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Comparative Study
. 2011 Apr;9(2):184-90.
doi: 10.1089/adt.2010.0298. Epub 2010 Nov 4.

A High-Throughput Screening Strategy to Overcome Virus Instability

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Free PMC article
Comparative Study

A High-Throughput Screening Strategy to Overcome Virus Instability

Lynn Rasmussen et al. Assay Drug Dev Technol. .
Free PMC article

Abstract

Respiratory syncytial virus (RSV) is a widely distributed pathogen that causes severe disease in children, the elderly, and immunocompromised individuals. Both vaccine development and drug discovery have been hampered by the inherent instability of the virus itself. Drug discovery efforts have had limited success due, at least in part, to the lack of an antiviral assay robust enough for high-throughput screening. Instability of the purified virus has long been recognized as a problem in RSV research and has been a major hurdle to producing a virus-based screening assay. Using frozen RSV-infected cells as the source of infectious material, we have overcome the problem of virus instability and validated a cell-based high-throughput screening assay to screen for inhibitors of RSV-induced cytopathic effect. The assay was validated with 1,280 compounds identified as potentially active against RSV (Long strain) in a virus-based screen. To date over 300,000 compounds have been screened over several months with minimal variability in cell or virus controls. Long-term assay stability studies are still in progress.

Figures

Fig. 1.
Fig. 1.
Plot of the number of virus-positive wells out of 192 wells tested versus dilution for TCID50 determination. TCID50 is the amount of virus that produces a CPE in 50% of the wells infected. Excel XLfit formula 504 was used to plot the data and calculate the dilution at which 96 positive wells (50% of the 192 tested) would occur. Log10 of this dilution is defined as the TCID50. TCID50 = 3.34. CPE, cytopathic effect; TCID50, tissue culture infectious dose 50%.
Fig. 2.
Fig. 2.
Seven individual vials of respiratory syncytial virus stock were evaluated for infectivity. Two rows correspond to one vial of virus stock. Cell controls with no virus are in the bottom two rows.
Fig. 3.
Fig. 3.
Comparison of assay formats by EC50 values derived for Ribavirin. Data are plotted as % Inhibition of CPE versus log [μM]. (A) EC50 is 36.7 ± 1.7 μM for Ribavirin using the FIC assay. (B) EC50 31.7 ± 1.8 μM for Ribavirin using the virus assay. EC50 values and standard deviations were calculated based on % Inhibition of CPE at 10 concentrations, n = 5, using the four-parameter Levenburg-Marquardt algorithm with the minimum and maximum parameters locked at 0 and 100, respectively. EC50, effective concentration 50%; FIC, frozen infected cell.
Fig. 4.
Fig. 4.
Mean of cell and virus controls from 40 compound plates run in both assay formats for comparison. Data are plotted as luminescence values/Z′ versus plate number. (A) shows the plate controls and Z′ values for the FIC assay. (B) shows plate controls and Z′ values for the virus assay. Error bars represent standard deviation calculated from n = 32 values per plate for each cell and virus controls.
Fig. 5.
Fig. 5.
Dose–response curves of selected compounds from both assay formats. Assay plates were prepared from a single compound dilution plate on the same day, using the same uninfected cells and the same medium to directly compare assay performance and minimize variables. Single replicate 10 point dose–response was done for each compound in both assay formats. Curves in (A) were derived from the FIC-based assay and curves in (B) were from the virus assay. EC50 values and standard deviations were calculated based on % Inhibition of CPE at 10 concentrations, n = 1, for each substance using the four parameter Levenburg-Marquardt algorithm with the minimum and maximum parameters locked at 0 and 100, respectively.
Fig. 6.
Fig. 6.
Correlation analysis of the respiratory syncytial virus assay done using FICs versus virus as the source of infectious material. A Pearson's correlation of 0.84 was calculated from this EC50 data for 92 compounds.

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