To investigate whether currently circulating H9N2 avian influenza viruses (AIVs) in domestic poultry have evolved in Korean poultry since 2007, genetic and serological comparisons were conducted of H9N2 isolates from poultry slaughterhouses from January 2008 to December 2009. The isolation rate was relatively low in 2008 but increased gradually from January 2009 onwards. Genetic and phylogenetic analyses revealed that reassortant viruses had emerged, generating at least five novel genotypes, mostly containing segments of a previously prevalent domestic H9N2 virus lineage (Ck/Korea/04116/04-like). It was noteworthy that the N2 genes of some H9N2 isolates (genotypes D, E and F) were derived from those of H3N2-like viruses commonly isolated among domestic ducks in live-poultry markets. Animal challenge studies demonstrated that the pathogenicity of Ck/Korea/SH0906/09 (genotype B) and Ck/Korea/SH0912/09 (genotype F) in domestic avian species was altered due to reassortment. Furthermore, serological analysis revealed that the isolates were antigenically distinct from previous Korean H9N2 viruses including Ck/Korea/01310/01. Such antigenic diversity was illustrated further in experiments using H9N2-immunized chickens, which could not inhibit the replication and transmission of challenge viruses from each genotype. These results suggest that H9N2 viruses from domestic poultry have undergone substantial evolution since 2007 by immune selection as a result of vaccinal and natural immunity, coupled with reassortment. Taken together, this study demonstrates that periodical updating of vaccine strains, based on continuous surveillance data, is an important issue in order to provide sufficient protectivity against AIV infections.


