This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gubareva, L. V. Articles by Webster, R. G. Search for Related Content PubMed PubMed Citation Articles by Gubareva, L. V. Articles by Webster, R. G.

 Previous Article  |  Next Article 

J. Virol., 03 1996, 1818-1827, Vol 70, No. 3
Copyright © 1996, American Society for Microbiology

Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en

LV Gubareva, R Bethell, GJ Hart, KG Murti, CR Penn and RG Webster
Department of Virology/Molecular Biology, St Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.

The development of viral resistance to the neuraminidase (NA) inhibitor, 4-guanidino-Neu5Ac2en, of influenza viruses was studied by serial passage of A/Turkey/Minnesota/833/80 (H4N2) in Madin-Darby canine kidney cells in the presence of increasing concentrations of inhibitor. Resistant mutants selected after eight passages, had a 10,000-fold reduction in sensitivity to the inhibitor in plaque assays, but their affinity (1/Kd) to the inhibitor was similar to that of the parental virus. Electron microscopic analysis revealed aggregation of the mutant virus at the cell surface in the presence of the inhibitor. Sequence analysis established that a substitution had occurred in the NA (Arg-249 to Lys) and in the HA2 subunit of the hemagglutinin (Gly-75 to Glu), in the vicinity of the proposed second sialic acid binding site. The change of residue 249 appears to be a chance mutation, for we were unable to reisolate this mutant, whereas subsequent experiments indicate changes in the hemagglutinin. After 13 passages of the parental virus, mutants that were resistant to the high concentrations of inhibitor tested were obtained. These viruses retained their drug- resistant phenotype even after five passages without the inhibitor. Electron microscopic analysis revealed no aggregation of virus on the surface of infected cells in the presence of the inhibitor. Sequence analysis of the NA gene from these drug-resistant mutants revealed an additional substitution of Glu to Ala at the conserved amino acid residue 119. This substitution is responsible for reducing the affinity of the inhibitor to the NA. Our findings suggest that the emergence of mutants resistant to 4-guanidine-Neu5Ac2en is a multistep process requiring prolonged exposure to the inhibitor.

This article has been cited by other articles:

