Mapping the adaptive landscape of H9N2 hemagglutinin links hotspot substitutions to cross-species potential and thermostability

Abstract

H9N2 avian influenza virus poses a persistent threat to poultry and public health because of its widespread circulation and role in generating novel viruses. To investigate the genetic determinants of HA antigenic diversity and adaptation, we constructed a hotspot library targeting 179 substitution hotspots identified through large-scale sequence analysis of 13,984 HA sequences distributed across the HA protein. The highly variable residues 145 and 168 represent prominent substitution hotspots that correlate with host-specific lineages and highlight poultry as a key adaptation reservoir. The library was subjected to replication in multiple host-cell systems, as well as immune and thermal selection. Functional screening revealed that N115H enhances viral replication in mammalian cells, residue 164 is critical for antigenic escape, and I134L, L230S, and V306L collectively increase HA thermostability. These results link natural sequence variation to functional phenotypes, demonstrating that H9N2 HA antigenic diversity and cross-species potential are shaped by mutation hotspots. Our integrated computational and experimental framework provides a platform for probing viral fitness landscapes, with implications for risk assessment and the rational design of next-generation vaccines.

Keywords

Avian influenza virus / Hotspot library / Antigenic diversity / HA hotspots / Vaccine development

References

[1] Bi Y, Li J, Li S, Fu G, Jin T, Zhang C, Yang Y, Ma Z, Tian W, Li J, et al. . Dominant subtype switch in avian influenza viruses during 2016-2019 in China. Nature Communications, 2020, 11(1): 5909

[2] Burton, T. D., and N. S. Eyre. 2021. Applications of Deep Mutational Scanning in Virology. Viruses 13 (6):1020. https://doi.org/10.3390/v13061020.

[3] Carnaccini, S., and D. R. Perez. 2020. H9 Influenza Viruses: An Emerging Challenge. Cold Spring Harb Perspect Med 10 (6):a038588. https://doi.org/10.1101/cshperspect.a038588.

[4] Cotter CR, Jin H, Chen Z. A single amino acid in the stalk region of the H1N1pdm influenza virus HA protein affects viral fusion, stability and infectivity. PLoS Pathogens, 2014, 10(1): e1003831

[5] Cui H, De Jong MC, Beerens N, Van Oers MM, Teng Q, Li L, Li X, Liu Q, Li Z. Vaccination with inactivated virus against low pathogenic avian influenza subtype H9N2 does not prevent virus transmission in chickens. Journal of Virus Eradication, 2021, 7(3): 100055

[6] Ding X, Liu J, Jiang T, Wu A. Transmission restriction and genomic evolution coshape the genetic diversity patterns of influenza A virus. Virologica Sinica, 2024, 39(4): 525-536

[7] Dong G, Luo J, Zhang H, Wang C, Duan M, Deliberto TJ, Nolte DL, Ji G, He H. Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis. PLoS ONE, 2011, 6(2): e17212

[8] Dong, J., Y. Zhou, J. Pu, and L. Liu. 2022. Status and Challenges for Vaccination against Avian H9N2 Influenza Virus in China. Life (Basel) 12 (9):1326. https://doi.org/10.3390/life12091326.

[9] Doud, M. B., and J. D. Bloom. 2016. Accurate measurement of the effects of all amino-acid mutations on influenza hemagglutinin. Viruses 8 (6):155. https://doi.org/10.3390/v8060155.

[10] Doud MB, Lee JM, Bloom JD. How single mutations affect viral escape from broad and narrow antibodies to H1 influenza hemagglutinin. Nature Communications, 2018, 9(1): 1386

[11] Fulton, B. O., W. Sun, N. S. Heaton, P. Palese. 2018. The Influenza B Virus Hemagglutinin Head Domain Is Less Tolerant to Transposon Mutagenesis than That of the Influenza A Virus. J Virol 92 (16):e00754–18. https://doi.org/10.1128/JVI.00754-18.

