Laboratory methodologies for testing the antiviral susceptibility of influenza viruses:
Neuraminidase inhibitor (NAI)

November 2012

An overview of amino acid substitutions in influenza neuraminidase associated with resistance or reduced susceptibility to NAIs

The neuraminidase amino acid substitutions that have been detected in respiratory specimens from clinical settings and associated with reduced neuraminidase inhibitor (NAI) susceptibility are listed in the table below. The table is divided into two sections.

The first section lists substitutions that are considered clinically relevant, due to their frequency of occurrence and the availability of clinical data to demonstrate a reduced treatment efficacy, making it cost-effective for screening using a SNP-based assay.

The second section lists substitutions observed infrequently and causing reduced susceptibility in vitro but with clinical significance being less clear. These substitutions are best screened for using Sanger sequencing of full-length NA genes, ideally following phenotypic testing of virus isolates. Viruses carrying these less frequent substitutions or displaying reduced susceptibility/resistance by phenotypic assays, should be forwarded to a WHO Collaborating Centre for Reference and Research on Influenza (WHO CC) for further analysis.

Currently, SNP-based assays should be used to screen for H275Y substitution in N1 neuraminidases, i.e. A(H1N1)pdm09 and A(H5N1) viruses infecting humans. Mutations causing this substitution are the only ones consistently associated with clinical resistance that occurs with sufficient frequency to make SNP-based testing worthwhile.

In general National Influenza Centres in GISRS (NICs) are not recommended to set up SNP-based assays for screening of rare substitutions. The WHO Collaborating Centres in GISRS will continue to monitor and analyse the frequencies of these rare substitutions and their effects on NAI susceptibility. If a particular variant becomes more prevalent, additional substitution(s) will be recommended to NICs as the target(s) for SNP-based assays. Relevant protocols will be made available on the WHO website.

The approximate fold increases in IC50 (equating to reductions in susceptibility) relative to sensitive wild-type viruses for three licensed NAIs are given in the table below. Published data are derived from both fluorescent-based and chemiluminescence-based NAI-susceptibility assays. The approximate fold increases in IC50 reported here serve as a guide only.

Substitutions in influenza neuraminidases associated with resistance or reduced susceptibility to NAIs