A Cell Based Assay for the Identification of Probe candidate Compounds with Anti- Venezuelan Equine Encephalitis virus.
This functional assay was developed for detection of compounds inhibiting the replication of Venezuelan Equine Encephalitis virus(VEEV), strain V3526-luc. The virus expresses a functional luciferase while it is actively replication in host cells and the signal is proportional to the extent of the virus replication. ..more
BioActive Compounds: 29
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
This functional assay was developed for detection of compounds inhibiting the replication of Venezuelan Equine Encephalitis virus(VEEV), strain V3526-luc. The virus expresses a functional luciferase while it is actively replication in host cells and the signal is proportional to the extent of the virus replication.
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 probe
candidates for anti-WNV compounds. The assay measures a luciferase activity expressed in Vero 76 cells by V3526-luc virus infection using luminescence as the end point. A luciferase gene is encoded in the nsP3 gene in-frame so the activity is proportional to virus replication in the system. The screen for inhibitors of a luciferase expression in V3526-luc infected cells involves challenging 4,500 cells with V3526-luc virus at a multiplicity of infection (MOI) of 0.06 in the presence of the compounds to be screened. The V3526-luc virus was prepared from BHK cells transfected with genomic infectious RNA at the SRI BSL-2 facility. The assay includes both cell and virus controls. The activity was measured 24 h post virus addition using BrightGlo luciferase reagent (Promega). Percent luciferase activity was calculated by using mean luminescence values of the virus-infected cells in the presence of compound divided by the virus-infected cell control x 100. The percent cell viability was taken as an assessment of antiviral activity.
Cell Culture: Vero 76 cells were cultured in Eagles modified MEM (E-MEM) with 2 mM L-glutamine, 10% FBS and 100 U of penicillin, 100 ug of streptomycin per mL (culture media). The cells are maintained at 37C, 5.0% CO2 to 100% confluence being passaged every four days. For cell plating, cells were detached from flask bottom by using Trypsin-EDTA solution and then re-suspended in a growth media
VEEV culture: VEEV strain,V3526-luc was used for screening. The V3526-luc stock was prepared in BHK-21 cells. The plasmid which encodes whole genome of V3526 and luciferase gene has been created by Dr. Brett Beitzel at USAMRIID. The stock virus was prepared by a process of rescuing virus from infectious RNAs. The infectious genomic RNA was generated from the linearized plasmid DNA, pV3526-luc, in conjunction with 7mG cap analogue by using in vitro RNA transcription. The RNA was then delivered into BHK C-21 cells by electrophoration and the cells were incubated at 37 degrees C with 5% CO2 until cytopathic effect was seen (4 days). Culture media was then collected and clarified by centrifugation. 1.5 mL aliquots of the media were stored at -80 degrees C as stock virus.
Dose Response Compound Preparation and Controls: The compounds were tested in a dose response format using a 1:2 serial dilution with the highest concentrations starting at 133.3uM and 66.6uM depending on the stock concentrations available. A total of ten concentrations were generated for the dose response curve with the lowest concentrations of 0.26uM and 0.13uM, respectively. 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 0.33%
Cell Plating: Once controls and compounds had been dispensed to the assay plates, 4,500 cells/well were plated in 15 uL using a Matrix WellMate. Plates were incubated overnight at 37oC, 5.0% CO2 and high humidity.
Virus Addition: V3526-luc was diluted in the culture media to 30000 TCID50/ml and 10 uL was added to the compound wells and the virus control wells (0.06 TCID50/ cell). Media only (mock virus) was added to the cell control wells. All additions were done using a Matrix WellMate housed in a class II Biosafety Cabinet within the BSL-2 laboratory. The plates were incubated in an actively humidified incubator with 5.0% CO2 at 37C for 24 hrs.
Endpoint Read: The assay plates were equilibrated to room temperature for 30 minutes and then an equal volume of Bright-Glo reagent (Promega Inc.) was added to each well. Plates were incubated for 10 min at room temperature and luminescence was measured using a Perkin Elmer Envision multi-label reader with an integration time of 0.1 s. This step was also performed within the BSL-3 facility.
Data Analysis: Thirty two control wells containing cells only (Cell control), twenty four wells containing cells and virus (Virus control) 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. And also eight wells containing cells, virus and control drug, Mycophenolic Acid at 5 uM were included on each assay plate. The percent inhibition of virus induced cytopathic effect (CPE) was calculated as: 100*(Cmpd Lum - Median Virus Ctrl)/(Median Cell Ctrl - Med Virus Ctrl). Percent inhibition values were used to calculate EC50 values for the compounds by using4 parameter regression curve analysis with ActivityBase.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
If the maximum inhibition at any concentration was less than 30%, the compound was considered Inactive. Compounds that showed a maximum inhibition of at least 30% were defined as Active and has IC50 values calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0.
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)