A Cell Based Assay for the Identification of Probe candidate Compounds with Anti- Respiratory Syncytial virus
Assay Rationale and Summary: This functional assay was developed for detection of compounds inhibiting the replication of Respiratory syncytial virus (RSV). The assay measures CPE induced in HEp-2 cells by RSV infection strain Long, using a luminescent-based detection system for signal endpoint.The goal of the proposed project is to find probes specific to the West Nile viruses, therefore it is more ..
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dr. Marintha Heil, Southern Research Institute
Award: 1R03 MH084847-01
Assay Rationale and Summary: This functional assay was developed for detection of compounds inhibiting the replication of Respiratory syncytial virus (RSV). The assay measures CPE induced in HEp-2 cells by RSV infection strain Long, using a luminescent-based detection system for signal endpoint.The goal of the proposed project is to find probes specific to the West Nile viruses, therefore it is important to confirm the antiviral spectrum of the confirmed compounds to other viruses at a later stage. Antiviral compounds show different activity spectra to related or unrelated viruses depending on the specificity and mechanism of action of the compounds. Hence, the assay was implemented as a counter screen to the West Nile Virus anti-viral assay. This would provide the characteristics of the probes.
Probe candidates for anti-WNV compounds were tested in the assay. The assay measures cytopathic effect (CPE) induced in HEp-2 cells by RSV infection using cell viability as the end point.
Cell Culture: HEp-2 cells (ATCC CCL-23, American Tissue Culture Type) were maintained as adherent cell lines in Optimem 1 with 2 mM L-glutamine and 5% fetal bovine serum (FBS) at 37oC in a humidified 5% CO2 atmosphere. Cells were passaged as needed and harvested from flasks using 0.25% trypsin-EDTA.
Assay Media - Preparation of Complete DMEM/F12: 50 mL Pen/Strep/Glutamine (Gibco, Cat No. 10378) was added to four liters of room temperature DMEM/F12 (Sigma, Cat No. D6434) and the pH adjusted to 7.5 using 1N NaOH. The medium was sterile filtered through a 0.2 um filter and 10 mL of HI-FBS was added per 500 mL of media.
Infectious material - Frozen Infected Virus Cell Preparation: Two vials of RSV (strain Long) containing 1 x 107 pfu/mL was thawed using an Eppendorf thermomixer for 13 min at 15 degrees C, with shaking at 350 rpm. Two mL of the virus stock was added to a T-225 flask containing 3.0 x 108 HEp-2 cells in 30 mL Complete DMEM/F12. The cells were incubated for 18 - 20 h at 37 degrees C, 5% C02, 90% relative humidity. The medium was aspirated and the cells washed with 10 mL PBS without Mg2+ or Ca2+. Cells were harvested from flasks using 0.25% trypsin-EDTA. The cells were resuspended in a freezing medium of 95% fetal calf serum and 5% DMSO to a final cell density of 2 x 106 cells/mL. One mL aliquots of this virus infected cell suspension were dispensed to cryovials and cells were rate frozen to -80 degrees C. Frozen infected cells were then transferred to -150 degrees C for long term storage.
Dose Response Compound Preparation: The compounds were tested in a dose response format using a 1:2 serial dilution with the highest concentrations starting at 100uM. A total of ten concentrations were generated for the dose response curve with the lowest concentrations of 0.195uM. Carrier control (DMSO) and compounds were diluted in assay media to 6x and 5uL was dispensed to assay plates. The final DMSO in the assay for all screening concentrations was 0.5%.
Control Drug: The positive control drug for this assay, ribavirin  (No. 196066, MP Biomedicals, Solon, OH) was solubilized in DMSO. It was diluted and added to the assay plates as described for test compounds. Final concentration for ribavirin was 35uM (at 0.5% DMSO).
Preparation of HEp-2 cells: Cells were harvested and resuspended to 80,000 cells per ml in Complete DMEM/F12.
Frozen infected HEp-2 cells: Cells were thawed in a room temperature water bath with gentle agitation. The tube was inverted 5-10. Cells were diluted to 80,000 cells per ml by adding the contents of the vial to 24 ml of cold (4 degrees C) media.
Assay Set up: Twenty five ul of uninfected HEp-2 cells were plated in the cell control wells. Frozen infected cells were combined with uninfected HEp-2 cells at a 1:100 ratio. Twenty five ul of the cell mixture was added to the virus control and compound wells. All cell plating was conducted using a Matrix WellMate and cells were maintained at room temperature with stirring during the plating process. The assay plates were incubated for six days at 37 degrees C, 5% CO2 and 90% relative humidity.
Endpoint Read: Following the six day incubation period, the assay plates were equilibrated to room temperature for 30 min and an equal volume (30 uL) of Cell Titer-Glo reagent (Promega Inc.) was added to each well using a WellMate (Matrix, Hudson, NH) and plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Perkin Elmer EnvisionTM multi-label reader (PerkinElmer, Wellesley, MA) with an integration time of 0.1 s.
Data Analysis: Data was analyzed using ActivityBase software (IDBS, Inc, Guilford, UK). thirty two control wells containing cells only and twenty four wells containing cells and virus were included on each assay plate and used to calculate Z value for each plate and to normalize the data on a per plate basis. The overall Z score for the campaign were >=0.7. Eight ribavirin positive control wells were included on each plate for quality control purposes, but were not used in Z calculations. Results are reported as percent (%) CPE inhibition and were calculated using the following formula: % CPE inhibition = 100*(Test Cmpd - Med Virus)/(Med Cells - Med Virus). To quantify the viral cytopathic effect, IC50s were calculated for each substance using the 4 parameter Levenburg-Marquardt algorithm with the minimum and maximum parameters locked at 0 and 100, respectively.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Outcome: Compounds that showed a maximum inhibition of at least 50% were defined as Active. If the maximum inhibition at any concentration was less than 50%, the compound was considered Inactive. In this particular assay, no compounds showed activity of greater than 50%.
Score: Because of the inherent error in all high throughput screens including the fallacy of over-interpreting single dose data, the following tiered scoring system has been implemented at SRBCSC. Compounds in the single dose primary screen are scored on a scale of 0-40, based on % CPE inhibition. Active compounds in the confirmatory screen are scored based on EC50 results on a tier of 40-80 with compounds failing confirmation scoring 0. Synthesized/Analog active compounds are also scored based on EC50 results and fall into the most reliable tier where actives will be scored from 80-100. Inactive compounds show a score of 0.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)