Abstract
Influenza A viruses must undergo adaptation to acquire virulence in new host species. In mouse models, host adaptation for virulence is generally performed through 5 to 20 lung-to-lung passages. However, highly pathogenic avian influenza viruses (e.g., H5N1 and H7N7 subtypes) have been observed to acquire virulence in mice after only a few in vivo passages. In this study, a low-pathogenic avian influenza H5N2 virus, A/Aquatic Bird/Korea/CN2/2009, which was a prevalent subtype in South Korea in 2009, was serially passaged in mice to evaluate its potential to become highly pathogenic. Unexpectedly, the virus became highly pathogenic in mice after a single lung-to-lung passage, resulting in 100% lethality with a mean death time (MDT) of 6.1 days postinfection (DPI). Moreover, the pathogenicity gradually increased after subsequent in vivo passages with an MDT of 5.2 and 4.2 DPI after the second and third passage, respectively. Our molecular analysis revealed that two amino acid changes in the polymerase complex (a glutamate-to-lysine substitution at position 627 of PB2 and a threonine-to-isoleucine substitution at position 97 of PA) were associated with the increased pathogenicity; the PB2 E627K mutation was responsible for the initial virulence conversion (0 to 100% lethality), while the PA T97I mutation acted as an accessory for the increased virulence.
Similar content being viewed by others
References
Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiol Rev 56:152–179
Slemons RD, Johnson DC, Osborn JS, Hayes F (1974) Type-A influenza viruses isolated from wild free-flying ducks in California. Avian Dis 18:119–124
Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, Chen LM, Recuenco S, Ellison JA, Davis CT, York IA, Turmelle AS, Moran D, Rogers S, Shi M, Tao Y, Weil MR, Tang K, Rowe LA, Sammons S, Xu X, Frace M, Lindblade KA, Cox NJ, Anderson LJ, Rupprecht CE, Donis RO (2012) A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci USA 109:4269–4274
Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, Yang H, Chen X, Recuenco S, Gomez J, Chen LM, Johnson A, Tao Y, Dreyfus C, Yu W, 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 9:e1003657
de Jong JC, Claas EC, Osterhaus AD, Webster RG, Lim WL (1997) A pandemic warning? Nature 389:554
Taubenberger JK, Morens DM (2006) 1918 Influenza: the mother of all pandemics. Emerg Infect Dis 12:15–22
Swayne DE, Suarez DL (2000) Highly pathogenic avian influenza. Rev Sci Tech 19:463–482
Organization WH Cumulative number of confirmed human cases for avian influenza A (H5N1) reported to WHO, 2003–2015 [cited 2015 Jan 19]
Nam JH, Kim EH, Song D, Choi YK, Kim JK, Poo H (2011) Emergence of mammalian species-infectious and -pathogenic avian influenza H6N5 virus with no evidence of adaptation. J Virol 85:13271–13277
Song MS, Pascua PN, Lee JH, Baek YH, Lee OJ, Kim CJ, Kim H, Webby RJ, Webster RG, Choi YK (2009) The polymerase acidic protein gene of influenza a virus contributes to pathogenicity in a mouse model. J Virol 83:12325–12335
Kim HR, Park CK, Oem JK, Bae YC, Choi JG, Lee OS, Lee YJ (2010) Characterization of H5N2 influenza viruses isolated in South Korea and their influence on the emergence of a novel H9N2 influenza virus. J Gen Virol 91:1978–1983
Naffakh N, Tomoiu A, Rameix-Welti MA, van der Werf S (2008) Host restriction of avian influenza viruses at the level of the ribonucleoproteins. Annu Rev Microbiol 62:403–424
Subbarao EK, London W, Murphy BR (1993) A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J Virol 67:1761–1764
Steel J, Lowen AC, Mubareka S, Palese P (2009) Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N. PLoS Pathog 5:e1000252
Hatta M, Gao P, Halfmann P, Kawaoka Y (2001) Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science (New York, NY) 293:1840–1842
Jonges M, Welkers MR, Jeeninga RE, Meijer A, Schneeberger P, Fouchier RA, de Jong MD, Koopmans M (2014) Emergence of the virulence-associated PB2 E627K substitution in a fatal human case of highly pathogenic avian influenza virus A(H7N7) infection as determined by Illumina ultra-deep sequencing. J Virol 88:1694–1702
Chen GW, Lai MM, Wu SC, Chang SC, Huang LM, Shih SR (2013) Is avian influenza A (H7N9) virus staggering its way to humans? J Formos Med Assoc 112:312–318
Ward AC (1997) Virulence of influenza A virus for mouse lung. Virus Genes 14:187–194
Narasaraju T, Sim MK, Ng HH, Phoon MC, Shanker N, Lal SK, Chow VT (2009) Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis. Microb Infect 11:2–11
