Epidemiological, phylogenetic, and pathogenicity analysis of H4 subtype avian influenza viruses in China, 2011-2022

Qiuyan Mao; Junfeng Zhu; Shuo Liu; Cheng Peng; Tiantian Wu; Jie Tian; Xiaoqi Li; Jizhe Yang; Jinping Li; Guangyu Hou; Wenming Jiang; Hualei Liu

doi:10.1016/j.virs.2025.09.005

October 2025

Citation: Qiuyan Mao, Junfeng Zhu, Shuo Liu, Cheng Peng, Tiantian Wu, Jie Tian, Xiaoqi Li, Jizhe Yang, Jinping Li, Guangyu Hou, Wenming Jiang, Hualei Liu. Epidemiological, phylogenetic, and pathogenicity analysis of H4 subtype avian influenza viruses in China, 2011-2022 .VIROLOGICA SINICA, 2025, 40(5) : 722-734. http://dx.doi.org/10.1016/j.virs.2025.09.005

Epidemiological, phylogenetic, and pathogenicity analysis of H4 subtype avian influenza viruses in China, 2011-2022

Qiuyan Mao ^a ,
Junfeng Zhu ^b ,
Shuo Liu ^a ,
Cheng Peng ^a ,
Tiantian Wu ^c ,
Jie Tian ^a ,
Xiaoqi Li ^a ,
Jizhe Yang ^a ,
Jinping Li ^a ,
Guangyu Hou ^a ,
Wenming Jiang ^{a
,,} ,
Hualei Liu ^{a
,,}

a. Avian Diseases Surveillance Laboratory, China Animal Health and Epidemiology Center, Qingdao 266032, China;
b. China Institute of Veterinary Drug Control, Beijing 100081, China;
c. Agricultural Technology Comprehensive Service Center of Fangshan District, Beijing 102488, China

Corresponding author: Wenming Jiang, civcul@163.com
Hualei Liu, liuhualei@cahec.cn
Received Date: 07 May 2025
Accepted Date: 19 September 2025
Available online: 23 September 2025

Abstract

The H4 subtype of avian influenza virus (AIV) is prevalent worldwide, but only receives little attention due to its low pathogenicity in poultry. Consequently, it remains largely unclear whether H4 AIVs pose a potential threat to the poultry industry and public health. During the period from 2011 to 2022, we conducted an active surveillance programm. A total of 154,762 swab samples were collected across various provinces, and 427 H4 viruses were detected, resulting in a positivity rate of 0.28%. All H4 viruses were isolated from poultry, primarily from ducks in live poultry markets. We further investigated the genetic evolutionary characteristics and pathogenicity of 20 H4Nx viruses isolated in our program. Phylogenetic analysis revealed that the 20 H4Nx viruses belonged to the Eurasian lineage and exhibited significant genetic diversity, with 19 distinct genotypes identified. Molecular characterization indicated that these viruses were low-pathogenicity AIVs with limited binding affinity to human receptors, yet they contained mutations associated with enhanced viral replication and pathogenicity in mammals. Pathogenicity tests conducted in ducks demonstrated that H4 viruses were weakly pathogenic, exhibiting limited replication and transmission capabilities. However, some viruses were able to replicate effectively in mice and induce weight loss. For instance, DK/AH/AG61/11(H4N6) can replicate efficiently in MDCK cells, indicating a potential threat to mammals. These findings underscore the importance of ongoing surveillance of H4 AIVs to better understand their evolution and transmission dynamics and to prevent potential public health risks.
- H4 avian influenza viruses (H4 AIVs)
- , Epidemiology
- , Phylogenetic
- , Pathogenicity
- , Ducks
- , Mice

