Protocol

Influenza Virus

Volume 865 of the series Methods in Molecular Biology pp 95-113

Date:

The Chemiluminescent Neuraminidase Inhibition Assay: A Functional Method for Detection of Influenza Virus Resistance to the Neuraminidase Inhibitors

  • Margaret Okomo-AdhiamboAffiliated withInfluenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and PreventionWHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza
  • , Aeron C. HurtAffiliated withVictorian Infectious Diseases Reference Laboratory, WHO Collaborating Centre for Reference and Research on Influenza
  • , Larisa V. GubarevaAffiliated withInfluenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and PreventionWHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza Email author 

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Abstract

Neuraminidase inhibitors (NAIs) represent a newer class of anti-influenza drugs. Widespread natural or acquired resistance to NAIs is a major public health concern as it limits pharmaceutical options available for managing seasonal and pandemic influenza virus infections. Molecular-based methods, such as pyrosequencing, sequencing, and PCR are rapid techniques for detecting known genetic markers of resistance, but they are unable to identify novel mutations that may confer resistance, or subtle differences in the susceptibility of viruses to the NAIs. This chapter describes the chemiluminescent neuraminidase (NA) inhibition (NI) assay, a functional method used for assessing influenza virus susceptibility to NAIs. The assay generates IC50 values (drug concentration needed to reduce the NA enzymatic activity by 50%) which are determined by curve-fitting analysis. Test viruses showing elevated IC50 values relative to those of NAI-sensitive reference viruses of the same antigenic type and subtype are further analyzed by pyrosequencing or conventional sequencing to identify known markers of NAI resistance or new changes in the NA. The criteria for NAI resistance are currently not well defined and tend to vary by laboratory and NI assay, therefore harmonization of NI assay conditions and interpretation of results across surveillance laboratories is necessary to improve the NAI susceptibility testing and analysis.

Key words

Neuraminidase inhibitor Oseltamivir Zanamivir Chemiluminescence Inhibition Sialidase