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  1. Aiyar, A.(2000). The use of clustalw and clustalx for multiple sequence alignment. Methods Mol Biol 132, 221–241. [Google Scholar]
  2. Alexander, D. J.(2000). A review of avian influenza in different bird species. Vet Microbiol 74, 3–13.[CrossRef] [Google Scholar]
  3. Brown, I. H., Banks, J., Manvell, R. J., Essen, S. C., Shell, W., Slomka, M., Londt, B. & Alexander, D. J.(2006). Recent epidemiology and ecology of influenza A viruses in avian species in Europe and the Middle East. Dev Biol (Basel) 124, 45–50. [Google Scholar]
  4. Chang, H. K., Park, J. H., Song, M. S., Oh, T. K., Kim, S. Y., Kim, C. J., Kim, H., Sung, M. H., Han, H. S. & other authors(2008). Development of multiplex RT-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens. J Microbiol Biotechnol 18, 1164–1169. [Google Scholar]
  5. Choi, Y. K., Seo, S. H., Kim, J. A., Webby, R. J. & Webster, R. G.(2005). Avian influenza viruses in Korean live poultry markets and their pathogenic potential. Virology 332, 529–537.[CrossRef] [Google Scholar]
  6. Choi, J. G., Lee, Y. J., Kim, Y. J., Lee, E. K., Jeong, O. M., Sung, H. W., Kim, J. H. & Kwon, J. H.(2008). An inactivated vaccine to control the current H9N2 low pathogenic avian influenza in Korea. J Vet Sci 9, 67–74.[CrossRef] [Google Scholar]
  7. Eggert, D., Thomas, C., Spackman, E., Pritchard, N., Rojo, F., Bublot, M. & Swayne, D. E.(2010). Characterization and efficacy determination of commercially available Central American H5N2 avian influenza vaccines for poultry. Vaccine 28, 4609–4615.[CrossRef] [Google Scholar]
  8. Fouchier, R. A., Munster, V., Wallensten, A., Bestebroer, T. M., Herfst, S., Smith, D., Rimmelzwaan, G. F., Olsen, B. & Osterhaus, A. D.(2005). Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79, 2814–2822.[CrossRef] [Google Scholar]
  9. Gambaryan, A., Tuzikov, A., Pazynina, G., Bovin, N., Balish, A. & Klimov, A.(2006). Evolution of the receptor binding phenotype of influenza A (H5) viruses. Virology 344, 432–438.[CrossRef] [Google Scholar]
  10. González, S., Zürcher, T. & Ortín, J.(1996). Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2 and PA subunits: a model for the viral RNA polymerase structure. Nucleic Acids Res 24, 4456–4463.[CrossRef] [Google Scholar]
  11. 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? Proc Natl Acad Sci U S A 96, 9363–9367.[CrossRef] [Google Scholar]
  12. Gubareva, L. V., Robinson, M. J., Bethell, R. C. & Webster, R. G.(1997). Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en. J Virol 71, 3385–3390. [Google Scholar]
  13. Hatta, M., Gao, P., Halfmann, P. & Kawaoka, Y.(2001). Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293, 1840–1842.[CrossRef] [Google Scholar]
  14. Hay, A. J., Wolstenholme, A. J., Skehel, J. J. & Smith, M. H.(1985). The molecular basis of the specific anti-influenza action of amantadine. EMBO J 4, 3021–3024. [Google Scholar]
  15. 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. Arch Virol 146, 2275–2289.[CrossRef] [Google Scholar]
  16. Holsinger, L. J., Nichani, D., Pinto, L. H. & Lamb, R. A.(1994). Influenza A virus M2 ion channel protein: a structure–function analysis. J Virol 68, 1551–1563. [Google Scholar]
  17. Homme, P. J. & Easterday, B. C.(1970). Avian influenza virus infections. IV. Response of pheasants, ducks, and geese to influenza A-turkey-Wisconsin-1966 virus. Avian Dis 14, 285–290.[CrossRef] [Google Scholar]
  18. Kaverin, N. V., Rudneva, I. A., Ilyushina, N. A., Lipatov, A. S., Krauss, S. & Webster, R. G.(2004). Structural differences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture: analysis of H9 escape mutants. J Virol 78, 240–249.[CrossRef] [Google Scholar]
  19. Kim, H. R., Park, C. K., Oem, J. K., Bae, Y. C., Choi, J. G., Lee, O. S. & Lee, Y. J.(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.[CrossRef] [Google Scholar]
  20. King, D. J.(1991). Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum. Avian Dis 35, 505–514.[CrossRef] [Google Scholar]
  21. Kiso, M., Mitamura, K., Sakai-Tagawa, Y., Shiraishi, K., Kawakami, C., Kimura, K., Hayden, F. G., Sugaya, N. & Kawaoka, Y.(2004). Resistant influenza A viruses in children treated with oseltamivir: descriptive study. Lancet 364, 759–765.[CrossRef] [Google Scholar]