Electronic Supplementary Material

10.1016j.virs.2025.09.005-ESM1.docx
References
1. Abente, E.J., Gauger, P.C., Walia, R.R., Rajao, D.S., Zhang, J., Harmon, K.M., Killian, M.L.,Vincent, A.L., 2017. Detection and characterization of an H4N6 avian-lineage influenza A virus in pigs in the Midwestern United States. Virology, 511, 56-65.
2. Abubakar, U., Amrani, L., Kamarulzaman, F.A., Karsani, S.A., Hassandarvish, P.,Khairat, J.E., 2023. Avian Influenza Virus Tropism in Humans. Viruses, 15, 833.
3. Alexander,J., D., 2007. An overview of the epidemiology of avian influenza. Vaccine, 25, 5637-5644.
4. Blagodatski, A., Trutneva, K., Glazova, O., Mityaeva, O., Shevkova, L., Kegeles, E., Onyanov, N., Fede, K., Maznina, A., Khavina, E., Yeo, S.-J., Park, H.,Volchkov, P., 2021. Avian Influenza in Wild Birds and Poultry: Dissemination Pathways, Monitoring Methods, and Virus Ecology. Pathogens, 10, 630.
5. Brown, E.G., Liu, H., Kit, L.C., Baird, S.,Nesrallah, M., 2001. Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: Identification of functional themes. Proceedings of the National Academy of Sciences, 98, 6883-6888.
6. Bui, V.N., Ogawa, H., Xininigen, Karibe, K., Matsuo, K., Awad, S.S.A., Minoungou, G.L., Yoden, S., Haneda, H., Ngo, L.H., Tamaki, S., Yamamoto, Y., Nakamura, K., Saito, K., Watanabe, Y., Runstadler, J., Huettman, F., Happ, G.M.,Imai, K., 2012. H4N8 subtype avian influenza virus isolated from shorebirds contains a unique PB1 gene and causes severe respiratory disease in mice. Virology, 423, 77-88.
7. Chauhan, R.P.,Gordon, M.L., 2022. An overview of influenza A virus genes, protein functions, and replication cycle highlighting important updates. Virus Genes, 58, 255-269.
8. Chen, W., Lu, S., Xiong, H., Xiang, Z., Wang, Y., Hu, J., Pan, Y., Li, Y., Gao, Q., Chen, Q., Hu, S., Wang, W., Xiong, C., 2025. Gene flow and its sporadic spillover: H10 and N5 avian influenza viruses from wild birds and the H10N5 human cases in in China. Virol Sin 40, 15-23.
9. Cui, P., Shi, J., Yan, C., Wang, C., Zhang, Y., Zhang, Y., Xing, X., Chen, Y., Zhang, J., Liu, L., Zeng, X., Tian, G., Li, C., Suzuki, Y., Deng, G.,Chen, H., 2023. Analysis of avian influenza A (H3N8) viruses in poultry and their zoonotic potential, China, September 2021 to May 2022. Eurosurveillance, 28, 2200871.
10. De Bruin, A.C.M., Funk, M., Spronken, M.I., Gultyaev, A.P., Fouchier, R.a.M.,Richard, M. 2022. Hemagglutinin Subtype Specificity and Mechanisms of Highly Pathogenic Avian Influenza Virus Genesis. Viruses [Online], 14.
11. Deng, G., Tan, D., Shi, J., Cui, P., Jiang, Y., Liu, L., Tian, G., Kawaoka, Y., Li, C.,Chen, H., 2013. Complex Reassortment of Multiple Subtypes of Avian Influenza Viruses in Domestic Ducks at the Dongting Lake Region of China. Journal of Virology, 87, 9452-9462.
12. Donis, R.O., Bean, W.J., Kawaoka, Y.,Webster, R.G., 1989. Distinct lineages of influenza virus H4 hemagglutinin genes in different regions of the world. Virology, 169, 408-417.
13. Fan, S., Deng, G., Song, J., Tian, G., Suo, Y., Jiang, Y., Guan, Y., Bu, Z., Kawaoka, Y.,Chen, H., 2009. Two amino acid residues in the matrix protein M1 contribute to the virulence difference of H5N1 avian influenza viruses in mice. Virology, 384, 28-32.
14. Fereidouni, S., Starick, E., Karamendin, K., Di Genova, C., Scott, S.D., Khan, Y., Harder, T.,Kydyrmanov, A., 2023. Genetic characterization of a new candidate hemagglutinin subtype of influenza A viruses. Emerging Microbes & Infections, 12, 2225645.
15. Gao, R., Gu, M., Shi, L., Liu, K., Li, X., Wang, X., Hu, J., Liu, X., Hu, S., Chen, S., Peng, D., Jiao, X.,Liu, X., 2021. N-linked glycosylation at site 158 of the HA protein of H5N6 highly pathogenic avian influenza virus is important for viral biological properties and host immune responses. Veterinary Research, 52, 8.
