[Source: Journal of Infectious Diseases, full page: (LINK). Abstract, edited.]


A novel I221 L substitution in neuraminidase confers high level resistance to oseltamivir in influenza B viruses

Vanessa Escuret 1,2,*, Patrick J. Collins 5,*, Jean-Sébastien Casalegno 1,2,*, Sebastien G. Vachieri 5,6,*, Nicholas Cattle 4, Olivier Ferraris 2, Murielle Sabatier 2, Emilie Frobert 1,2, Valérie Caro 7, John J. Skehel 5, Steve Gamblin 6, Frédéric Valla 3, Martine Valette 1, Michèle Ottmann 2, John W. McCauley 4, Rodney S. Daniels 4 and Bruno Lina 1,2

Author Affiliations: <SUP>1</SUP>Hospices Civils de Lyon, Groupement Hospitalier Est, Laboratoire de Virologie et Centre National de Référence virus influenzae, F-69677, Bron, France <SUP>2</SUP>Université de Lyon, Université Claude Bernard Lyon 1, Faculté de Médecine Lyon Est, EA 4610, F-69372, Lyon, France <SUP>3</SUP>Hospices Civils de Lyon, Groupement Hospitalier Est, Hôpital Femme Mère Enfant, Service de Réanimation Pédiatrique, , F-69677, Bron, France <SUP>4</SUP>WHO Collaborating Centre for Reference and Research on Influenza, Division of Virology, Medical Research Council National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom <SUP>5</SUP>Division of Virology, Medical Research Council National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom <SUP>6</SUP>Division of Molecular Structure, Medical Research Council National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom <SUP>7</SUP>Institut Pasteur, Genotyping of Pathogens and Public Health Platform, 28 rue du Docteur Roux , 75724 Paris Cedex 15, France

Corresponding author: Dr Vanessa Escuret or Pr. Bruno Lina, Laboratoire de Virologie - Bât A3, 59 Boulevard Pinel, F-69677 Bron Cedex, France, Téléphone : 04 72 12 96 17, Fax : 04 72 12 95 00, Email : vanessa.escuret@chu-lyon.fr, bruno.lina@chu-lyon.fr

* These authors contributed equally to the work


Abstract

Introduction.

Influenza B viruses with a novel I221 L 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 (wt) or mutated (I221 L) NA genes were evaluated. Proportions of wt and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wt and I221 L variant NA were performed.


Results.

The Km and Ki revealed that the I221 L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir respectively compared to wt NA.

Viruses with a wt or I221 L variant NA had similar growth kinetics in MDCK cells and five passages in MDCK cells revealed no reversion of the I221 L substitution. The crystal structure of the I221 L 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 resistance to oseltamivir, while retaining good virus fitness of viruses carrying I221 L variant NA.


Received February 10, 2014. Revision received April 17, 2014. Accepted April 17, 2014.

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/ ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.


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