[12] Gamblin, S. J., S. G. Vachieri, X. Xiong, J. Zhang, S. R. Martin, and J. J. Skehel. 2021. Hemagglutinin Structure and Activities. Cold Spring Harb Perspect Med 11 (10):a038638. https://doi.org/10.1101/cshperspect.a038638.

[13] Gawronski JD, Wong SM, Giannoukos G, Ward DV, Akerley BJ. Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(38): 16422-16427

[14] Guan Y, Shortridge KF, Krauss S, Chin PS, Dyrting KC, Ellis TM, Webster RG, Peiris M. H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in southeastern China. Journal of Virology, 2000, 74(20): 9372-9380

[15] Hu Z, Ai H, Wang Z, Huang S, Sun H, Xuan X, Chen M, Wang J, Yan W, Sun J, et al. . Impact of inactivated vaccine on transmission and evolution of H9N2 avian influenza virus in chickens. NPJ Vaccines, 2025, 10(1): 67

[16] Jones JE, Le Sage V, Lakdawala SS. Viral and host heterogeneity and their effects on the viral life cycle. Nature Reviews Microbiology, 2021, 19(4): 272-282

[17] Lee, J. M., R. Eguia, S. J. Zost, S. Choudhary, P. C. Wilson, T. Bedford, T. Stevens-Ayers, M. Boeckh, A. C. Hurt, S. S. Lakdawala, et al. 2019. Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin. Elife 8:e49324. https://doi.org/10.7554/eLife.49324.

[18] Lee JM, Huddleston J, Doud MB, Hooper KA, Wu NC, Bedford T, Bloom JD. Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(35): E8276-E8285

[19] Ma L, Zheng H, Ke X, Gui R, Yao Z, Xiong J, Chen Q. Mutual antagonism of mouse-adaptation mutations in HA and PA proteins on H9N2 virus replication. Virology Sinica, 2024, 39(1): 56-70

[20] Mosaad Z, Hagag NM, Elsayed MM, Mady WH, Zanaty AM, El-Badiea ZA, Amer F, Said M, Selim A, Farghally E, et al. . Isolation, characterization and phylogenetic analyses of avian influenza A (H9N2) viruses isolated from poultry between 2019 and 2023 in Egypt. BMC Veterinary Research, 2025, 21(1): 455

[21] Ping J, Li C, Deng G, Jiang Y, Tian G, Zhang S, Bu Z, Chen H. Single-amino-acid mutation in the HA alters the recognition of H9N2 influenza virus by a monoclonal antibody. Biochemical and Biophysical Research Communications, 2008, 371(1): 168-171

[22] Pu J, Wang S, Yin Y, Zhang G, Carter RA, Wang J, Xu G, Sun H, Wang M, Wen C, et al. . Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(2): 548-553

[23] Song JH, Son SE, Kim H, Kim SJ, An SH, Lee CY, Choi KS, Kwon HJ. Development of an H9N2 vaccine strain with enhanced neuraminidase immunogenicity via dual hemagglutinin and neuraminidase modification. Journal of Veterinary Science, 2025, 26(6): e81

[24] Teng Q, Xu D, Shen W, Liu Q, Rong G, Li X, Yan L, Yang J, Chen H, Yu H, et al. . A single mutation at position 190 in hemagglutinin enhances binding affinity for human type sialic acid receptor and replication of H9N2 avian influenza virus in mice. Journal of Virology, 2016, 90(21): 9806-9825

[25] Wan H, Perez DR. Amino acid 226 in the hemagglutinin of H9N2 influenza viruses determines cell tropism and replication in human airway epithelial cells. Journal of Virology, 2007, 81(10): 5181-5191

[26] Wang L, Liu SY, Chen HW, Xu J, Chapon M, Zhang T, Zhou F, Wang YE, Quanquin N, Wang G, et al. . Generation of a live attenuated influenza vaccine that elicits broad protection in mice and ferrets. Cell Host & Microbe, 2017, 21(3): 334-343