16. Ge, Y., Liu, J., Li, Y., Peng, P., Zhou, Y., Yu, J., Huo, M., Liang, X., Gao, Y.,Yao, Q., 2025. Geographical distribution and evolutionary dynamics of H4Nx avian influenza viruses. Frontiers in Microbiology, 15, 1505203.
17. Guan, Y., Shortridge, K.F., Krauss, S.,Webster, R.G., 1999. Molecular characterization of H9N2 influenza viruses: Were they the donors of the “internal” genes of H5N1 viruses in Hong Kong? Proceedings of the National Academy of Sciences, 96, 9363-9367.
18. He, J., Deng, J., Wen, X., Yan, M., Liu, Y., Zhou, Y., Du, X., Yang, H.,Peng, X., 2024. Isolation and genetic characteristics of Novel H4N1 Avian Influenza viruses in ChongQing, China. Virology Journal, 21, 85.
19. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G.,Perez, D.R., 2001. Universal primer set for the full-length amplification of all influenza A viruses. Archives of Virology, 146, 2275-2289.
20. Hu, Y., Liu, X., Li, S., Guo, X., Yang, Y.,Jin, M., 2012. Complete Genome Sequence of a Novel H4N1 Influenza Virus Isolated from a Pig in Central China. Journal of Virology, 86, 13879-13879.
21. Jiao, P., Tian, G., Li, Y., Deng, G., Jiang, Y., Liu, C., Liu, W., Bu, Z., Kawaoka, Y.,Chen, H., 2008. A Single-Amino-Acid Substitution in the NS1 Protein Changes the Pathogenicity of H5N1 Avian Influenza Viruses in Mice. Journal of Virology, 82, 1146-1154.
22. Jing, J., Wang, L., Wang, G., Dai, Z., Ren, W., Yi, C., Wei, J.,Xu, C., 2021. A human infection case with avian-origin H10N3 influenza virus. Quantitative Imaging in Medicine and Surgery, 11, 4508-4510.
23. Kamal, R., Alymova, I.,York, I., 2017. Evolution and Virulence of Influenza A Virus Protein PB1-F2. International Journal of Molecular Sciences, 19, 96.
24. Karakus, U., Mena, I., Kottur, J., El Zahed, S.S., Seoane, R., Yildiz, S., Chen, L., Plancarte, M., Lindsay, L., Halpin, R., Stockwell, T.B., Wentworth, D.E., Boons, G.-J., Krammer, F., Stertz, S., Boyce, W., De Vries, R.P., Aggarwal, A.K.,Garcia-Sastre, A., 2024. H19 influenza A virus exhibits species-specific MHC class II receptor usage. Cell Host & Microbe, 32, 1089-1102.e1010.
25. Kayali, G., Ortiz, E.J., Chorazy, M.L.,Gray, G.C., 2009. Evidence of Previous Avian Influenza Infection among US Turkey Workers. Zoonoses and Public Health, 57, 265-272.
26. Kim, E.-H., Kim, Y.-L., Kim, S.M., Yu, K.-M., Casel, M.a.B., Jang, S.-G., Pascua, P.N.Q., Webby, R.J.,Choi, Y.K., 2021. Pathogenic assessment of avian influenza viruses in migratory birds. Emerging Microbes & Infections, 10, 565-577.
27. Kode, S.S., Pawar, S.D., Tare, D.S., Keng, S.S., Hurt, A.C.,Mullick, J., 2019. A novel I117T substitution in neuraminidase of highly pathogenic avian influenza H5N1 virus conferring reduced susceptibility to oseltamivir and zanamivir. Veterinary Microbiology, 235, 21-24.
28. Li, T., Zhao, C., Guo, Y., Dong, J., Du, F., Zhou, Y., Shu, S., Liu, Y., Cheng, Y., Cao, Z., Cao, Q., Shi, S., Huang, Y., Pu, J.,Liu, L., 2024. Genetic and Biological Characteristics of Duck-Origin H4N6 Avian Influenza Virus Isolated in China in 2022. Viruses, 16, 207.
29. Liang, L., Deng, G., Shi, J., Wang, S., Zhang, Q., Kong, H., Gu, C., Guan, Y., Suzuki, Y., Li, Y., Jiang, Y., Tian, G., Liu, L., Li, C., Chen, H.,Dermody, T.S., 2016. Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China. Journal of Virology, 90, 1455-1469.
30. Lin, S., Zhang, Y., Yang, J., Yang, L., Li, X., Bo, H., Liu, J., Tan, M., Zhu, W., Wang, D.,Shu, Y., 2024. Cross-Species Transmission Potential of H4 Avian Influenza Viruses in China: Epidemiological and Evolutionary Study. Viruses, 16, 353.
