Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Mouse adaptation of the H9N2 avian influenza virus causes the downregulation of genes related to innate immune responses and ubiquitin-mediated proteolysis in mice

  • 88 Accesses


H9N2 avian influenza viruses sporadically infect humans worldwide. These viruses have also contributed internal genes to H5N1, H5N6, H7N9, and H10N8 viruses, which have been isolated from humans with infections and are a substantial public health threat. To investigate the potential pathogenic mechanism of the H9N2 virus, we performed serial lung-to-lung passage of an avirulent H9N2 avian influenza virus (A/Chicken/Shandong/416/2016 [SD/416]) in mice to increase the pathogenicity of this virus. We generated a mouse-adapted (MA) virus that exhibited increased viral titers in the lungs, caused severe lung damage in mice, and induced body weight loss in mice; however, the avirulent parental virus did not cause any clinical symptoms in infected mice. Global gene expression analysis was performed and indicated that the transcriptional responses of these viruses were distinct. The lungs of mice infected with the MA virus exhibited the downregulation of genes related to innate immunity and ubiquitin-mediated proteolysis, which was not seen in infections with the avirulent parental virus. These data indicated that the MA virus might evade immune surveillance and changed its replication capacity to increase the viral replication level and pathogenicity. Our study demonstrates that host factors play an important role in the adaptive evolution of influenza virus in new hosts.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. 1.

    Tong SX, Zhu XY, Li Y, Shi M, Zhang J, Bourgeois M, Yang H, Chen XF, Recuenco S, Gomez J, Chen LM, Johnson A, Tao Y, Dreyfus C, Yu WL, McBride R, Carney PJ, Gilbert AT, Chang J, Guo Z, Davis CT, Paulson JC, Stevens J, Rupprecht CE, Holmes EC, Wilson IA, Donis RO (2013) New world bats harbor diverse influenza A viruses. Plos Pathog. https://doi.org/10.1371/journal.ppat.1003657

  2. 2.

    Wu Y, Wu Y, Tefsen B, Shi Y, Gao GF (2014) Bat-derived influenza-like viruses H17N10 and H18N11. Trends Microbiol 22(4):183–191. https://doi.org/10.1016/j.tim.2014.01.010

  3. 3.

    Olsen B, Munster VJ, Wallensten A, Waldenstrom J, Osterhaus AD, Fouchier RA (2006) Global patterns of influenza a virus in wild birds. Science 312(5772):384–388. https://doi.org/10.1126/science.1122438

  4. 4.

    Taubenberger JK, Kash JC (2010) Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe 7(6):440–451. https://doi.org/10.1016/j.chom.2010.05.009

  5. 5.

    Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Li X, Yang L, Guo J, Dong J, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Zhang Y, Han J, Yu H, Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu Y (2013) Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med 368(20):1888–1897. https://doi.org/10.1056/NEJMoa1304459

  6. 6.

    Peiris M, Yuen KY, Leung CW, Chan KH, Ip PL, Lai RW, Orr WK, Shortridge KF (1999) Human infection with influenza H9N2. Lancet 354(9182):916–917

  7. 7.

    Claas EC, de Jong JC, van Beek R, Rimmelzwaan GF, Osterhaus AD (1998) Human influenza virus A/HongKong/156/97 (H5N1) infection. Vaccine 16(9–10):977–978

  8. 8.

    Homme PJ, Easterday BC (1970) Avian influenza virus infections. I. characteristics of influenza A-Turkey-Wisconsin-1966 virus. Avian Dis 14(1):66–74

  9. 9.

    Li X, Shi J, Guo J, Deng G, Zhang Q, Wang J, He X, Wang K, Chen J, Li Y, Fan J, Kong H, Gu C, Guan Y, Suzuki Y, Kawaoka Y, Liu L, Jiang Y, Tian G, Li Y, Bu Z, Chen H (2014) Genetics, receptor binding property, and transmissibility in mammals of naturally isolated H9N2 Avian Influenza viruses. Plos Pathog 10(11):e1004508. https://doi.org/10.1371/journal.ppat.1004508

  10. 10.

