1887

Abstract

Influenza A virus causes significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. It has been shown that conversion of the haemagglutinin (HA) cleavage site from a trypsin-sensitive motif to an elastase-sensitive motif resulted in attenuated viruses in mouse models. However, application of this attenuation approach in a natural host has not been achieved yet. Here, we report that using reverse genetics, we generated two mutant SIVs derived from strain A/SW/SK/18789/02 (H1N1). Mutant A/SW/SK-R345V carries a mutation from arginine to valine at aa 345 of HA. Similarly, mutant A/SW/SK-R345A encodes alanine instead of arginine at aa 345 of HA. Our data showed that both mutants are solely dependent on neutrophil elastase cleavage in tissue culture. These tissue culture-grown mutant SIVs showed similar growth properties in terms of plaque size and growth kinetics to the wild-type virus. In addition, SIV mutants were able to maintain their genetic information after multiple passaging on MDCK cells. Furthermore, mutant SIVs were highly attenuated in pigs. Thus, these mutants may have the potential to serve as live attenuated vaccines.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.005447-0
2009-02-01
2019-11-17
Loading full text...

Full text loading...

/deliver/fulltext/jgv/90/2/375.html?itemId=/content/journal/jgv/10.1099/vir.0.005447-0&mimeType=html&fmt=ahah

