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Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals
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Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

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Systematic identification of H274Y compensatory mutations in influenza A virus neuraminidase by high-throughput

Nicholas C Wu1, Arthur P Young, Sugandha Dandekar

  • 1Department of Molecular and Medical Pharmacology, University of California, Los Angeles David Geffen School of Medicine, Los Angeles, CA, USA.

Journal of Virology
|November 16, 2012
PubMed

Insights

Compensatory mutations help oseltamivir resistance in H1N1 influenza. Researchers identified four mutations that restore fitness to the H274Y oseltamivir resistance substitution in the neuraminidase gene.

Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • Oseltamivir resistance in H1N1 influenza is often mediated by the H274Y substitution in the neuraminidase (NA) gene.
  • This substitution can lead to a fitness cost for the virus, which may be overcome by compensatory mutations.
  • Understanding these compensatory mutations is crucial for predicting the evolution of antiviral resistance.

Purpose of the Study:

  • To develop and apply a high-throughput screening method to identify compensatory mutations for the H274Y substitution in the NA gene.
  • To characterize the fitness effects of identified compensatory mutations.
  • To assess the applicability of compensatory mutations across different influenza virus strains.

Main Methods:

  • Utilized error-prone PCR and next-generation sequencing for comprehensive screening of NA genes.
  • Designed a high-throughput screening assay to detect H274Y compensatory mutations.
  • Tested the compensatory effects of identified mutations in both seasonal and pandemic influenza virus strains.

Main Results:

  • Identified four mutations (R194G, E214D, L250P, F239Y) that compensate for the H274Y substitution's fitness defect.
  • R194G and E214D fully compensated, while L250P and F239Y partially compensated for the fitness deficiency.
  • The E214D mutation demonstrated efficacy in both seasonal (A/New Caledonia/20/1999) and pandemic (A/California/04/2009) H1N1 strains.

Conclusions:

  • High-throughput screening effectively identifies compensatory mutations influencing antiviral resistance.
  • Compensatory mutations play a significant role in the evolutionary dynamics of oseltamivir resistance.
  • The developed method offers predictive power for emerging viral mutant species by profiling gene mutation space and fitness.

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