    He J, Liu BY, Gong L, Chen Z, Chen XL, Hou S, Yu JL, Wu JB, Xia ZC, Latif A, Gao R, Su B, Liu Y (2018) Genetic characterization of the first detected human case of avian influenza A (H5N6) in Anhui Province, East China. Sci Rep 8(1):15282. https://doi.org/10.1038/s41598-018-33356-4

  11. 11.

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

  12. 12.

    Zhang Q, Shi J, Deng G, Guo J, Zeng X, He X, Kong H, Gu C, Li X, Liu J, Wang G, Chen Y, Liu L, Liang L, Li Y, Fan J, Wang J, Li W, Guan L, Li Q, Yang H, Chen P, Jiang L, Guan Y, Xin X, Jiang Y, Tian G, Wang X, Qiao C, Li C, Bu Z, Chen H (2013) H7N9 influenza viruses are transmissible in ferrets by respiratory droplet. Science 341(6144):410–414. https://doi.org/10.1126/science.1240532

  13. 13.

    Chen H, Yuan H, Gao R, Zhang J, Wang D, Xiong Y, Fan G, Yang F, Li X, Zhou J, Zou S, Yang L, Chen T, Dong L, Bo H, Zhao X, Zhang Y, Lan Y, Bai T, Dong J, Li Q, Wang S, Zhang Y, Li H, Gong T, Shi Y, Ni X, Li J, Zhou J, Fan J, Wu J, Zhou X, Hu M, Wan J, Yang W, Li D, Wu G, Feng Z, Gao GF, Wang Y, Jin Q, Liu M, Shu Y (2014) Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study. Lancet 383(9918):714–721. https://doi.org/10.1016/S0140-6736(14)60111-2

  14. 14.

    Li X, Cui P, Zeng X, Jiang Y, Li Y, Yang J, Pan Y, Gao X, Zhao C, Wang J, Wang K, Deng G, Guo J (2019) Characterization of avian influenza H5N3 reassortants isolated from migratory waterfowl and domestic ducks in China from 2015 to 2018. Transbound Emerg Dis 66(6):2605–2610. https://doi.org/10.1111/tbed.13324

  15. 15.

    Li XY, Liu BT, Ma SJ, Cui P, Liu WQ, Li YB, Guo J, Chen HL (2018) High frequency of reassortment after co-infection of chickens with the H4N6 and H9N2 influenza A viruses and the biological characteristics of the reassortants. Vet Microbiol 222:11–17. https://doi.org/10.1016/j.vetmic.2018.06.011

  16. 16.

    Wang J, Sun Y, Xu Q, Tan Y, Pu J, Yang H, Brown EG, Liu J (2012) Mouse-adapted H9N2 influenza A virus PB2 protein M147L and E627K mutations are critical for high virulence. PLoS ONE 7(7):e40752. https://doi.org/10.1371/journal.pone.0040752

  17. 17.

    Kamiki H, Matsugo H, Kobayashi T, Ishida H, Takenaka-Uema A, Murakami S, Horimoto T (2018) A PB1-K577E mutation in H9N2 influenza virus increases polymerase activity and pathogenicity in mice. Viruses. https://doi.org/10.3390/v10110653

  18. 18.

    Park KJ, Song MS, Kim EH, Kwon HI, Baek YH, Choi EH, Park SJ, Kim SM, Kim YI, Choi WS, Yoo DW, Kim CJ, Choi YK (2015) Molecular characterization of mammalian-adapted Korean-type avian H9N2 virus and evaluation of its virulence in mice. J Microbiol 53(8):570–577. https://doi.org/10.1007/s12275-015-5329-4

  19. 19.

    Wright GW, Simon RM (2003) A random variance model for detection of differential gene expression in small microarray experiments. Bioinformatics 19(18):2448–2455

  20. 20.

