New low-viscosity overlay medium for viral plaque assays.
BACKGROUND
Plaque 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/CLtrade mark as overlay media in the plaque and plaque-inhibition assay of influenza viruses.


RESULTS
Significantly 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.


CONCLUSIONS
Plaque 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.
Matrosovich
Mikhail
M
Institute of Virology, Philipps University, Hans-Meerwein-Str, 3, 35043 Marburg, Germany. M.Matrosovich@gmail.com
Matrosovich
Tatyana
T
Garten
Wolfgang
W
Klenk
Hans-Dieter
HD
D000362:Agar; D000818:Animals; D002460:Cell Line; D002482:Cellulose; D003470:Culture Media / Q000737:chemistry*; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000145:classification / Q000254:growth & development*; D008747:Methylcellulose; D010948:Viral Plaque Assay / Q000379:methods*; D014783:Viscosity
D003470:Culture Media; D000362:Agar; D002482:Cellulose; D008747:Methylcellulose