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Methodology

Neuraminidase inhibitors (NAIs)

Neuraminidase inhibition assay  Robust and reproducible assays of drug susceptibility are needed to generate meaningful data on NAI resistance.  Whilst there are a number of different laboratory methods currently available to measure NAI susceptibility, the enzyme inhibition assay is the simplest and most definitive method.  This method of phenotypic characterisation is used to determine the concentration of the inhibitor that results in a 50% inhibition of enzyme activity (IC50) and the relative antiviral susceptibility of the NAs of different viruses.  To help standardise results, a reference panel of resistant and sensitive viruses is available from the AVG.

Cell culture-based assays (e.g. plaque reduction, yield reduction or EIA) are not currently recommended for reliable assessment of NAI susceptibility due to specific technical problems which limit their usefulness (reviewed by Tisdale, 2000).  A modified Madin-Darby canine kidney (MDCK) cell line, MDCK-SIAT1, which is stably transfected with the human CMP-N-acetylneuraminate:β-galactoside α-2,6-sialyltransferase gene for enhanced expression of Neu5Ac2-6Gal-terminated oligosaccharides (Matrosovich et al., 2003) has given more consistent results (Matrosovich et al., 2006) and is more suitable than the parent cell line.

 

The clinical significance of increased IC50 values or associated mutations in the NA is not known in many cases. Furthermore, there is no clear relationship between the phenotype and genotype of viruses.  To substantiate phenotypic resistance, the virus NA should also be sequenced to identify the mutations responsible for the change in phenotype.  Optimally, sequencing should be performed on original clinical samples.

 

 

M2 inhibitors

Plaque and yield reduction assays can be used to determine the IC50s of the M2 inhibitors (amantadine and rimantadine) and an ELISA assay is useful in identifying resistant variants (Belshe
 et al., 1988).  In view of the few well-established resistance mutations, detection of these mutations is a definitive and widely accepted method for surveillance.

 


Pyrosequencing

Pyrosequencing is a sequence based technology which is useful for analysing short to medium stretches of DNA.  This technique is being more widely used to identify common resistance mutations and analyse polymorphisms indicative of mixed sensitive-resistant virus populations.

 


References:

Belshe, R. B., M. H. Smith, C. B. Hall, R. Betts, and A. J. Hay. 1988. Genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection. J Virol 62:1508-12.

Matrosovich, M., T. Matrosovich, J. Carr, N. A. Roberts, and H. D. Klenk. 2003. Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors. J Virol 77:8418-25.

Matrosovich, M., T. Matrosovich, W. Garten, and H. D. Klenk. 2006. New low-viscosity overlay medium for viral plaque assays. Virol J 3:63.

Tisdale, M. 2000. Monitoring of viral susceptibility: new challenges with the development of influenza NA inhibitors. Rev Med Virol 10:45-55.

 

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