    Clarke R, Ressom HW, Wang A, Xuan J, Liu MC, Gehan EA, Wang Y (2008) The properties of high-dimensional data spaces: implications for exploring gene and protein expression data. Nat Rev Cancer 8(1):37–49. https://doi.org/10.1038/nrc2294

  21. 21.

    da Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4(1):44–57. https://doi.org/10.1038/nprot.2008.211

  22. 22.

    Zhang H, Li XY, Guo J, Li L, Chang C, Li YY, Bian C, Xu K, Chen HL, Sun B (2014) The PB2 E627K mutation contributes to the high polymerase activity and enhanced replication of H7N9 influenza virus. J Gen Virol 95:779–786. https://doi.org/10.1099/vir.0.061721-0

  23. 23.

    Cheng K, Yu Z, Chai H, Sun W, Xin Y, Zhang Q, Huang J, Zhang K, Li X, Yang S, Wang T, Zheng X, Wang H, Qin C, Qian J, Chen H, Hua Y, Gao Y, Xia X (2014) PB2-E627K and PA-T97I substitutions enhance polymerase activity and confer a virulent phenotype to an H6N1 avian influenza virus in mice. Virology 468-470C:207–213. https://doi.org/10.1016/j.virol.2014.08.010

  24. 24.

    Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C (2004) Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303(5663):1529–1531. https://doi.org/10.1126/science.1093616

  25. 25.

    Le Goffic R, Balloy V, Lagranderie M, Alexopoulou L, Escriou N, Flavell R, Chignard M, Si-Tahar M (2006) Detrimental contribution of the toll-like receptor (TLR)3 to influenza A virus-induced acute pneumonia. PloS Pathog 2(6):e53. https://doi.org/10.1371/journal.ppat.0020053

  26. 26.

    Friedman CS, O'Donnell MA, Legarda-Addison D, Ng A, Cardenas WB, Yount JS, Moran TM, Basler CF, Komuro A, Horvath CM, Xavier R, Ting AT (2008) The tumour suppressor CYLD is a negative regulator of RIG-I-mediated antiviral response. EMBO Rep 9(9):930–936. https://doi.org/10.1038/embor.2008.136

  27. 27.

    Qian S, Fan W, Liu T, Wu M, Zhang H, Cui X, Zhou Y, Hu J, Wei S, Chen H, Li X, Qian P (2017) Seneca valley virus suppresses host type I interferon production by targeting adaptor proteins MAVS, TRIF, and TANK for cleavage. J Virol. https://doi.org/10.1128/JVI.00823-17

  28. 28.

    Ding S, Mooney N, Li B, Kelly MR, Feng N, Loktev AV, Sen A, Patton JT, Jackson PK, Greenberg HB (2016) Comparative proteomics reveals strain-specific beta-TrCP degradation via rotavirus NSP1 hijacking a host Cullin-3-Rbx1 complex. PloS Pathog 12(10):e1005929. https://doi.org/10.1371/journal.ppat.1005929

  29. 29.

    Paparisto E, Woods MW, Coleman MD, Moghadasi SA, Kochar DS, Tom SK, Kohio HP, Gibson RM, Rohringer TJ, Hunt NR, Di Gravio EJ, Zhang JY, Tian M, Gao Y, Arts EJ, Barr SD (2018) Evolution-guided structural and functional analyses of the HERC family reveal an ancient marine origin and determinants of antiviral activity. J Virol. https://doi.org/10.1128/JVI.00528-18

  30. 30.

    Ma-Lauer Y, Carbajo-Lozoya J, Hein MY, Mueller MA, Deng W, Lei J, Meyer B, Kusov Y, von Brunn B, Bairad DR, Hunten S, Drosten C, Hermeking H, Leonhardt H, Mann M, Hilgenfeld R, von Brunn A (2016) p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1. Proc Natl Acad Sci USA 113(35):E5192–E5201. https://doi.org/10.1073/pnas.1603435113

  31. 31.

