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

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A summary of amino acid substitutions in the influenza neuraminidase associated with resistance or reduced susceptibility to NAIs

The amino acid substitutions and deletions in neuraminidase (NA) and their effects on NA enzyme activity inhibition by NAIs are shown in Table 1. These NA substitutions and deletions have been detected in respiratory specimens from clinical settings, virus isolates, or have been introduced into recombinant NA.

The amino acid substitution H275Y, shown in bold, is considered clinically relevant due to the frequency of occurrence and the availability of clinical data demonstrating reduced treatment efficacy against viruses with this NA amino acid substitution.

The other substitutions and deletions listed have been observed infrequently and caused reduced susceptibility in vitro, but clinical significance is less clear. These substitutions/deletions are best screened by sequencing the full-length NA gene using Sanger or Next Generation Sequencing (NGS, deep sequencing), ideally following phenotypic testing of virus isolates. Viruses carrying these less frequent substitutions/deletions 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.

The acquisition of resistance/reduced susceptibility to NAIs by avian influenza viruses due to amino acid substitutions in NA is of serious public health concern. Monitoring the susceptibility of these viruses to currently available NAIs is an important part of surveillance studies and an informative aspect of risk assessment for pandemic preparedness. The NA amino acid substitutions that have been associated with reduced NAIs susceptibility in avian influenza virus samples and/or in experimental recombinant viruses are listed in Table 2. NA amino acid substitutions detected in influenza A(H5N1) and A(H7N9) viruses are presented in both Tables 1 and 2.

Currently, SNP-based assays should be used to screen for H275Y substitution in N1NAs, e.g., 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 analyze the frequencies of these rare substitutions and their effects on NAIs 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 the licensed NAIs are given in the table below. Published data are derived from both fluorescent- and chemiluminescent-based NAI-susceptibility assays. The approximate fold increases in IC50 reported here serve as a guide only.

Genotyping: molecular-based assays

Genotypic methods for detecting amino acid substitutions associated with influenza antiviral resistance

The advantages, disadvantages and challenges associated with the use of genotypic methods for detecting amino acid substitution associated with influenza antiviral resistance or reduced susceptibility are listed below.

Advantages

  • Molecular methods are relatively simple and rapid.
  • Methods can be implemented using existing molecular technologies in the laboratory.
  • The choice of a method or a combination of methods will depend on the testing capabilities of the laboratory.
  • Methods can be used directly with clinical material.

Disadvantages

  • Interpretation of mutations is difficult without phenotypic information.
    • Lack of known mutations associated with antiviral resistance is not a guarantee of susceptibility to a particular drug.
    • Interpretation of the significance of novel mutations is difficult.

Challenges

  • Understanding the relationship between virus drug susceptibility phenotype and genotype.
  • Determining what proportion, resistant vs sensitive, of a mixed virus population is required to confer resistance.

A variety of methods are currently available for the monitoring of substitutions associated with antiviral resistance.

The current laboratory protocols for Sanger sequencing, pyrosequencing and allelic discrimination by real-time RT-PCR are listed below.

SNP-based assays for the detection of neuraminidase inhibitor resistance are currently only used for the detection of H275Y in A(H1N1)pdm09 viruses. Therefore, it is essential to subtype influenza viruses prior to using the H275Y detection protocols listed here.

Should additional information or reference viruses be required, please contact the authors of the protocols, or any of the WHO Collaborating Centres within GISRS.

Laboratory protocols for the genotypic detection of the H275Y substitution

Assays

Sanger Sequencing - NA

Standard Operating Procedures (WHO CC, CDC Atlanta)

Primers and probes are available and described in the protocol.

Pyrosequencing - A(H1N1)pdm09 NA

Standard Operating Procedures (WHO CC, CDC Atlanta)

Primers and probes are available and described in the protocols.

Real time RT-PCR allelic discrimination - A(H1N1)pdm09 NA

Standard Operating Procedures (WHO CC, NIID Tokyo) updated July 2023

Primers and probes are available and described in the protocol.

Phenotyping: neuraminidase inhibition assays

Two types of neuraminidase (NA) inhibition assays are commonly used for determining influenza susceptibility to the NA inhibitor (NAI) antivirals: fluorescence-based (FL) and chemiluminescence-based (CL) assays.

Both types of assays have advantages and disadvantages (see summary below). For practical reasons, the FL assays are more cost-effective and applicable, in particular for National Influenza Centres (NICs) in the process of establishing NA inhibition assays.

NICs are encouraged to contact any of the WHO Collaborating Centres in GISRS when establishing NA inhibition assays, to ensure continuous support, including provision/updates of protocols, control viruses, etc.

Protocols available for NA inhibition FL assays are:

  • an ‘in-house’ FL method (see the WHO Manual for the laboratory diagnosis and virological surveillance of influenza);
  • commercially available kits (see summary below).

    • Software for calculating IC50 values from NA inhibition assay data

    Various software programs are available for the calculation of IC50 values from NA enzyme inhibition assay data. Variations in IC50 values may occur as a result of differences in the software programs.

    • GraphPad – commercially available software: http://www.graphpad.com
    • GraFit – commercially available software: http://www.erithacus.com/grafit
    • JASPR – freely available software developed by the Centers for Disease Control and Prevention (CDC), Atlanta, United States of America. To request the software please contact FLUANTIVIRAL@CDC.GOV or contact Dr. Larisa Gubareva (lqg3@cdc.gov) for any further assistance.
    • PHE point-to-point – freely available analysis developed by the Health Protection Agency, London, United Kingdom of Great Britain and Northern Ireland. Please contact Dr. Angie Lackenby (Angie.Lackenby@PHE.gov.uk) to request the template and protocol.

Laboratory methodologies for testing the antiviral susceptibility of influenza viruses: Reference viruses for validation and controls

Reference viruses for NAI susceptibility assays

Reference viruses are necessary for the quality control of phenotyping neuraminidase (NA) enzyme inhibition assays and genotyping molecular-based assays in determining the NAI susceptibility of viruses. Although the same reference influenza viruses can be used for both methodologies, there are certain differences that need to be considered.

Phenotyping neuraminidase enzyme inhibition assays

To assist in the establishment of neuraminidase inhibition assays and standardization of IC50 values, the isirv-Antiviral Group (the isirv-AVG, formerly NISN) has assembled a panel of eight reference viruses. This panel includes type A (subtypes H1N1, H1N1pdm09 and H3N2) and type B viruses.

The reference virus panels are available without a material transfer agreement (MTA) and are free of charge, although shipping expenses must be covered by the recipient laboratory. All requests should be submitted through the isirv-AVG web site:

Other reference viruses can be requested from WHO Collaborating Centres in GISRS.

Genotyping molecular-based assays

Reference virus variants for genotyping molecular-based assays are not currently available. To facilitate the validation of genotypic assays used for the detection of the H275Y substitution in A(H1N1)pdm09 viruses, WHO is exploring the possibility of including virus variants in the WHO GISRS External Quality Assessment Project for virus detection by PCR.