AAC Accepts, published online ahead of print on 3 August 2009

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Antimicrob. Agents Chemother. doi:10.1128/AAC.00334-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

In vitro generation of neuraminidase inhibitor resistance in A(H5N1) influenza viruses

Aeron C Hurt^*, Jessica K Holien, and Ian G Barr

WHO Collaborating Centre for Reference and Research on Influenza, North Melbourne, Victoria 3051, Australia; Monash University, School of Applied Sciences, Churchill, Victoria 3842, Australia; Structural Biology Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia

^* To whom correspondence should be addressed. Email: aeron.hurt{at}influenzacentre.org.

To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged in increasing levels of either oseltamivir or zanamivir. Under oseltamivir pressure, both A(H5N1) viruses developed a H274Y neuraminidase mutation, although in one strain the mutation occurred in combination with a I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900- to 2500-fold compared to wildtype). However the dual H274Y/I222M neuraminidase mutation caused an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8000-fold compared to wildtype). A similar affect on oseltamivir susceptibility was observed when the dual H274Y/I222M mutations were introduced, by reverse genetics, into a recombinant seasonal human A(H1N1) virus, and also when an alternative I222 substitution (I222V) was generated in combination with H274Y in A(H5N1) and A(H1N1) viruses. These viruses remained fully susceptible to zanamivir but demonstrated reduced susceptibility to peramivir. Following passage of the A(H5N1) viruses in the presence of zanamivir, strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also a E119G neuraminidase mutation which demonstrated significantly reduced zanamivir susceptibility (1400-fold compared to wildtype). Mutations in haemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study has identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure, and the potential for dual NA mutations to result in dramatically reduced drug susceptibility.

This article has been cited by other articles:

• Hurt, A. C., Holien, J. K., Parker, M., Kelso, A., Barr, I. G. (2009). Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation. J. Virol. 83: 10366-10373 [Abstract] [Full Text]