Infectious Disease Research
Virology
Herpes Virus Research and Development
Primary Drug Screening Assays
- HSV-1 and HSV-2: An established, high capacity anti-HSV drug screening assay is available for the primary analysis of compounds in a 96 well microtiter assay. The basic assay involves infection of Vero cells with HSV-1 or HSV-2 in the presence of test compounds. The ability of the compounds to inhibit HSV-induced cell killing is measured five days post-infection using the tetrazolium dye MTS (CellTiter 96 Aqueous One Solution, Promega). Mitochondrial enzymes of viable (surviving) cells convert MTS to a soluble, colored formazan product. Quantitation of the amount of the formazan product present in each well of the microtiter plate is determined spectrophotometrically at 490/650 nm. The toxicity of the test compounds to host cells is measured concurrently in the same microtiter plate. Data are analyzed using a statistical software program developed at Southern and are provided in both tabular and graphical forms along with determinations of the efficacy (IC50), toxicity (TC50) and selectivity (therapeutic index, TI) of the compounds. Standard Syncytia/Plaque Reduction assays are also available.
- HCMV: Several primary screens are currently available for the evaluation of compounds against HCMV. The first of these is a high capacity drug screening assay similar to the one described for HSV-1 and HSV-2. MRC-5 cells are infected with HCMV in the presence of drugs for a period of seven days. Compound efficacy and toxicity is then measured using MTS (see description of HSV-1 and HSV-2 screen above). Also available is a standard Plaque Reduction assay (moderate to low throughput) as well as an ELISA (moderate throughput).
- VZV: The primary screen currently available for VZV is a standard Plaque Reduction assay. We are currently developing a higher throughput assay based upon the methodologies described above for the HSV-1 and HSV-2 primary screens.
- EBV: We have recently developed a moderate throughput PCR-based assay to identify inhibitors of EBV. This assay is performed using P3HR1 cells, a cell line that is latently infected with EBV. Lytic virus replication spontaneously occurs in approximately 5% of the cell population resulting in the release of virus particles from the cells. P3HR1 cells are incubated with compounds for a period of six days. Supernatant virus is collected and quantitated using TaqMan (PE Applied Biosystems) PCR methodology. Compound toxicity is evaluated in parallel using MTS (see description of HSV-1 and HSV-2 assays above). Higher throughput acute infection assay systems are currently in development.
- HHV-6: A virus induced cytopathic effects (CPE)-inhibition assay is employed to evaluate compounds against HHV-6. Uninfected HSB-2 cells are co-cultured with HHV-6 infected HSB-2 cells in the presence of test compounds. After six days incubation, CPE inhibition is determined by microscopic inspection of the cultures. Upon infection and replication of HHV-6 in HSB-2 cells, the cells increase in size and become light refractory. These changes are readily apparent by microscopic examination of the wells which allows for the quantitation of the numbers of infected cells in each of the cultures. Compound toxicity is evaluated in parallel using MTS (see description of HSV-1 and HSV-2 assays above). An HHV-6 ELISA is also available upon request.
- HHV-7: Screening assay currently under development.
- HHV-8: We have recently developed a moderate throughput PCR-based assay to identify inhibitors of HHV-8. This assay is performed using BCBL-1 cells, a cell line that is latently infected with HHV-8. Lytic virus replication is induced using the phorbol ester TPA, resulting in the release of virus particles from the cells. BCBL-1 cells are incubated with compounds for a period of four days. Supernatant virus is collected and quantitated using TaqMan (PE Applied Biosystems) PCR methodology. Compound toxicity is evaluated in parallel using MTS (see description of HSV-1 and HSV-2 assays above). Higher throughput acute infection assay systems are currently in development.
Primary Drug Screening Assays
Secondary Testing
Mechanism of Action Testing
Herpesvirus Web Resources
