Virological Methods for the Generation and Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en
The compound 4-guanidino-Neu5Ac2en (zanamivir) has been described as a selective inhibitor of the influenza virus neuraminidase (NA) (1). Viruses that are less sensitive to this inhibitor can be isolated after several passages in MDCK cells in the presence of the inhibitor. Variants isolated so far have had mutations predominantly in the hemagglutinin (HA) gene (2). Many of these mutations appear to lower the affinity of the HA for the cellular receptor, so that there is less requirement for significant NA activity for the newly synthesized progeny virus to elute. There are three reports of a mutation in the NA gene, all at the same conserved site, glu 119 (3, 4, 5). In this chapter, the authors describe methods for the isolation of the mutants, and for their characterization in cell culture based assays.
KeywordsMDCK Cell Maintenance Medium Drug Sensitivity Assay Carbol Fuchsin Passage Virus
- 1.von Itzstein, M., Wu, W. Y., Kok, G. B., Pegg, M. S., Dyason, J. C., Phan, T. V., Smythe, M. L., White, H. F., Oliver, S. W., Colman, P. M., Varghese, J. N., Ryan, D. M., Woods, J. M., Bethell, R. C., Hotham, V. J., Cameron, J. M., and Penn, C. R. (1993) Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363, 418–423.CrossRefGoogle Scholar
- 2.McKimm-Breschkin, J. L., Blick, T. J., Sahasrabudhe, A. V., Tiong, T., Marshall, D., Hart, G. J., Bethell, R. C., and Penn, C. R. (1996) Generation and characterization of variants of the NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en. Antimicrob. Agents Chemother. 40, 40–46.PubMedGoogle Scholar
- 3.Blick, T. J., Tiong, T., Sahasrabudhe, A., Varghese, J. N., Colman, P. M., Hart, G. J., Bethell, R. C., and McKimm-Breschkin, J. L. (1995) Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en. Virology 214, 475–484.CrossRefPubMedGoogle Scholar