31. Liu, J., Liang, Z., Sun, W., Hua, W., Huang, S.,Wen, F., 2024. The H4 subtype of avian influenza virus: a review of its historical evolution, global distribution, adaptive mutations and receptor binding properties. Poultry Science, 103, 103913.
32. Liu, M., He, S., Walker, D., Zhou, N., Perez, D.R., Mo, B., Li, F., Huang, X., Webster, R.G.,Webby, R.J., 2003. The Influenza Virus Gene Pool in a Poultry Market in South Central China. Virology, 305, 267-275.
33. Luo, S., Xie, Z., Li, M., Li, D., Xie, L., Huang, J., Zhang, M., Zeng, T., Wang, S., Fan, Q., Zhang, Y., Xie, Z., Deng, X.,Liu, J., 2021. Survey of low pathogenic avian influenza viruses in live poultry markets in Guangxi Province, Southern China, 2016-2019. Scientific Reports, 11, 23223.
34. Luo, S., Xie, Z., Xie, Z., Xie, L., Huang, L., Huang, J., Deng, X., Zeng, T., Wang, S., Zhang, Y.,Liu, J., 2017. Surveillance of Live Poultry Markets for Low Pathogenic Avian Influenza Viruses in Guangxi Province, Southern China, from 2012-2015. Scientific Reports, 7, 17577.
35. Lupiani, B.,Reddy, S.M., 2009. The history of avian influenza. Comparative Immunology, Microbiology and Infectious Diseases, 32, 311-323.
36. Murcia, P.R., Hill, N.J., Bishop, M.A., Trovao, N.S., Ineson, K.M., Schaefer, A.L., Puryear, W.B., Zhou, K., Foss, A.D., Clark, D.E., Mackenzie, K.G., Gass, J.D., Borkenhagen, L.K., Hall, J.S.,Runstadler, J.A., 2022. Ecological divergence of wild birds drives avian influenza spillover and global spread. PLOS Pathogens, 18, e1010062.
37. Naguib, M.M., Verhagen, J.H., Mostafa, A., Wille, M., Li, R., Graaf, A., Jarhult, J.D., Ellstrom, P., Zohari, S., Lundkvist, A.,Olsen, B., 2019. Global patterns of avian influenza A (H7): virus evolution and zoonotic threats. FEMS Microbiology Reviews, 43, 608-621.
38. Olsen, B., Munster, V.J., Wallensten, A., Waldenstrom, J., Osterhaus, A.D.M.E.,Fouchier, R.a.M., 2006. Global Patterns of Influenza A Virus in Wild Birds. Science, 312, 384-388.
39. Parums, D.V., 2023. Editorial: Global Surveillance of Highly Pathogenic Avian Influenza Viruses in Poultry, Wild Birds, and Mammals to Prevent a Human Influenza Pandemic. Medical Science Monitor, 29, e939968.
40. Shi, Y., Cui, H., Wang, J., Chi, Q., Li, X., Teng, Q., Chen, H., Yang, J., Liu, Q.,Li, Z., 2016. Characterizations of H4 avian influenza viruses isolated from ducks in live poultry markets and farm in Shanghai. Scientific Reports, 6, 37843.
41. Simancas-Racines, A., Cadena-Ullauri, S., Guevara-Ramirez, P., Zambrano, A.K.,Simancas-Racines, D., 2023. Avian Influenza: Strategies to Manage an Outbreak. Pathogens, 12, 610.
42. Smith, G.J.D., Kayali, G., Barbour, E., Dbaibo, G., Tabet, C., Saade, M., Shaib, H.A., Debeauchamp, J.,Webby, R.J., 2011. Evidence of Infection with H4 and H11 Avian Influenza Viruses among Lebanese Chicken Growers. PLoS ONE, 6, e26818.
43. Song, H., Qi, J., Xiao, H., Bi, Y., Zhang, W., Xu, Y., Wang, F., Shi, Y.,Gao, G.F., 2017. Avian-to-Human Receptor-Binding Adaptation by Influenza A Virus Hemagglutinin H4. Cell Reports, 20, 1201-1214.
44. Song, J., Xu, J., Shi, J., Li, Y.,Chen, H., 2015. Synergistic Effect of S224P and N383D Substitutions in the PA of H5N1 Avian Influenza Virus Contributes to Mammalian Adaptation. Scientific Reports, 5, 10510.
45. Song, W.,Qin, K., 2020. Human-infecting influenza A (H9N2) virus: A forgotten potential pandemic strain? Zoonoses and Public Health, 67, 203-212.
46. Song, X., Tian, J., Li, M., Bai, X., Zhao, Z., Shi, J., Zeng, X., Tian, G., Guan, Y., Chai, H., Li, Y.,Chen, H., 2024a. Epidemiology and biological characteristics of influenza A (H4N6) viruses from wild birds. Emerging Microbes & Infections, 13, 2418909.
