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

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.


  • 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.


  • 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.


  • 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


Sanger Sequencing - NA

Standard Operating Procedures (WHO CC, CDC Atlanta)

Pyrosequencing - A(H1N1)pdm09 NA

Standard Operating Procedures (WHO CC, CDC Atlanta)

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

Standard Operating Procedures (WHO CC, NIID Tokyo)