title: A novel I221L substitution in neuraminidase confers high-level resistance to oseltamivir in influenza B viruses.
abstract: Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment.
  
  
  METHODS
  Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type or mutated (I221L) NA genes were evaluated. Proportions of wild-type and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wild-type and I221L variant NA were performed.
  
  
  RESULTS
  The Km and Ki revealed that the I221L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir, respectively, compared with wild-type NA. Viruses with a wild-type or I221L variant NA had similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, and 5 passages in MDCK cells revealed no reversion of the I221L substitution. The crystal structure of the I221L NA and oseltamivir complex showed that the leucine side chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir.
  
  
  CONCLUSIONS
  Enzyme kinetic and NA structural analyses provide an explanation for the high level of resistance to oseltamivir while retaining good fitness of viruses carrying I221L variant NA.
authors.affiliation: Laboratoire de Virologie et Centre National de Référence virus influenzae Laboratoire Virpath EA4610, Faculté de Médecine Lyon Est, Université Claude Bernard Lyon 1, Université de Lyon, and.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Escuret
authors.affiliation: Division of Virology.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Collins
authors.affiliation: Laboratoire de Virologie et Centre National de Référence virus influenzae Laboratoire Virpath EA4610, Faculté de Médecine Lyon Est, Université Claude Bernard Lyon 1, Université de Lyon, and.
authors.forename: Jean-Sébastien
authors.initials: JS
authors.lastname: Casalegno
authors.affiliation: Division of Virology Division of Molecular Structure.
authors.forename: Sebastien G
authors.initials: SG
authors.lastname: Vachieri
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza, Division of Virology, Medical Research Council National Institute for Medical Research, London, United Kingdom.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Cattle
authors.affiliation: Laboratoire Virpath EA4610, Faculté de Médecine Lyon Est, Université Claude Bernard Lyon 1, Université de Lyon, and.
authors.forename: Olivier
authors.initials: O
authors.lastname: Ferraris
authors.affiliation: Laboratoire Virpath EA4610, Faculté de Médecine Lyon Est, Université Claude Bernard Lyon 1, Université de Lyon, and.
authors.forename: Murielle
authors.initials: M
authors.lastname: Sabatier
authors.affiliation: Laboratoire de Virologie et Centre National de Référence virus influenzae Laboratoire Virpath EA4610, Faculté de Médecine Lyon Est, Université Claude Bernard Lyon 1, Université de Lyon, and.
authors.forename: Emilie
authors.initials: E
authors.lastname: Frobert
authors.affiliation: Genotyping of Pathogens and Public Health Platform, Institut Pasteur, Paris, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Caro
authors.affiliation: Division of Virology.
authors.forename: John J
authors.initials: JJ
authors.lastname: Skehel
authors.affiliation: Division of Molecular Structure.
authors.forename: Steve
authors.initials: S
authors.lastname: Gamblin
authors.affiliation: Service de Réanimation Pédiatrique, Hôpital Femme Mère Enfant, Groupement Hospitalier Est, Hospices Civils de Lyon, Bron.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Valla
authors.affiliation: Laboratoire de Virologie et Centre National de Référence virus influenzae.
authors.forename: Martine
authors.initials: M
authors.lastname: Valette
authors.affiliation: Laboratoire Virpath EA4610, Faculté de Médecine Lyon Est, Université Claude Bernard Lyon 1, Université de Lyon, and.
authors.forename: Michèle
authors.initials: M
authors.lastname: Ottmann
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza, Division of Virology, Medical Research Council National Institute for Medical Research, London, United Kingdom.
authors.forename: John W
authors.initials: JW
authors.lastname: McCauley
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza, Division of Virology, Medical Research Council National Institute for Medical Research, London, United Kingdom.
authors.forename: Rodney S
authors.initials: RS
authors.lastname: Daniels
authors.affiliation: Laboratoire de Virologie et Centre National de Référence virus influenzae Laboratoire Virpath EA4610, Faculté de Médecine Lyon Est, Université Claude Bernard Lyon 1, Université de Lyon, and.
authors.forename: Bruno
authors.initials: B
authors.lastname: Lina
mesh_terms: D000293:Adolescent; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004285:Dogs; D024882:Drug Resistance, Viral / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009981:Influenza B virus / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D008297:Male; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism*; D053139:Oseltamivir / Q000494:pharmacology*; D010948:Viral Plaque Assay
chemical_list: D000998:Antiviral Agents; D006388:Hemagglutinins; D053139:Oseltamivir; D009439:Neuraminidase
keywords: influenza B virus; neuraminidase substitution I221L; oseltamivir resistance