• Sheu, T. G., Deyde, V. M., Okomo-Adhiambo, M., Garten, R. J., Xu, X., Bright, R. A., Butler, E. N., Wallis, T. R., Klimov, A. I., Gubareva, L. V. (2008). Surveillance for Neuraminidase Inhibitor Resistance among Human Influenza A and B Viruses Circulating Worldwide from 2004 to 2008. Antimicrob. Agents Chemother. 52: 3284-3292 [Abstract] [Full Text]  
• Alymova, I. V., Taylor, G., Mishin, V. P., Watanabe, M., Murti, K. G., Boyd, K., Chand, P., Babu, Y. S., Portner, A. (2008). Loss of the N-Linked Glycan at Residue 173 of Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Exposes a Second Receptor-Binding Site. J. Virol. 82: 8400-8410 [Abstract] [Full Text]  
• Tsiodras, S., Mooney, J. D, Hatzakis, A. (2007). Role of combination antiviral therapy in pandemic influenza and stockpiling implications. BMJ 334: 293-294 [Full Text]  
• Zurcher, T., Yates, P. J., Daly, J., Sahasrabudhe, A., Walters, M., Dash, L., Tisdale, M., McKimm-Breschkin, J. L. (2006). Mutations conferring zanamivir resistance in human influenza virus N2 neuraminidases compromise virus fitness and are not stably maintained in vitro. J Antimicrob Chemother 58: 723-732 [Abstract] [Full Text]  
• Yen, H.-L., Hoffmann, E., Taylor, G., Scholtissek, C., Monto, A. S., Webster, R. G., Govorkova, E. A. (2006). Importance of neuraminidase active-site residues to the neuraminidase inhibitor resistance of influenza viruses.. J. Virol. 80: 8787-8795 [Abstract] [Full Text]  
• Mishin, V. P., Hayden, F. G., Gubareva, L. V. (2005). Susceptibilities of Antiviral-Resistant Influenza Viruses to Novel Neuraminidase Inhibitors. Antimicrob. Agents Chemother. 49: 4515-4520 [Abstract] [Full Text]  
• Yen, H.-L., Herlocher, L. M., Hoffmann, E., Matrosovich, M. N., Monto, A. S., Webster, R. G., Govorkova, E. A. (2005). Neuraminidase Inhibitor-Resistant Influenza Viruses May Differ Substantially in Fitness and Transmissibility. Antimicrob. Agents Chemother. 49: 4075-4084 [Abstract] [Full Text]  
• Mishin, V. P., Novikov, D., Hayden, F. G., Gubareva, L. V. (2005). Effect of Hemagglutinin Glycosylation on Influenza Virus Susceptibility to Neuraminidase Inhibitors. J. Virol. 79: 12416-12424 [Abstract] [Full Text]  
• Lu, B., Zhou, H., Ye, D., Kemble, G., Jin, H. (2005). Improvement of Influenza A/Fujian/411/02 (H3N2) Virus Growth in Embryonated Chicken Eggs by Balancing the Hemagglutinin and Neuraminidase Activities, Using Reverse Genetics. J. Virol. 79: 6763-6771 [Abstract] [Full Text]  
• Jackson, D., Barclay, W., Zurcher, T. (2005). Characterization of recombinant influenza B viruses with key neuraminidase inhibitor resistance mutations. J Antimicrob Chemother 55: 162-169 [Abstract] [Full Text]  
• Macdonald, S. J. F., Watson, K. G., Cameron, R., Chalmers, D. K., Demaine, D. A., Fenton, R. J., Gower, D., Hamblin, J. N., Hamilton, S., Hart, G. J., Inglis, G. G. A., Jin, B., Jones, H. T., McConnell, D. B., Mason, A. M., Nguyen, V., Owens, I. J., Parry, N., Reece, P. A., Shanahan, S. E., Smith, D., Wu, W.-Y., Tucker, S. P. (2004). Potent and Long-Acting Dimeric Inhibitors of Influenza Virus Neuraminidase Are Effective at a Once-Weekly Dosing Regimen. Antimicrob. Agents Chemother. 48: 4542-4549 [Abstract] [Full Text]  
• McSharry, J. J., McDonough, A. C., Olson, B. A., Drusano, G. L. (2004). Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors. CVI 11: 21-28 [Abstract] [Full Text]  
• Wang, M. Z., Tai, C. Y., Mendel, D. B. (2002). Mechanism by Which Mutations at His274 Alter Sensitivity of Influenza A Virus N1 Neuraminidase to Oseltamivir Carboxylate and Zanamivir. Antimicrob. Agents Chemother. 46: 3809-3816 [Abstract] [Full Text]  
• Molla, A., Kati, W., Carrick, R., Steffy, K., Shi, Y., Montgomery, D., Gusick, N., Stoll, V. S., Stewart, K. D., Ng, T. I., Maring, C., Kempf, D. J., Kohlbrenner, W. (2002). In Vitro Selection and Characterization of Influenza A (A/N9) Virus Variants Resistant to a Novel Neuraminidase Inhibitor, A-315675. J. Virol. 76: 5380-5386 [Abstract] [Full Text]  
