tetano
Editor, Senior Moderator
J. Antimicrob. Chemother. (2013) doi: 10.1093/jac/dkt205 First published online: June 11, 2013
Reduced susceptibility to all neuraminidase inhibitors of influenza H1N1 viruses with haemagglutinin mutations and mutations in non-conserved residues of the neuraminidase
Jennifer L. McKimm-Breschkin1,*,
Janelle Williams1,
Susan Barrett1,
Kim Jachno1,†,
Mandy McDonald1,
Peter G. Mohr2,
Takehiko Saito3,‡ and
Masato Tashiro3
+ Author Affiliations
1CSIRO Materials Science and Engineering, 343 Royal Parade Parkville, 3052 Australia
2CSIRO Australian Animal Health Laboratories, Geelong, 3219 Australia
3WHO Collaborating Centre for Reference and Research on Influenza, Influenza Virus Research Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan
↵*Corresponding author. Tel: +613-9662-7257; Fax: +613-9662-7101; E-mail: mck245@csiro.au
Received January 25, 2013.
Revision requested March 5, 2013.
Revision received April 15, 2013.
Accepted April 30, 2013.
Abstract
Objectives We characterized human H1N1 influenza isolate A/Hokkaido/15/02, which has haemagglutinin and neuraminidase mutations that reduce drug susceptibility to oseltamivir, zanamivir and peramivir.
Methods One wild-type and three mutant viruses were isolated by plaque purification. Viruses were tested in MUNANA-based enzyme assays, cell culture and receptor binding assays.
Results Two viruses had a neuraminidase Y155H mutation that reduced susceptibility in the enzyme inhibition assay to all inhibitors by 30-fold to >100-fold. The Y155H mutation reduced plaque size and affected the stability, Km and pH activity profile of the enzyme. In contrast to previous mutants, this neuraminidase demonstrated a slower rate of inhibitor binding in the IC50 kinetics assay. One virus had both the Y155H mutation and a haemagglutinin D225G mutation that rescued the small-plaque phenotype of the Y155H virus and affected receptor binding and drug susceptibility in cell culture and binding assays. We also isolated a third mutant virus, with both neuraminidase V114I and haemagglutinin D225N mutations, which affected susceptibility in the enzyme inhibition assay and receptor binding, respectively, but to lesser extents than the Y155H and D225G mutations.
Conclusions Neither Y155 nor V114 is conserved across neuraminidase subtypes. Furthermore, Y155 is not conserved even among avian and swine N1 viruses. Structurally, both residues reside far from the neuraminidase active site. D225 forms part of the receptor binding site of the haemagglutinin. We believe this is the first demonstration of a specific haemagglutinin mutation correlating with reduced drug susceptibility in plaque assays in both Madin Darby Canine Kidney and SIAT cells.
http://jac.oxfordjournals.org/content/early/2013/06/10/jac.dkt205.short
Reduced susceptibility to all neuraminidase inhibitors of influenza H1N1 viruses with haemagglutinin mutations and mutations in non-conserved residues of the neuraminidase
Jennifer L. McKimm-Breschkin1,*,
Janelle Williams1,
Susan Barrett1,
Kim Jachno1,†,
Mandy McDonald1,
Peter G. Mohr2,
Takehiko Saito3,‡ and
Masato Tashiro3
+ Author Affiliations
1CSIRO Materials Science and Engineering, 343 Royal Parade Parkville, 3052 Australia
2CSIRO Australian Animal Health Laboratories, Geelong, 3219 Australia
3WHO Collaborating Centre for Reference and Research on Influenza, Influenza Virus Research Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan
↵*Corresponding author. Tel: +613-9662-7257; Fax: +613-9662-7101; E-mail: mck245@csiro.au
Received January 25, 2013.
Revision requested March 5, 2013.
Revision received April 15, 2013.
Accepted April 30, 2013.
Abstract
Objectives We characterized human H1N1 influenza isolate A/Hokkaido/15/02, which has haemagglutinin and neuraminidase mutations that reduce drug susceptibility to oseltamivir, zanamivir and peramivir.
Methods One wild-type and three mutant viruses were isolated by plaque purification. Viruses were tested in MUNANA-based enzyme assays, cell culture and receptor binding assays.
Results Two viruses had a neuraminidase Y155H mutation that reduced susceptibility in the enzyme inhibition assay to all inhibitors by 30-fold to >100-fold. The Y155H mutation reduced plaque size and affected the stability, Km and pH activity profile of the enzyme. In contrast to previous mutants, this neuraminidase demonstrated a slower rate of inhibitor binding in the IC50 kinetics assay. One virus had both the Y155H mutation and a haemagglutinin D225G mutation that rescued the small-plaque phenotype of the Y155H virus and affected receptor binding and drug susceptibility in cell culture and binding assays. We also isolated a third mutant virus, with both neuraminidase V114I and haemagglutinin D225N mutations, which affected susceptibility in the enzyme inhibition assay and receptor binding, respectively, but to lesser extents than the Y155H and D225G mutations.
Conclusions Neither Y155 nor V114 is conserved across neuraminidase subtypes. Furthermore, Y155 is not conserved even among avian and swine N1 viruses. Structurally, both residues reside far from the neuraminidase active site. D225 forms part of the receptor binding site of the haemagglutinin. We believe this is the first demonstration of a specific haemagglutinin mutation correlating with reduced drug susceptibility in plaque assays in both Madin Darby Canine Kidney and SIAT cells.
http://jac.oxfordjournals.org/content/early/2013/06/10/jac.dkt205.short