    Su WC, Chen YC, Tseng CH, Hsu PW, Tung KF, Jeng KS, Lai MM (2013) Pooled RNAi screen identifies ubiquitin ligase itch as crucial for influenza A virus release from the endosome during virus entry. Proc Natl Acad Sci USA 110(43):17516–17521. https://doi.org/10.1073/pnas.1312374110

  32. 32.

    Shi Y, Yuan B, Zhu W, Zhang R, Li L, Hao X, Chen S, Hou F (2017) Ube2D3 and Ube2N are essential for RIG-I-mediated MAVS aggregation in antiviral innate immunity. Nat Commun 8:15138. https://doi.org/10.1038/ncomms15138

  33. 33.

    Feng T, Deng L, Lu X, Pan W, Wu Q, Dai J (2018) Ubiquitin-conjugating enzyme UBE2J1 negatively modulates interferon pathway and promotes RNA virus infection. Virol J 15(1):132. https://doi.org/10.1186/s12985-018-1040-5

  34. 34.

    Westrich JA, Warren CJ, Klausner MJ, Guo K, Liu CW, Santiago ML, Pyeon D (2018) Human papillomavirus 16 E7 stabilizes APOBEC3A protein by inhibiting cullin 2-dependent protein degradation. J Virol. https://doi.org/10.1128/JVI.01318-17

  35. 35.

    Holscher C, Sonntag F, Henrich K, Chen Q, Beneke J, Matula P, Rohr K, Kaderali L, Beil N, Erfle H, Kleinschmidt JA, Muller M (2015) The SUMOylation pathway restricts gene transduction by adeno-associated viruses. Plos Pathog 11(12):e1005281. https://doi.org/10.1371/journal.ppat.1005281

  36. 36.

    Wang M, Fu CX, Zheng BJ (2009) Antibodies against H5 and H9 Avian influenza among poultry workers in China. New Engl J Med 360(24):2583–2584. https://doi.org/10.1056/NEJMc0900358

  37. 37.

    Pawar SD, Tandale BV, Raut CG, Parkhi SS, Barde TD, Gurav YK, Kode SS, Mishra AC (2012) Avian influenza H9N2 seroprevalence among poultry workers in Pune, India, 2010. PLoS ONE. https://doi.org/10.1371/journal.pone.0036374

  38. 38.

    Blair PJ, Putnam SD, Krueger WS, Chum C, Wierzba TF, Heil GL, Yasuda CY, Williams M, Kasper MR, Friary JA, Capuano AW, Saphonn V, Peiris M, Shao HX, Perez DR, Gray GC (2013) Evidence for avian H9N2 influenza virus infections among rural villagers in Cambodia. J Infect Public Heal 6(2):69–79. https://doi.org/10.1016/j.jiph.2012.11.005

  39. 39.

    Okoye J, Eze D, Krueger WS, Heil GL, Friary JA, Gray GC (2013) Serologic evidence of avian influenza virus infections among Nigerian agricultural workers. J Med Virol 85(4):670–676. https://doi.org/10.1002/jmv.23520

  40. 40.

    Coman A, Maftei DN, Krueger WS, Heil GL, Friary JA, Chereches RM, Sirlincan E, Bria P, Dragnea C, Kasler I, Gray GC (2013) Serological evidence for avian H9N2 influenza virus infections among Romanian agriculture workers. J Infect Public Heal 6(6):438–447. https://doi.org/10.1016/j.jiph.2013.05.003

  41. 41.

    Gray GC, Ferguson DD, Lowther PE, Heil GL, Friary JA (2011) A national study of US bird banders for evidence of avian influenza virus infections. J Clin Virol 51(2):132–135. https://doi.org/10.1016/j.jcv.2011.03.011

  42. 42.

    Wang Q, Ju L, Liu P, Zhou J, Lv X, Li L, Shen H, Su H, Jiang L, Jiang Q (2015) Serological and virological surveillance of avian influenza A virus H9N2 subtype in humans and poultry in Shanghai, China, between 2008 and 2010. Zoonoses Public Hlth 62(2):131–140. https://doi.org/10.1111/zph.12133

  43. 43.