References

  1. Bosch, F. X., Garten, W., Klenk, H. D. & Rott, R. ( 1981; ). Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of Avian influenza viruses. Virology 113, 725–735.[CrossRef]
    [Google Scholar]
  2. Brown, G. & McMillen, J. (1994). Maxivac-Flu: evaluation of the safety and efficacy of a swine influenza vaccine. Proc Am Ass Swine Prac 25, 37–39.
  3. Castillo, M. J., Nakajima, K., Zimmerman, M. & Powers, J. C. ( 1979; ). Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases. Anal Biochem 99, 53–64.[CrossRef]
    [Google Scholar]
  4. Cox, R. J., Brokstad, K. A. & Ogra, P. ( 2004; ). Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines. Scand J Immunol 59, 1–15.[CrossRef]
    [Google Scholar]
  5. Cross, K. J., Burleigh, L. M. & Steinhauer, D. A. ( 2001; ). Mechanisms of cell entry by influenza virus. Expert Rev Mol Med 6, 1–18.
    [Google Scholar]
  6. Gabriel, G., Garn, H., Wegmann, M., Renz, H., Herwig, A., Klenk, H. D. & Stech, J. ( 2008; ). The potential of a protease activation mutant of a highly pathogenic avian influenza virus for a pandemic live vaccine. Vaccine 26, 956–965.[CrossRef]
    [Google Scholar]
  7. Garten, W., Bosch, F. X., Linder, D., Rott, R. & Klenk, H. D. ( 1981; ). Proteolytic activation of the influenza virus hemagglutinin: the structure of the cleavage site and the enzymes involved in cleavage. Virology 115, 361–374.[CrossRef]
    [Google Scholar]
  8. Gertler, A. & Hofmann, T. ( 1970; ). Acetyl-L-alanyl-L-alanyl-L-alanine methyl ester: a new highly specific elastase substrate. Can J Biochem 48, 384–386.
    [Google Scholar]
  9. Gorse, G. J., Campbell, M. J., Otto, E. E., Powers, D. C., Chambers, G. W. & Newman, F. K. ( 1995; ). Increased anti-influenza A virus cytotoxic T cell activity following vaccination of the chronically ill elderly with live attenuated or inactivated influenza virus vaccine. J Infect Dis 172, 1–10.[CrossRef]
    [Google Scholar]
  10. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. ( 2000; ). A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97, 6108–6113.[CrossRef]
    [Google Scholar]
  11. 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]
  12. Karasin, A. I., West, K., Carman, S. & Olsen, C. W. ( 2004; ). Characterization of avian H3N3 and H1N1 influenza A viruses isolated from pigs in Canada. J Clin Microbiol 42, 4349–4354.[CrossRef]
    [Google Scholar]
  13. Klenk, H. D., Rott, R., Orlich, M. & Blodorn, J. ( 1975; ). Activation of influenza A viruses by trypsin treatment. Virology 68, 426–439.[CrossRef]
    [Google Scholar]
  14. Lamb R. A. & Krug, R. M. ( 2001; ). Orthomyxoviridae: the viruses and their replication. In Fields Virology, pp 1488–1532. Edited by B. N. Fields, D. M. Knipe, P. M. Howley & D. E. Griffin. Philadelphia, PA: Lipincott, Williams & Wilkins.
  15. Lazarowitz, S. G., Compans, R. W. & Choppin, P. W. ( 1973; ). Proteolytic cleavage of the hemagglutinin polypeptide of influenza virus. Function of the uncleaved polypeptide HA. Virology 52, 199–212.[CrossRef]
    [Google Scholar]
  16. Ma, W., Vincent, A. L., Gramer, M. R., Brockwell, C. B., Lager, K. M., Janke, B. H., Gauger, P. C., Patnayak, D. P., Webby, R. J. & Richt, J. A. ( 2007; ). Identification of H2N3 influenza A viruses from swine in the United States. Proc Natl Acad Sci U S A 104, 20949–20954.[CrossRef]
    [Google Scholar]
  17. Macklin, M. D., McCabe, D., McGregor, M. W., Neumann, V., Meyer, T., Callan, R., Hinshaw, V. S. & Swain, W. F. ( 1998; ). Immunization of pigs with a particle-mediated DNA vaccine to influenza A virus protects against challenge with homologous virus. J Virol 72, 1491–1496.
    [Google Scholar]
  18. Narayanan, A. S. & Anwar, R. A. ( 1969; ). The specificity of purified porcine pancreatic elastase. Biochem J 114, 11–17.
    [Google Scholar]
  19. Olsen, C. W. ( 2002; ). The emergence of novel swine influenza viruses in North America. Virus Res 85, 199–210.[CrossRef]
    [Google Scholar]
  20. Paillot, R., Hannant, D., Kydd, J. H. & Daly, J. M. ( 2006; ). Vaccination against equine influenza: quid novi? Vaccine 24, 4047–4061.[CrossRef]
    [Google Scholar]
  21. Reed, L. J. & Muench, H. ( 1938; ). A simple method of estimating fifty percent endpoints. Am J Hyg 27, 493–497.
    [Google Scholar]
  22. Richt, J. A., Lager, K. M., Janke, B. H., Woods, R. D., Webster, R. G. & Webby, R. J. ( 2003; ). Pathogenic and antigenic properties of phylogenetically distinct reassortant H3N2 swine influenza viruses cocirculating in the United States. J Clin Microbiol 41, 3198–3205.[CrossRef]
    [Google Scholar]
  23. Richt, J. A., Lekcharoensuk, P., Lager, K. M., Vincent, A. L., Loiacono, C. M., Janke, B. H., Wu, W. H., Yoon, K. J., Webby, R. J. & other authors ( 2006; ). Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine. J Virol 80, 11009–11018.[CrossRef]
    [Google Scholar]
  24. Shin, Y. K., Li, Y., Liu, Q., Anderson, D. H., Babiuk, L. A. & Zhou, Y. ( 2007a; ). SH3 binding motif 1 in influenza A virus NS1 protein is essential for PI3K/Akt signaling pathway activation. J Virol 81, 12730–12739.[CrossRef]
    [Google Scholar]
  25. Shin, Y. K., Liu, Q., Tikoo, S. K., Babiuk, L. A. & Zhou, Y. ( 2007b; ). Effect of the phosphatidylinositol 3-kinase/Akt pathway on influenza A virus propagation. J Gen Virol 88, 942–950.[CrossRef]
    [Google Scholar]
  26. Shin, Y. K., Liu, Q., Tikoo, S. K., Babiuk, L. A. & Zhou, Y. ( 2007c; ). Influenza A virus NS1 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by direct interaction with the p85 subunit of PI3K. J Gen Virol 88, 13–18.[CrossRef]
    [Google Scholar]
  27. Skehel, J. J. & Wiley, D. C. ( 2000; ). Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69, 531–569.[CrossRef]
    [Google Scholar]
  28. Solorzano, A., Webby, R. J., Lager, K. M., Janke, B. H., Garcia-Sastre, A. & Richt, J. A. ( 2005; ). Mutations in the NS1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs. J Virol 79, 7535–7543.[CrossRef]
    [Google Scholar]
  29. Stech, J., Garn, H., Wegmann, M., Wagner, R. & Klenk, H. D. ( 2005; ). A new approach to an influenza live vaccine: modification of the cleavage site of hemagglutinin. Nat Med 11, 683–689.[CrossRef]
    [Google Scholar]
  30. Straw, B. E., D'Allaire, S., Mengeling, W. L. & Taylor, D. J. ( 1999; ). Diseases of Swine. Ames, IA: Iowa State University Press.
  31. Vincent, A. L., Ma, W., Lager, K. M., Janke, B. H., Webby, R. J., Garcia-Sastre, A. & Richt, J. A. ( 2007; ). Efficacy of intranasal administration of a truncated NS1 modified live influenza virus vaccine in swine. Vaccine 25, 7999–8009.[CrossRef]
    [Google Scholar]
  32. Wells, D. L., Hopfensperger, D. J., Arden, N. H., Harmon, M. W., Davis, J. P., Tipple, M. A. & Schonberger, L. B. ( 1991; ). Swine influenza virus infections. Transmission from ill pigs to humans at a Wisconsin agricultural fair and subsequent probable person-to-person transmission. JAMA 265, 478–481.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.005447-0
Loading
/content/journal/jgv/10.1099/vir.0.005447-0
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error