  22. Lee, C. W., Song, C. S., Lee, Y. J., Mo, I. P., Garcia, M., Suarez, D. L. & Kim, S. J.(2000). Sequence analysis of the hemagglutinin gene of H9N2 Korean avian influenza viruses and assessment of the pathogenic potential of isolate MS96. Avian Dis 44, 527–535.[CrossRef] [Google Scholar]
  23. Lee, C. W., Senne, D. A. & Suarez, D. L.(2004). Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. J Virol 78, 8372–8381.[CrossRef] [Google Scholar]
  24. Lee, Y. J., Shin, J. Y., Song, M. S., Lee, Y. M., Choi, J. G., Lee, E. K., Jeong, O. M., Sung, H. W., Kim, J. H. & other authors(2007). Continuing evolution of H9 influenza viruses in Korean poultry. Virology 359, 313–323.[CrossRef] [Google Scholar]
  25. Lee, Y. J., Choi, Y. K., Kim, Y. J., Song, M. S., Jeong, O. M., Lee, E. K., Jeon, W. J., Jeong, W., Joh, S. J. & other authors(2008). Highly pathogenic avian influenza virus (H5N1) in domestic poultry and relationship with migratory birds, South Korea. Emerg Infect Dis 14, 487–490.[CrossRef] [Google Scholar]
  26. Matrosovich, M. N., Krauss, S. & Webster, R. G.(2001). H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity. Virology 281, 156–162.[CrossRef] [Google Scholar]
  27. Mo, I. P., Song, C. S., Kim, K. S. & Rhee, J. C.(1997). An occurrence of non-highly pathogenic avian influenza in Korea. In Proceeding of the Fourth International Symposium on Avian Influenza United States Animal Health Association, pp. 379–383. Edited by D. Swayne & R. Slemons. FL: Rose Printing Company.
  28. Moon, H. J., Song, M. S., Cruz, D. J., Park, K. J., Pascua, P. N., Lee, J. H., Baek, Y. H., Choi, D. H., Choi, Y. K. & Kim, C. J.(2010). Active reassortment of H9 influenza viruses between wild birds and live-poultry markets in Korea. Arch Virol 155, 229–241.[CrossRef] [Google Scholar]
  29. Palmer, D. F., Dowdle, W. R., Coleman, M. T. & Schild, G. C.(1975). Advanced laboratory techniques for influenza diagnosis. Immunol Ser 6, 25–45. [Google Scholar]
  30. Perrière, G. & Gouy, M.(1996). WWW-query: an on-line retrieval system for biological sequence banks. Biochimie 78, 364–369.[CrossRef] [Google Scholar]
  31. Reed, L. J. & Muench, H.(1938). A simple method of estimating fifty per cent endpoints. Am J Hyg 27, 493–497. [Google Scholar]
  32. Song, M. S., Oh, T. K., Moon, H. J., Yoo, D. W., Lee, E. H., Lee, J. S., Kim, C. J., Yoo, G. J., Kim, H. & Choi, Y. K.(2008). Ecology of H3 avian influenza viruses in Korea and assessment of their pathogenic potentials. J Gen Virol 89, 949–957.[CrossRef] [Google Scholar]
  33. Suarez, D. L., Lee, C. W. & Swayne, D. E.(2006). Avian influenza vaccination in North America: strategies and difficulties. Dev Biol (Basel) 124, 117–124. [Google Scholar]
  34. Swayne, D. E. & Beck, J. R.(2004). Heat inactivation of avian influenza and Newcastle disease viruses in egg products. Avian Pathol 33, 512–518.[CrossRef] [Google Scholar]
  35. Swayne, D. E. & Kapczynski, D.(2008). Strategies and challenges for eliciting immunity against avian influenza virus in birds. Immunol Rev 225, 314–331.[CrossRef] [Google Scholar]
  36. Thomas, C. & Swayne, D. E.(2007). Thermal inactivation of H5N1 high pathogenicity avian influenza virus in naturally infected chicken meat. J Food Prot 70, 674–680. [Google Scholar]
  37. Thomas, C., King, D. J. & Swayne, D. E.(2008). Thermal inactivation of avian influenza and Newcastle disease viruses in chicken meat. J Food Prot 71, 1214–1222. [Google Scholar]
  38. Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M. & Kawaoka, Y.(1992). Evolution and ecology of influenza A viruses. Microbiol Rev 56, 152–179. [Google Scholar]
  39. Widjaja, L., Ilyushina, N., Webster, R. G. & Webby, R. J.(2006). Molecular changes associated with adaptation of human influenza A virus in embryonated chicken eggs. Virology 350, 137–145.[CrossRef] [Google Scholar]
  40. Xu, K. M., Smith, G. J., Bahl, J., Duan, L., Tai, H., Vijaykrishna, D., Wang, J., Zhang, J. X., Li, K. S. & other authors(2007). The genesis and evolution of H9N2 influenza viruses in poultry from southern China, 2000 to 2005. J Virol 81, 10389–10401.[CrossRef] [Google Scholar]
  41. Zamarin, D., Ortigoza, M. B. & Palese, P.(2006). Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice. J Virol 80, 7976–7983.[CrossRef] [Google Scholar]

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vol. , part 1, pp. 36 - 50

Molecular analysis

Rate of change of the H9 HA protein from 2001 to 2009 relative to that of the vaccine strain [Single PDF file](18 KB)


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