Genetic diversity of H9N2 avian influenza viruses in Iran over the past two decades

Document Type : Original Article

Authors

Department of Avian Disease Research and Diagnostics, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran

Abstract
Numerous studies have explored the molecular epidemiology of H9N2 viruses in Iran; however, continuous monitoring remains vital for timely interventions to mitigate potential damage. This study examined the molecular characteristics and evolutionary features of Iranian H9N2 viruses by sequencing the complete genomes of two viruses, Marand and Baneh, isolated in 1998 and 2022, respectively, alongside other Iranian strains from GenBank. All Iranian viruses were identified as low-pathogenic avian influenza viruses, as evidenced by the presence of the di-basic motif K/RSSR cleavage site. Notably, all Iranian viruses isolated from 2009 onward had an L at position 216 in the hemagglutinin receptor binding site, whereas earlier viruses exhibited a Q/L at the same position, an essential mutation that enhances replication in mammalian cells. The molecular evolutionary rates for the Iranian hemagglutinin (HA) and neuraminidase (NA) genes were estimated at 4.50 × 10-3 and 3.60 × 10-3 substitutions per site per year, respectively. Error-prone replication of H9N2 viruses has resulted in the continuous evolution of Iranian strains over two decades, characterized by three phases of population growth. Maximum likelihood phylogenetic analysis revealed that the HA and NA genes of H9N2 viruses from domestic chickens belonged to the G1 sublineage. Additionally, the internal genes of some viruses displayed evidence of reassortment with other subtypes, indicating potential gene exchange with other viruses. These findings underscored the importance of ongoing surveillance of H9N2 viruses in both domestic and wild bird populations, given the human-like receptor-binding preference and the possibility of genetic reassortment with various viral subtypes.

