Abstract
BackgroundPlaque assays in cell culture monolayers under solid or semisolid overlay media are commonly used for quantification of viruses and antiviral substances. To overcome the pitfalls of known overlays, we tested suspensions of microcrystalline cellulose Avicel RC/CL™ as overlay media in the plaque and plaque-inhibition assay of influenza viruses.ResultsSignificantly larger plaques were formed under Avicel-containing media, as compared to agar and methylcellulose (MC) overlay media. The plaque size increased with decreasing Avicel concentration, but even very diluted Avicel overlays (0.3%) ensured formation of localized plaques. Due to their low viscosity, Avicel overlays were easier to use than methylcellulose overlays, especially in the 96-well culture plates. Furthermore, Avicel overlay could be applied without prior removal of the virus inoculum thus facilitating the assay and reducing chances of cross-contamination. Using neuraminidase inhibitor oseltamivir carboxylate, we demonstrated applicability of the Avicel-based plaque reduction assay for testing of antiviral substances.ConclusionPlaque assay under Avicel-containing overlay media is easier, faster and more sensitive than assays under agar- and methylcellulose overlays. The assay can be readily performed in a 96-well plate format and seems particularly suitable for high-throughput virus titrations, serological studies and experiments on viral drug sensitivity. It may also facilitate work with highly pathogenic agents performed under hampered conditions of bio-safety labs.
Highlights
Plaque assays in cell culture monolayers under solid or semisolid overlay media are commonly used for quantification of viruses and antiviral substances
We recently developed a focus reduction assay of influenza virus sensitivity to neuraminidase inhibitors in 96well format under liquid medium [2]
Comparison of Avicel and agar overlays We first compared Avicel-based overlay media with standard agar overlay in the plaque assay in 6-well plates
Summary
Plaque assays in cell culture monolayers under solid or semisolid overlay media are commonly used for quantification of viruses and antiviral substances. Plaque assays in cell culture monolayers represent the most common method for quantification of infectious viruses and antiviral substances (see reference 1 for a review) In these assays, each infectious virus particle multiplies under conditions that result in a localized area of infected cells known as plaque. The plaques are revealed either as area of dead/destroyed cells detected by general cellular stains or as area of infected cells detected by immuno-staining Some viruses, such as herpes viruses and poxviruses, may be plaque-assayed under standard liquid culture medium, because direct cell-to-cell spread of these viruses ensures formation of localized plaques. Many viruses, including the influenza virus, do not form localized plaques under liquid medium This is because the progeny of these viruses efficiently detach from infected cells and spread over the cell monolayer with convectional flow of the liquid medium, which results from the temperature gradients in the culture vessels. Large (page number not for citation purposes)
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