    Uyeki TM, Nguyen DC, Rowe T, Lu XH, Hu-Primmer J, Huynh LP, Hang NLK, Katz JM (2012) Seroprevalence of antibodies to avian influenza A (H5) and A (H9) viruses among market poultry workers, Hanoi, Vietnam, 2001. PLoS ONE. https://doi.org/10.1371/journal.pone.0043948

  44. 44.

    Mehle A, Doudna JA (2009) Adaptive strategies of the influenza virus polymerase for replication in humans. Proc Natl Acad Sci USA 106(50):21312–21316. https://doi.org/10.1073/pnas.0911915106

  45. 45.

    Min JY, Santos C, Fitch A, Twaddle A, Toyoda Y, DePasse JV, Ghedin E, Subbarao K (2013) Mammalian adaptation in the PB2 gene of avian H5N1 influenza virus. J Virol 87(19):10884–10888. https://doi.org/10.1128/JVI.01016-13

  46. 46.

    Zhang T, Wang TC, Zhao PS, Liang M, Gao YW, Yang ST, Qin C, Wang CY, Xia XZ (2011) Antisense oligonucleotides targeting the RNA binding region of the NP gene inhibit replication of highly pathogenic avian influenza virus H5N1. Int Immunopharmacol 11(12):2057–2061. https://doi.org/10.1016/j.intimp.2011.08.019

  47. 47.

    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. J Virol 82(3):1146–1154. https://doi.org/10.1128/JVI.01698-07

  48. 48.

    Ma W, Belisle SE, Mosier D, Li X, Stigger-Rosser E, Liu Q, Qiao C, Elder J, Webby R, Katze MG, Richt JA (2011) 2009 pandemic H1N1 influenza virus causes disease and upregulation of genes related to inflammatory and immune responses, cell death, and lipid metabolism in pigs. J Virol 85(22):11626–11637. https://doi.org/10.1128/JVI.05705-11

  49. 49.

    Zou W, Chen D, Xiong M, Zhu J, Lin X, Wang L, Zhang J, Chen L, Zhang H, Chen H, Chen M, Jin M (2013) Insights into the increasing virulence of the swine-origin pandemic H1N1/2009 influenza virus. Sci Rep 3:1601. https://doi.org/10.1038/srep01601

  50. 50.

    Fernandez-Sesma A (2007) The influenza virus NS1 protein: inhibitor of innate and adaptive immunity. Infect Disord Drug Targets 7(4):336–343

  51. 51.

    Voeten JT, Bestebroer TM, Nieuwkoop NJ, Fouchier RA, Osterhaus AD, Rimmelzwaan GF (2000) Antigenic drift in the influenza A virus (H3N2) nucleoprotein and escape from recognition by cytotoxic T lymphocytes. J Virol 74(15):6800–6807

  52. 52.

    Luo H (2016) Interplay between the virus and the ubiquitin-proteasome system: molecular mechanism of viral pathogenesis. Curr Opin Virol 17:1–10. https://doi.org/10.1016/j.coviro.2015.09.005

Download references


We thank the National Natural Science Foundation of China, the Natural Science Foundation of Shandong Province, and the State Key Laboratory of Veterinary Biotechnology. This work was supported by the National Natural Science Foundation of China (31702262 and 31702261), the Natural Science Foundation of Shandong Province (ZR2017BC057 and ZR2017BC048), and the State Key Laboratory of Veterinary Biotechnology (SKLVBF201906).

Author information

Correspondence to Jing Guo or Xuyong Li.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by Gülsah Gabriel.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Guo, J., Gao, X., Liu, B. et al. Mouse adaptation of the H9N2 avian influenza virus causes the downregulation of genes related to innate immune responses and ubiquitin-mediated proteolysis in mice. Med Microbiol Immunol (2020). https://doi.org/10.1007/s00430-020-00656-4

Download citation


  • Influenza virus
  • Adaptation
  • Pathogenicity
  • Gene expression analysis