Keywords

Subjects

1.     Black EJ, Powell CS, Dempsey DM, et al. Virus taxonomy: the database of the International Committee on Taxonomy of Viruses. Nucleic Acids Res 2026; 54(D1): D776-D789.
2.     Swayne ED, Suarez DL, Sims LD. Influenza. In: Swayne DE, Boulianne M, Logue CM, et al. (Eds). Diseases of poultry. 14th ed. New York, USA: John Wiley & Sons 2020; 210-256.
3.     Peacock THP, James J, Sealy JE, et al. A global perspective on H9N2 avian influenza virus. Viruses 2019; 11(7): 620. doi: 10.3390/v11070620.
4.     Zhang J, Huang L, Liao M, et al. H9N2 avian influenza viruses: challenges and the way forward. Lancet Microbe 2023; 4(2): e70-e71.
5.     Peiris M, Yuen KY, Leung CW, et al. Human infection with influenza H9N2. Lancet 1999; 354(9182): 916-917.
6.     Xu C, Fan W, Wei R, et al. Isolation and identification of swine influenza recombinant A/Swine/Shandong/1/ 2003 (H9N2) virus. Microbes Infect 2004; 6(10):919-925.
7.     Fusaro A, Monne I, Salviato A, et al. Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications. J Virol 2011; 85(16): 8413-8421.
8.     Bashashati M, Chung DH, Fallah Mehrabadi MH, et al. Evolution of H9N2 avian influenza viruses in Iran, 2017-2019. Transbound Emerg Dis 2021; 68(6): 3405-3414.
9.     Bashashati M, Vasfi Marandi M, Sabouri F. Genetic diversity of early (1998) and recent (2010) avian influenza H9N2 virus strains isolated from poultry in Iran. Arch Virol 2013; 158(10): 2089-2100.
10. Hoffmann E, Stech J, Guan Y, et al. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 2001; 146(12): 2275-2289.
11. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999; 41: 95-98.
12. Kumar S, Stecher G, Suleski M, et al. MEGA12: molecular evolutionary genetic analysis version 12 for adaptive and green computing. Mol Biol Evol 2024; 41(12): msae263. doi: 10.1093/molbev/msae263.
13. Martin DP, Murrell B, Golden M, et al. RDP4: Detection and analysis of recombination patterns in virus genomes. Virus Evol 2015; 1(1): vev003. doi: 10.1093/ ve/vev003.
14. Suchard MA, Lemey P, Baele G, et al. Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol 2018; 4(1): vey016. doi: 10.1093/ve/vey016.
15. Rambaut A, Drummond AJ, Xie D, et al. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst Biol 2018; 67(5): 901-904.
16. Wan H, Perez DR. Amino acid 226 in the hemagglutinin of H9N2 influenza viruses determines cell tropism and replication in human airway epithelial cells. J Virol 2007; 81(10): 5181-5191.
17. Wan H, Sorrell EM, Song H, et al. Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential. PloS One 2008; 3(8): e2923. doi: 10.1371/journal.pone.0002923.
18. Kaverin NV, Rudneva IA, Ilyushina NA, et al. Structural differences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture: analysis of H9 escape mutants. J Virol 2004; 78(1): 240-249.
19. Okamatsu M, Sakoda Y, Kishida N, et al. Antigenic structure of the hemagglutinin of H9N2 influenza viruses. Arch Virol 2008; 153(12): 2189-2195.
20. Peacock T, Reddy K, James J, et al. Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape. Sci Rep 2016; 6: 18745. doi: 10.1038/ srep18745.
21. Peacock TP, Harvey WT, Sadeyen JR, et al. The molecular basis of antigenic variation among A (H9N2) avian influenza viruses. Emerg Microbes Infect 2018; 7(1): 176. doi: 10.1038/s41426-018-0178-y.
22. Wan Z, Ye J, Xu L, et al. Antigenic mapping of the hemagglutinin of an H9N2 avian influenza virus reveals novel critical amino acid positions in antigenic sites J Virol 2014; 88(7): 3898-3901.
23. Bright RA, Shay DK, Shu B, et al. Adamantane resistance among influenza A viruses isolated early during the 2005-2006 influenza season in the United States. JAMA 2006; 295(8): 891-894.
24. Zhang M, Zhao C, Chen H, et al. Internal gene cassette from a human-origin H7N9 influenza virus promotes the pathogenicity of H9N2 avian influenza virus in mice. Front Microbiol 2020; 11: 1441. doi: 10.3389/ fmicb.2020.01441.
25. Naffakh N, Tomoiu A, Rameix-Welti MA, et al. Host restriction of avian influenza viruses at the level of the ribonucleoproteins. Annu Rev Microbiol 2008; 62: 403-424.
26. Yao Q, Liu J, Liu H, et al. One‐Health challenge in H9N2 avian influenza: novel human‐avian reassortment virus in Guangdong province, China. Transbound Emerg Dis 2024; 2024: 9913934. doi: 10.1155/2024/9913934.
27. Li X, Shi J, Guo J, et al. Genetics, receptor binding property, and transmissibility in mammals of naturally isolated H9N2 avian influenza viruses. PLoS Pathog 2014; 10(11): e1004508. doi: 10.1371/journal. ppat.1004508.
28. Bashashati M, Mojahedi Z, Roudsari AA, et al. Ongoing genetic evolution of H9N2 avian influenza viruses in Iranian industrial poultry farms. Acta Vet Hung 2020; 68(3): 328-335.
29. Moosakhani F, Shoshtari AH, Pourbakhsh SA, et al. Phylogenetic analysis of the hemagglutinin genes of 12 H9N2 influenza viruses isolated from chickens in Iran from 2003 to 2005. Avian Dis 2010; 54(2): 870-874.
30. Kim P, Jang YH, Kwon SB, et al. Glycosylation of hemagglutinin and neuraminidase of influenza A virus as signature for ecological spillover and adaptation among influenza reservoirs. Viruses 2018; 10(4): 183.
31. Shi WF, Dun AS, Zhang Z, et al. Selection pressure on haemagglutinin genes of H9N2 influenza viruses from different hosts. Virol Sin 2009; 24(1): 65-70.
32. Li W, Shi W, Qiao H, et al. Positive selection on hemagglutinin and neuraminidase genes of H1N1 influenza viruses. Virol J 2011; 8: 183. doi: 10.1186/ 1743-422X-8-183.
Volume 17, Issue 3
March 2026
Pages 207-215

  • Receive Date 22 January 2025
  • Revise Date 07 April 2025
  • Accept Date 22 April 2025