Brown EG, Bailly JE (1999) Genetic analysis of mouse-adapted influenza A virus identifies roles for the NA, PB1, and PB2 genes in virulence. Virus Res 61:63–76
Gabriel G, Dauber B, Wolff T, Planz O, Klenk HD, Stech J (2005) The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc Natl Acad Sci USA 102:18590–18595
Brown EG (1990) Increased virulence of a mouse-adapted variant of influenza A/FM/1/47 virus is controlled by mutations in genome segments 4, 5, 7, and 8. J Virol 64:4523–4533
Chin PS, Hoffmann E, Webby R, Webster RG, Guan Y, Peiris M, Shortridge KF (2002) Molecular evolution of H6 influenza viruses from poultry in Southeastern China: prevalence of H6N1 influenza viruses possessing seven A/Hong Kong/156/97 (H5N1)-like genes in poultry. J Virol 76:507–516
Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001) Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146:2275–2289
Higgins DG, Bleasby AJ, Fuchs R (1992) CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci 8:189–191
Perriere G, Gouy M (1996) WWW-query: an on-line retrieval system for biological sequence banks. Biochimie 78:364–369
Connor RJ, Kawaoka Y, Webster RG, Paulson JC (1994) Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205:17–23
Zhou B, Li Y, Halpin R, Hine E, Spiro DJ, Wentworth DE (2011) PB2 residue 158 is a pathogenic determinant of pandemic H1N1 and H5 influenza a viruses in mice. J Virol 85:357–365
Hay AJ, Wolstenholme AJ, Skehel JJ, Smith MH (1985) The molecular basis of the specific anti-influenza action of amantadine. EMBO J 4:3021–3024
Manz B, Schwemmle M, Brunotte L (2013) Adaptation of avian influenza A virus polymerase in mammals to overcome the host species barrier. J Virol 87:7200–7209
Gabriel G, Klingel K, Otte A, Thiele S, Hudjetz B, Arman-Kalcek G, Sauter M, Shmidt T, Rother F, Baumgarte S, Keiner B, Hartmann E, Bader M, Brownlee GG, Fodor E, Klenk HD (2011) Differential use of importin-alpha isoforms governs cell tropism and host adaptation of influenza virus. Nat Commun 2:156
Sang X, Wang A, Chai T, He X, Ding J, Gao X, Li Y, Zhang K, Ren Z, Li L, Yu Z, Wang T, Feng N, Zheng X, Wang H, Zhao Y, Yang S, Gao Y, Xia X (2015) Rapid emergence of a PB2-E627K substitution confers a virulent phenotype to an H9N2 avian influenza virus during adoption in mice. Arch Virol 160:1267–1277
Mase M, Tanimura N, Imada T, Okamatsu M, Tsukamoto K, Yamaguchi S (2006) Recent H5N1 avian influenza A virus increases rapidly in virulence to mice after a single passage in mice. J Gen Virol 87:3655–3659
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 368:1888–1897
Mok CK, Lee HH, Lestra M, Nicholls JM, Chan MC, Sia SF, Zhu H, Poon LL, Guan Y, Peiris JS (2014) Amino acid substitutions in polymerase basic protein 2 gene contribute to the pathogenicity of the novel A/H7N9 influenza virus in mammalian hosts. J Virol 88:3568–3576
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 468–470:207–213
Yuan P, Bartlam M, Lou Z, Chen S, Zhou J, He X, Lv Z, Ge R, Li X, Deng T, Fodor E, Rao Z, Liu Y (2009) Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature 458:909–913
Domingo E, Sheldon J, Perales C (2012) Viral quasispecies evolution. Microbiol Mol Biol Rev 76:159–216
Andino R, Domingo E (2015) Viral quasispecies. Virology 479–480:46–51. doi:10.1016/j.virol.2015.03.022
Wu H, Peng X, Peng X, Cheng L, Jin C, Lu X, Xie T, Yao H, Wu N (2016) Multiple amino acid substitutions involved in the adaptation of avian-origin influenza A (H10N7) virus in mice. Arch Virol 161:977–980
Acknowledgements
This work was supported by Grants from the Korea Institute for Planning and Evaluation of Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Animal Disease Management Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Grant no. 316042-03-2-HD020) and the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (Grant no. HD16A1487).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
All authors confirm that they have no conflict of interest.
Ethical approval
All animal experiments were performed in biosafety level 2 plus (BSL2+) facilities at Korea Research Institute of Bioscience and Biotechnology (KRIBB) (Daejeon, South Korea) and were approved by the Institutional Animal Use and Care Committee of the KRIBB in accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (KRIBB-AEC-15191).
Rights and permissions
About this article
Cite this article
Nam, JH., Shim, SM., Song, EJ. et al. Rapid virulence shift of an H5N2 avian influenza virus during a single passage in mice. Arch Virol 162, 3017–3024 (2017). https://doi.org/10.1007/s00705-017-3451-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00705-017-3451-9