A Cell Based Assay for the Identification of Lead Compounds with Anti-Viral Activity Against West Nile Virus
West Nile virus (WNV) is a mosquito borne infectious agent that causes febrile illness and occasionally encephalitis. Outbreaks had been reported in Africa, Asia, and Europe since 1937. In 1999, the first case of WNV was detected in New York City from which time the virus spread rapidly west resulting in the largest epidemics of neuroinvasive WNV disease ever reported in the US. Currently, more ..
BioActive Compounds: 62
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
West Nile virus (WNV) is a mosquito borne infectious agent that causes febrile illness and occasionally encephalitis. Outbreaks had been reported in Africa, Asia, and Europe since 1937. In 1999, the first case of WNV was detected in New York City from which time the virus spread rapidly west resulting in the largest epidemics of neuroinvasive WNV disease ever reported in the US. Currently, there is no vaccine for human use or antivirals that are available to treat WNV infections, leading to patient care which is mainly supportive. Hence this program will provide chemical probes from which to optimize leads for treatment of this emerging disease.
To screen large compound libraries, a CPE based assay was developed in 384-plate format. The assay measures cytopathic effect (CPE) induced in Vero E6 cells by WNV infection using cell viability as the end point. The screen for inhibitors of CPE caused by WNV infection involves challenging 3,000 cells with WNV virus at a multiplicity of infection (MOI) of 0.1 in the presence of the compounds to be screened. The WNV virus strain NY-99 stock was prepared from Vero E6 cells at the SRI BSL-3 facility. The assay includes both cell and virus controls and cell survival was measured 96 h post virus addition using CellTiterGlo viability reagent (Promega). Percent cell viability was calculated by using mean luminescence values of the virus-infected cells in the presence of compound divided by the uninfected cell control x 100. The percent cell viability was taken as an assessment of antiviral activity. Library compounds were screened at 10 uM.
Cell Culture: Vero E6 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 seven days. For cell plating, cells were detached from flask bottom by using Trypsin-EDTA solution and then re-suspended in a growth media
WNV culture: WNV strain NY-99 was used for screening. The WNV stock was prepared in Vero E6 cells using an initial stock obtained from Dr. Heil. Briefly, Vero E6 cells were grown in two T-150 plates to 90% confluence. After the supernatant was removed from the culture, 2ml of WNV at 10E7 pfu/ml was then added to the culture media. Virus was adsorbed to cells for 2 hours at 37C and then the fresh culture media was replaced. After four days incubation, the supernatant was harvested and the cell debris pelleted by low speed centrifugation. The cell debris was then spun down at 3,000 rpm for 10 minutes, and the supernatant was aliquoted (1ml per tube) and stored at -80C. These virus stocks were titrated in Vero E6 cells using agarose overlay plaque method and the titer was 8.3E7 pfu/ml.
Control and Drug Preparation: Carrier Control consisted of DMSO diluted in assay media to 0.6% and 5 uL was dispensed to both cell and virus control wells of 384 well black clear-bottom tissue culture treated plates. Test compounds were diluted in media to 60 uM with a DMSO concentration of 0.6%. 5 uL was also dispensed to the assay plate. The final DMSO concentration in the assay was 0.1% for the single dose screen and 0.5% for the dose response screen.
Cell Plating: Once controls and compounds had been dispensed to the assay plates, 3,000 cells/well were plated in 15 uL using a Matrix WellMate. Plates were incubated overnight at 37C, 5.0% CO2 and high humidity. Then the plates were transferred to a BSL-3 containment laboratory.
Virus Addition: WNV stock was diluted in the culture media to 60,000 pfu/ml and 10 uL was added to the compound wells and the virus control wells (viral MOI of 0.1 ~ 0.2). 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-3 laboratory. The plates were incubated in an actively humidified incubator with 5.0% CO2 at 37C for 96 h.
Endpoint Read: The assay plates were equilibrated to room temperature for 30 minutes and then an equal volume of CellTiter-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.
Compounds Screened: A single dose screen of the current full set of compounds from the MLSMR was performed at a concentration of 10 uM. Compounds in the single dose assay were evaluated on the basis of % inhibition of the virus compared to cell only control. The 601 most potent compounds were resupplied for a dose-response confirmatory assay. These compounds were tested in triplicate in a ten point stacked-plate dose response in concentrations diluted 1:2 beginning at 20 uM and ending at 0.039 uM.
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 average Z factor for the single dose campaign was 0.81. The percent inhibition of virus induced cytopathic effect (CPE) was calculated as: 100*(Cmpd Lum - Median Virus Ctrl)/(Median Cell Ctrl - Med Virus Ctrl). "Hits" in the single dose screen are those that show inhibition >= 3.42, defined as 3*standard deviation (1.419) above the mean (-0.008) of the % inhibition of the compounds in the full library screen.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Single dose assay: An activity threshold of >3.42 % CPE inhibition was calculated as greater than three standard deviations from the median compound inhibition. Compounds with an activity threshold of <3.42% inhibition were defined as "inactive". Compounds that exhibited >3.42% inhibition were considered for follow up testing. Compounds that exceeded the single dose screen activity threshold, but were not tested in dose response were given the outcome of "inconclusive".
Dose-response assay: Of 320 compounds selected for dose response testing, 311 were available. Compounds that showed >=30% CPE inhibition for at least one concentration were defined as Active. If Inhibition at all doses was <30%, the compound was defined as Inactive.
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 were scored on a scale of 0-40, based on % CPE inhibition, and based on the maximum value where multiple data values were returned. On this scale, 0 corresponds to no inhibitive activity and a score of 40 corresponds to 100% CPE inhibition. Active compounds in the confirmatory screen were scored based on EC50 results on a tier of 40-80 with compounds failing confirmation scoring 0. Resynthesized/analog active compounds screened in later stage probe development will fall into the most reliable tier where actives will be scored from 80-100.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)