread of Eurasian Highly Pathogenic Avian Influenza A(H5N1) to Senegal. Emerg Infect Dis 28, 234-237.
20. Peng, Y., Zou, Y., Li, H., Li, K., Jiang, T., 2014. Inferring the antigenic epitopes for highly pathogenic avian influenza H5N1 viruses. Vaccine 32, 671-676.
21. PIZZI, M., 1950. Sampling variation of the fifty percent end-point, determined by the Reed-Muench (Behrens) method. Hum Biol 22, 150-190.
22. Pyankova, O.G., Susloparov, I.M., Moiseeva, A.A., Kolosova, N.P., Onkhonova, G.S., Danilenko, A. V., Vakalova, E. V., Shendo, G.L., Nekeshina, N.N., Noskova, L.N., Demina, J. V., Frolova, N. V., Gavrilova, E. V., Maksyutov, R.A., Ryzhikov, A.B., 2021. Isolation of clade 2.3.4.4b A(H5N8), a highly pathogenic avian influenza virus, from a worker during an outbreak on a poultry farm, Russia, December 2020. Euro Surveill 26, 2100439.
23. Rambaut, A., Lam, T.T., Carvalho, L.M., Pybus, O.G., 2016. Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen). Virus Evol 2, vew007.
24. Samir, M., Hamed, M., Abdallah, F., Kinh Nguyen, V., Hernandez-Vargas, E.A., Seehusen, F., Baumgartner, W., Hussein, A., Ali, A.A.H., Pessler, F., 2018. An Egyptian HPAI H5N1 isolate from clade 2.2.1.2 is highly pathogenic in an experimentally infected domestic duck breed (Sudani duck). Transbound Emerg Dis 65, 859-873.
25. Sanogo, I.N., Djegui, F., Akpo, Y., Gnanvi, C., Dupre, G., Rubrum, A., Jeevan, T., McKenzie, P., Webby, R.J., Ducatez, M.F., 2022. Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Poultry, Benin, 2021. Emerg Infect Dis 28, 2534-2537.
26. Shi, J., Deng, G., Kong, H., Gu, C., Ma, S., Yin, X., Zeng, X., Cui, P., Chen, Y., Yang, H., Wan, X., Wang, X., Liu, L., Chen, P., Jiang, Y., Liu, J., Guan, Y., Suzuki, Y., Li, M., Qu, Z., Guan, L., Zang, J., Gu, W., Han, S., Song, Y., Hu, Y., Wang, Z., Gu, L., Yang, W., Liang, L., Bao, H., Tian, G., Li, Y., Qiao, C., Jiang, L., Li, C., Bu, Z., Chen, H., 2017. H7N9 virulent mutants detected in chickens in China pose an increased threat to humans. Cell Res 27, 1409-1421.
27. Shi, J., Deng, G., Ma, S., Zeng, X., Yin, X., Li, M., Zhang, B., Cui, P., Chen, Y., Yang, H., Wan, X., Liu, L., Chen, P., Jiang, Y., Guan, Y., Liu, J., Gu, W., Han, S., Song, Y., Liang, L., Qu, Z., Hou, Y., Wang, X., Bao, H., Tian, G., Li, Y., Jiang, L., Li, C., Chen, H., 2018. Rapid Evolution of H7N9 Highly Pathogenic Viruses that Emerged in China in 2017. Cell Host Microbe 24, 558-568.e7.
28. Shi, J., Zeng, X., Cui, P., Yan, C., Chen, H., 2023. Alarming situation of emerging H5 and H7 avian influenza and effective control strategies. Emerg Microbes Infect 12, 2155072.
29. Shi, W., Gao, G.F., 2021. Emerging H5N8 avian influenza viruses: The global spread of H5N8 avian influenza viruses is a public health concern. Science 372, 784-786.
30. Smith, D.J., Lapedes, A.S., de Jong, J.C., Bestobroer, T.M., Rimmelzwaan, G.F., Osterhaus, A.D.M.E., Fouchier, R.A.M., 2004. Mapping the antigenic and genetic evolution of influenza virus. Science 305, 371-374.
31. Stokstad, E., 2022. Deadly bird flu establishes a foothold in North America. Science 377, 912.
32. Swieton, E., Fusaro, A., Fusaro, A., Shittu, I., Niemczuk, K., Zecchin, B., Joannis, T., Bonfante, F., Mietanka, K., Terregino, C., 2020. Sub-Saharan Africa and Eurasia Ancestry of Reassortant Highly Pathogenic Avian Influenza A(H5N8) Virus, Europe, December 2019. Emerg Infect Dis 26, 1557-1561.
33. Verhagen, J.H., Fouchier, R.A.M., Lewis, N., 2021. Highly Pathogenic Avian Influenza Viruses at the Wild-Domestic Bird Interface in Europe: Future Directions for Research and Surveillance. Viruses 13, 212.
34. Weaver, S., Shank, S.D., Spielman, S.J., Li, M., Muse, S. V., Kosakovsky Pond, S.L., 2018. Datamonkey 2.0: A modern web application for characterizing selective and other evolutionary processes. Mol Biol Evol 35, 773-777.
35. Wiley, D.C., Wilson, I.A., Skehel, J.J., 1981. Structural identifcation of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation, 25. Lazarowitz, S. G. & Choppin, P. W. Virology 289, 2737-2740.
36. Wille, M., Barr, I.G., 2022. Resurgence of avian influenza virus. Science 376, 459-460.
37. Xiao, C., Xu, J., Lan, Y., Huang, Z., Zhou, L., Guo, Y., Li, X., Yang, L., Gao, G. F., Wang, D., Liu, W. J., Zhou, X., Yang, H., 2021. Five Independent Cases of Human Infection with Avian Influenza H5N6-Sichuan Province, China, 2021. China CDC Wkly 3, 751-756.
38. Yamashita, A., Kawashita, N., Kubota-Koketsu, R., Inoue, Y., Watanabe, Y., Ibrahim, M.S., Ideno, S., Yunoki, M., Okuno, Y., Takagi, T., Yasunaga, T., Ikuta, K., 2010. Highly conserved sequences for human neutralization epitope on hemagglutinin of influenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition. Biochem Biophys Res Commun 393, 614-618.
39. Zhang, C., Wang, Z.Y., Cui, H., Chen, L.G., Zhang, C.M., Chen, Z.L., Dong, S.S., Zhao, K., Fu, Y.Y., Liu, J.X., Guo, Z.D., 2023. Emergence of H5N8 avian influenza virus in domestic geese in a wild bird habitat, Yishui Lake, north central China. Virol Sin 38, 157-161.
40. Zhang, G., Li, B., Raghwani, J., Vrancken, B., Jia, R., Hill, S.C., Fournie, G., Cheng, Y., Yang, Q., Wang, Y., Wang, Z., Dong, L., Pybus, O.G., Tian, H., 2023. Bidirectional Movement of Emerging H5N8 Avian Influenza Viruses between Europe and Asia via Migratory Birds since Early 2020. Mol Biol Evol 40, msad019.
41. Zhang, J., Li, X., Wang, X., Ye, H., Li, B., Chen, Y., Chen, J., Zhang, T., Qiu, Z., Li, H., Jia, W., Liao, M., Qi, W., 2021. Genomic evolution, transmission dynamics, and pathogenicity of avian influenza A (H5N8) viruses emerging in China, 2020. Virus Evol 7, veab046.
42. Zhang J, Ye H, Liu Y, Liao M, QI W, 2022. Resurgence of H5N6 avian influenza virus in 2021 poses new threat to public health. Lancet Microbe 3, e558.
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