isirv

Phenotypic assays, such as the plaque reduction assay, can be used to determine amantadine and rimantadine IC₅₀ values and to determine neuraminidase inhibitor sensitivity.

In the plaque assay, each infectious virus particle multiplies under conditions that result in a localized area of infected cells or ‘plaque’. The plaques are revealed either as areas of dead/destroyed cells detected by general cellular stains or as areas of infected cells detected by immuno-staining. The pdf file below was presented at a ViRgil training course in October 2006 and describes the plaque reduction assay methodology and guidance on interpretation of results.

Micro-plaque assay of influenza virus sensitivity to NAI  

Protocol for plaque reduction assay

A micro-plaque assay methodology for quantifying neuraminidase inhibitor resistance has been developed by Matrosovich et al (2006) which overcomes some of the difficulties of conventional overlay methods such as agar, which cannot be used in 96-well plates. The method utilises Avicel™ (microcrystalline water insoluble cellulose) which produces larger and more distinct plaques. This has the advantage of increasing the sensitivity of the assay and reducing the incubation time. A simple, inexpensive method for quantitfying the results has recently been developed by Sullivan et al (2012).

Micro-plaque assay protocol  

(Provided by kind permission of Dr M Matrosovich, Philipps University, Marburg)

 
References:

Matrosovich M, Matrosovich T, Garten W, Klenk HD (2006). New low-viscosity overlay medium for viral plaque assays. Virol J. Aug 31;3:63.

Sullivan, K., J. Kloess, C. Qian, D. Bell, A. Hay, Y. P. Lin, and Y. Gu. (2012). High throughput virus plaque quantitation using a flatbed scanner. J Virol Methods 179:81-9.