47. Song, X., Tian, J., Li, M., Bai, X., Zhao, Z., Shi, J., Zeng, X., Tian, G., Guan, Y., Cui, P., Deng, G., Liu, L., Chai, H., Li, Y., Chen, H.,Chen, N.-H., 2024b. Genetics and Pathogenicity of Influenza A (H4N6) Virus Isolated from Wild Birds in Jiangsu Province, China, 2023. Transboundary and Emerging Diseases, 2024.
48. Stevaert, A., Dallocchio, R., Dessi, A., Pala, N., Rogolino, D., Sechi, M.,Naesens, L., 2013. Mutational Analysis of the Binding Pockets of the Diketo Acid Inhibitor L-742,001 in the Influenza Virus PA Endonuclease. Journal of Virology, 87, 10524-10538.
49. Su, S., Qi, W.-B., Chen, J.-D., Cao, N., Zhu, W.-J., Yuan, L.-G., Wang, H.,Zhang, G.-H., 2012. Complete Genome Sequence of an Avian-Like H4N8 Swine Influenza Virus Discovered in Southern China. Journal of Virology, 86, 9542-9542.
50. Sutton, T.C. 2018. The Pandemic Threat of Emerging H5 and H7 Avian Influenza Viruses. Viruses [Online], 10.
51. To, K.K.W., Tsang, A.K.L., Chan, J.F.W., Cheng, V.C.C., Chen, H.,Yuen, K.-Y., 2014. Emergence in China of human disease due to avian influenza A(H10N8) - Cause for concern? Journal of Infection, 68, 205-215.
52. 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.
53. Wang, X., Liu, K., Guo, Y., Pei, Y., Chen, X., Lu, X., Gao, R., Chen, Y., Gu, M., Hu, J., Liu, X., Hu, S., Jiao, X.-A., Liu, X.,Wang, X., 2023. Emergence of a new designated clade 16 with significant antigenic drift in hemagglutinin gene of H9N2 subtype avian influenza virus in eastern China. Emerging Microbes & Infections, 12, 2249558.
54. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M.,Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiological Reviews, 56, 152-179.
55. Wu, A., Su, C., Wang, D., Peng, Y., Liu, M., Hua, S., Li, T., Gao, George f., Tang, H., Chen, J., Liu, X., Shu, Y., Peng, D.,Jiang, T., 2013. Sequential Reassortments Underlie Diverse Influenza H7N9 Genotypes in China. Cell Host & Microbe, 14, 446-452.
56. Wu, H., Peng, X., Peng, X., Cheng, L., Lu, X., Jin, C., Xie, T., Yao, H.,Wu, N., 2015. Genetic characterization of natural reassortant H4 subtype avian influenza viruses isolated from domestic ducks in Zhejiang province in China from 2013 to 2014. Virus Genes, 51, 347-355.
57. Wu, Y., Wu, Y., Tefsen, B., Shi, Y.,Gao, G.F., 2014. Bat-derived influenza-like viruses H17N10 and H18N11. Trends in Microbiology, 22, 183-191.
58. Xiang, B., Chen, L., Song, J., Xie, P., Lin, Q., Liao, M., Xu, C.,Ren, T., 2019. Genetic characterization of H7N4 avian influenza virus in China in 2018. Journal of Infection, 79, 174-187.
59. Xie, J., Chen, Y., Cai, G., Cai, R., Hu, Z.,Wang, H., 2023. Tree Visualization By One Table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Research, 51, W587-W592.
60. Xu, X., Chen, Q., Tan, M., Liu, J., Li, X., Yang, L., Shu, Y., Wang, D.,Zhu, W., 2022. Epidemiology, evolution, and biological characteristics of H6 avian influenza viruses in China. Emerging Microbes & Infections, 12, 2151380.
61. Yuan, J., Zhang, L., Kan, X., Jiang, L., Yang, J., Guo, Z.,Ren, Q., 2013. Origin and Molecular Characteristics of a Novel 2013 Avian Influenza A(H6N1) Virus Causing Human Infection in Taiwan. Clinical Infectious Diseases, 57, 1367-1368.
62. Yuan, Z., Zhang, J., Jiang, D., Huang, G.,Qi, W., 2024. Epidemiology and evolution of human-origin H10N5 influenza virus. One Health, 19, 100893.
63. Zhang, H., Chen, Q.,Chen, Z., 2011. Characterization of an H4N2 avian influenza virus isolated from domestic duck in Dongting Lake wetland in 2009. Virus Genes, 44, 24-31.
64. Zhuang, Y., Wang, M., Liang, L., Mao, Y., Wang, K., Yang, S., Deng, A., Zeng, K., Zhang, Y., Zhang, G., Kang, M., Li, B., Zhang, M.,Ye, S., 2024. First Known Human Death After Infection With the Avian Influenza A/H3N8 Virus: Guangdong Province, China, March 2023. Clinical Infectious Diseases, 78, 646-650.
Proportional views