A microtiter virus yield reduction assay for the evaluation of antiviral compounds against human cytomegalovirus and herpes simplex virus

Abstract

Although the virus yield reduction assay is a powerful technique for evaluating the efficacy of antiviral compounds, it is not routinely utilized due to its labor-intensive nature. This procedure was modified, developed, thereby reducing greatly the time and effort required to perform yield reduction assays. Monolayer cultures of mammalian cells were grown in 96-well microtiter tissue culture plates and infected with virus. Test compounds were added and serially diluted directly with the plates. Following a cycle of virus replication, culture lysates were made and serially diluted in a separate set of uninfected cultures grown in microtiter plates. The cultures were incubated, plaques were enumerated in wells containing 5 to 20 plaques, and virus titers were calculated. To illustrate the use of the assay the known antiviral drugs acyclovir and ganciclovir were evaluated using this procedure. Ninety percent inhibitory concentrations for the respective drugs were 3 microM and 0.7 microM against herpes simplex virus type 1 and 60 microM and 1 microM against human cytomegalovirus.

Resistance of Human Cytomegalovirus to the Benzimidazole l -Ribonucleoside Maribavir Maps to UL27

“…Experiments were carried out in duplicate for all time points. After all cultures were collected, they were thawed, and titers were determined in duplicate across 96-well plates (30). Seven days later they were stained with crystal violet, and titers were calculated.…”

“…After virus adsorption, medium was replaced with fresh medium containing test compounds in eight 1:3 dilutions from a starting drug concentration of 100 M. Plates were incubated for 7 days and subjected to one cycle of freezing and thawing, and titers were determined by transferring 100-l aliquots from each of the wells to a fresh 96-well monolayer culture of HFF cells followed by serial dilution across the plate. Cultures were incubated for 7 days, cells were stained, and the number of plaques was determined (30,36). Dose-response relationships were used to quantify drug effects.…”

“…Plasmid pBRep-Cre was constructed by W. Brune (19), University of Wuerzburg, Wuerzburg, Germany. Plasmid pCGN71 was constructed in the laboratory of T. Shenk, Princeton University, Princeton, N.J. Stocks of HCMV were prepared by infecting HFF cells at a MOI of 0.01 PFU per cell, and stock viral titers were determined using monolayer cultures of HFF cells as described previously (30,36).…”

Serine Peptide Phosphoester Prodrugs of Cyclic Cidofovir: Synthesis, Transport, and Antiviral Activity

“…39 All viruses were titered using monolayer cultures of HFF cells. 40 Following incubation of three days (poxviruses) or 10-12 days (HCMV), cells were fixed and stained with 0.1% crystal violet in 20% methanol and macroscopic plaques (poxviruses) or microscopic plaques (HCMV) enumerated.…”

“…In KB cells, the effect of compounds during two population doublings of KB cells was determined by crystal violet staining and spectrophotometric quantization of dye eluted from stained cells as described earlier. 40 Briefly, 96-well cluster dishes were plated with KB cells at 5000 cells per well. After overnight incubation at 37 °C, test compound was added in triplicate at eight concentrations.…”