• Kobasa, D., Wells, K., Kawaoka, Y. (2001). Amino Acids Responsible for the Absolute Sialidase Activity of the Influenza A Virus Neuraminidase: Relationship to Growth in the Duck Intestine. J. Virol. 75: 11773-11780 [Abstract] [Full Text]  
• Murrell, M. T., Porotto, M., Greengard, O., Poltoratskaia, N., Moscona, A. (2001). A Single Amino Acid Alteration in the Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Glycoprotein Confers Resistance to the Inhibitory Effects of Zanamivir on Receptor Binding and Neuraminidase Activity. J. Virol. 75: 6310-6320 [Abstract] [Full Text]  
• Leneva, I. A., Goloubeva, O., Fenton, R. J., Tisdale, M., Webster, R. G. (2001). Efficacy of Zanamivir against Avian Influenza A Viruses That Possess Genes Encoding H5N1 Internal Proteins and Are Pathogenic in Mammals. Antimicrob. Agents Chemother. 45: 1216-1224 [Abstract] [Full Text]  
• Hayden, F. G., Gubareva, L. V., Monto, A. S., Klein, T. C., Elliott, M. J., Hammond, J. M., Sharp, S. J., Ossi, M. J., The Zanamivir Family Study Group, (2000). Inhaled Zanamivir for the Prevention of Influenza in Families. NEJM 343: 1282-1289 [Abstract] [Full Text]  
• Wagner, R., Wolff, T., Herwig, A., Pleschka, S., Klenk, H.-D. (2000). Interdependence of Hemagglutinin Glycosylation and Neuraminidase as Regulators of Influenza Virus Growth: a Study by Reverse Genetics. J. Virol. 74: 6316-6323 [Abstract] [Full Text]  
• Mitnaul, L. J., Matrosovich, M. N., Castrucci, M. R., Tuzikov, A. B., Bovin, N. V., Kobasa, D., Kawaoka, Y. (2000). Balanced Hemagglutinin and Neuraminidase Activities Are Critical for Efficient Replication of Influenza A Virus. J. Virol. 74: 6015-6020 [Abstract] [Full Text]  
• Hughes, M. T., Matrosovich, M., Rodgers, M. E., McGregor, M., Kawaoka, Y. (2000). Influenza A Viruses Lacking Sialidase Activity Can Undergo Multiple Cycles of Replication in Cell Culture, Eggs, or Mice. J. Virol. 74: 5206-5212 [Abstract] [Full Text]  
• Barnett, J. M., Cadman, A., Gor, D., Dempsey, M., Walters, M., Candlin, A., Tisdale, M., Morley, P. J., Owens, I. J., Fenton, R. J., Lewis, A. P., Claas, E. C. J., Rimmelzwaan, G. F., De Groot, R., Osterhaus, A. D. M. E. (2000). Zanamivir Susceptibility Monitoring and Characterization of Influenza Virus Clinical Isolates Obtained during Phase II Clinical Efficacy Studies. Antimicrob. Agents Chemother. 44: 78-87 [Abstract] [Full Text]  
• Basler, C. F., Garcia-Sastre, A., Palese, P. (1999). Mutation of Neuraminidase Cysteine Residues Yields Temperature-Sensitive Influenza Viruses. J. Virol. 73: 8095-8103 [Abstract] [Full Text]  
• Rowley, K. V., Harvey, R., Barclay, W. S. (1999). Isolation and characterization of a transfectant influenza B virus altered in RNA segment 6. J. Gen. Virol. 80: 2353-2359 [Abstract] [Full Text]  
• Tai, C. Y., Escarpe, P. A., Sidwell, R. W., Williams, M. A., Lew, W., Wu, H., Kim, C. U., Mendel, D. B. (1998). Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071. Antimicrob. Agents Chemother. 42: 3234-3241 [Abstract] [Full Text]  
• Fodor, E., Palese, P., Brownlee, G. G., Garcia-Sastre, A. (1998). Attenuation of Influenza A Virus mRNA Levels by Promoter Mutations. J. Virol. 72: 6283-6290 [Abstract] [Full Text]  
• Bantia, S., Ghate, A. A., Ananth, S. L., Babu, Y. S., Air, G. M., Walsh, G. M. (1998). Generation and Characterization of a Mutant of Influenza A Virus Selected with the Neuraminidase Inhibitor BCX-140. Antimicrob. Agents Chemother. 42: 801-807 [Abstract] [Full Text]  
• McKimm-Breschkin, J. L., Sahasrabudhe, A., Blick, T. J., McDonald, M., Colman, P. M., Hart, G. J., Bethell, R. C., Varghese, J. N. (1998). Mutations in a Conserved Residue in the Influenza Virus Neuraminidase Active Site Decreases Sensitivity to Neu5Ac2en-Derived Inhibitors. J. Virol. 72: 2456-2462 [Abstract] [Full Text]