title: Detection of influenza virus resistance to neuraminidase inhibitors by an enzyme inhibition assay.
abstract: We previously characterized influenza viruses whose selection in the presence of neuraminidase (NA) inhibitors resulted in a substituted residue (position 119,152,274, or 292) in the NA active center. To identify the most favorable conditions for detecting NA inhibitor-resistant viruses we compared the results of four modifications of the NA inhibition assay utilizing a fluorogenic substrate. The IC50 values were highly dependent upon assay conditions, and most mutant enzymes were more sensitive to changes in assay conditions (e.g. addition of PO4(3-), Ca2+, DMSO, or EDTA) than wild-type enzymes or a mutant NA with an Arg292-->Lys substitution. Although the levels of resistance to zanamivir, oseltamivir carboxylate, and BCX-1812 (RWJ-270201) for each mutant varied among assays, mutants with substitutions at framework residues 119 or 274 exhibited sensitivity to at least one inhibitor. Viruses with substitutions at catalytic residues 152 or 292 were resistant to each inhibitor in all assays. Monitoring resistance in a clinical setting will require a panel of resistant viruses to ensure that assay conditions are favorable for detecting variants with substituted residues in the NA active center. Because variants selected in the presence of one NA inhibitor could be variably resistant to other inhibitors, all three inhibitors should be used in drug susceptibility testing.
authors.affiliation: Department of Internal Medicine, Division of Epidemiology and Virology, University of Virginia School of Medicine, P.O. Box 800473, Charlottesville, VA 22908-0473, USA. lvg9b@virginia.edu
authors.forename: Larisa V
authors.initials: LV
authors.lastname: Gubareva
authors.forename: Robert G
authors.initials: RG
authors.lastname: Webster
authors.forename: Frederick G
authors.initials: FG
authors.lastname: Hayden
mesh_terms: D000081:Acetamides / Q000494:pharmacology; D000146:Acids, Carbocyclic; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D003517:Cyclopentanes / Q000494:pharmacology; D004285:Dogs; D024882:Drug Resistance, Viral; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006146:Guanidines; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests / Q000379:methods; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D053139:Oseltamivir; D011714:Pyrans; D012794:Sialic Acids / Q000494:pharmacology; D053243:Zanamivir
chemical_list: D000081:Acetamides; D000146:Acids, Carbocyclic; D000998:Antiviral Agents; D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D006146:Guanidines; D011714:Pyrans; D012794:Sialic Acids; D053139:Oseltamivir; D004492:Edetic Acid; D009439:Neuraminidase; D053243:Zanamivir; C414210:peramivir