[27] Wang X, Liu K, Guo Y, Pei Y, Chen X, Lu X, Gao R, Chen Y, Gu M, Hu J, et al. . 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, 2023, 12(2): 2249558

[28] Wen F, Yan Z, Chen G, Chen Y, Wang N, Li Z, Guo J, Yu H, Liu Q, Huang S. Recent H9N2 avian influenza virus lost hemagglutination activity due to a K141N substitution in hemagglutinin. Journal of Virology, 2024, 98(4): e0024824

[29] Wu J, Wan Z, Qian K, Shao H, Ye J, Qin A. The amino acid variation at hemagglutinin sites 145, 153, 164 and 200 modulate antigenicity andreplication of H9N2 avian influenza virus. Veterinary Microbiology, 2024, 296: 110188

[30] Wu, N. C., and I. A. Wilson. 2020. Influenza Hemagglutinin Structures and Antibody Recognition. Cold Spring Harb Perspect Med 10 (8):a038778. https://doi.org/10.1101/cshperspect.a038778.

[31] Wu NC, Young AP, Dandekar S, Wijersuriya H, Al-Mawsawi LQ, Wu TT, Sun R. Systematic identification of H274Y compensatory mutations in influenza A virus neuraminidase by high-throughput screening. Journal of Virology, 2013, 87(2): 1193-1199

[32] Yan W, Cui H, Engelsma M, Beerens N, Van Oers MM, De Jong MCM, Li X, Liu Q, Yang J, Teng Q, et al. . Molecular and antigenic characterization of avian H9N2 viruses in Southern China. Microbiology Spectrum, 2022, 10(1): e0082221

[33] Yang J, Li J, Sun J, Li J, Fu G, Tian T, Yang Y, Lu X, Li S, Wang L, et al. . Genetic diversity of H9N2 avian influenza viruses in poultry across China and implications for zoonotic transmission. Nature Microbiology, 2025, 10(6): 1378-1392

[34] Yang R, Sun H, Gao F, Luo K, Huang Z, Tong Q, Song H, Han Q, Liu J, Lan Y, et al. . Human infection of avian influenza A H3N8 virus and the viral origins: A descriptive study. The Lancet Microbe, 2022, 3(11): e824-e834

[35] Ye J, Wen F, Xu Y, Zhao N, Long L, Sun H, Yang J, Cooley J, Todd Pharr G, Webby R, et al. . Error-prone pcr-based mutagenesis strategy for rapidly generating high-yield influenza vaccine candidates. Virology, 2015, 482: 234-243

[36] Zhai K, Dong J, Zeng J, Cheng P, Wu X, Han W, Chen Y, Qiu Z, Zhou Y, Pu J, et al. . Global antigenic landscape and vaccine recommendation strategy for low pathogenic avian influenza A (H9N2) viruses. Journal of Infection, 2024, 89(2): 106199

[37] Zhang J, Wang X, Chen Y, Ye H, Ding S, Zhang T, Liu Y, Li H, Huang L, Qi W, et al. . Mutational antigenic landscape of prevailing H9N2 influenza virus hemagglutinin spectrum. Cell Reports, 2023, 42(11): 113409

[38] Zhu R, Wu J, Chen R, Zhou M, Cao S, Wu Z, Wang L, Zhang L, Zhu S. HA198 mutations in H9N2 avian influenza: Molecular dynamics insights into receptor binding. Frontiers in Veterinary Science, 2024, 11: 1526600

Funding

the National Key Research and Development Program of China(2024YFE0106100)

the start-up fund from Hubei Hongshan Laboratory(11020107)

the National Natural Science Foundation of China(32573468)

National Postdoctoral Overseas Talent Introduction Program of China(590323070)

the fundamental research funds for the central universities of China(2662023PY005&2662025DKPY004)

RIGHTS & PERMISSIONS

The Author(s)