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Antimicrobial Agents and Chemotherapy, October 2009, p. 4433-4440, Vol. 53, No. 10
0066-4804/09/$08.00+0     doi:10.1128/AAC.00334-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

In Vitro Generation of Neuraminidase Inhibitor Resistance in A(H5N1) Influenza Viruses

Aeron C. Hurt,^1,2* Jessica K. Holien,³ and Ian G. Barr^1,2

WHO Collaborating Centre for Reference and Research on Influenza, North Melbourne, Victoria 3051, Australia,¹ Monash University, School of Applied Sciences, Churchill, Victoria 3842, Australia,² Structural Biology Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia³

Received 11 March 2009/ Returned for modification 9 April 2009/ Accepted 14 July 2009

To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged with 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 an I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900- to 2,500-fold compared to the wild type). However the dual H274Y/I222M neuraminidase mutation had an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8,000-fold compared to the wild type). 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, the strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also an E119G neuraminidase mutation, which demonstrated significantly reduced zanamivir susceptibility (1,400-fold compared to the wild type). Mutations in hemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure and the potential for dual neuraminidase mutations to result in dramatically reduced drug susceptibility.

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* Corresponding author. Mailing address: WHO Collaborating Centre for Reference and Research on Influenza, 10 Wreckyn St., North Melbourne, Victoria 3051, Australia. Phone: 613 93423914. Fax: 613 93423939. E-mail: aeron.hurt{at}influenzacentre.org

Published ahead of print on 3 August 2009.

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Antimicrobial Agents and Chemotherapy, October 2009, p. 4433-4440, Vol. 53, No. 10
0066-4804/09/$08.00+0     doi:10.1128/AAC.00334-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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]