A quantitative comet assay: imaging and analysis of virus plaques formed with a liquid overlay.
Although the plaque assay defines a "gold-standard" for measuring virus infectivity, its reliance on plaque counting limits its sensitivity. When the assay is performed with a liquid overlay, instead of agar overlay, spontaneous flows can promote a uni-directional spread of infection, creating elongated regions of cytopathology that resemble comets. As a model system comet and plaque cultures of vesicular stomatitis virus (VSV) on baby hamster kidney (BHK-21) cells were compared. Host-cell monolayers were infected with VSV particles, incubated 15 h in the presence of liquid or agar overlays and stained. VSV formed significantly larger comets than plaques, consistent with a mechanism of flow-enhanced spread. When antiviral drug (5-fluorouracil) was incorporated into the liquid overlay, comet sizes were reduced in a dose-dependent manner. Images of infected monolayers, acquired using a simple digital scanner, enabled a quantification of the inhibitory effect of the drug on infectivity. The resulting measure of drug susceptibility was found to be 18-fold more sensitive than the IC(50) measure attained by the traditional plaque-reduction assay. This quantitative comet assay has the potential to similarly enhance the sensitivity of infection measures for other plaque-forming viruses.
Zhu
Ying
Y
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706-1607, USA. yingzhu@wisc.edu
Yin
John
J
D000818:Animals; D002460:Cell Line; D020552:Comet Assay / Q000379:methods*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005472:Fluorouracil / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects* / Q000254:growth & development; D010948:Viral Plaque Assay / Q000379:methods*
D005472:Fluorouracil