Titer reduction assay for the selected compounds from the DR study as a confirmatory assay (for 55 compounds)
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 more ..
BioActive Compounds: 12
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.
After the initial primary and confirmatory screen (AID 1621) with a cell based HTS approach, active hit compounds were selected. Their inhibitory activities were confirmed and verified in a titer reduction assay format which measures the difference in viral titer between non-treated and treated group. A compound which inhibits viral replication during the replication process results in less progeny virus compared to control group. The titer reduction assay involves challenging 70~80% confluent Vero cells infected with WNV at a multiplicity of infection (MOI) of 0.01 in the presence of the compounds 48 hours. The WNV strain, NY-99 stock, is the same strain as used in the primary screen. The assay includes measuring the progeny viral titer using TCID50 method. The progeny viral was diluted in 10-fold serial dilution and the suspension transferred to 384-well plates in which Vero E6 were seeded. The plate was developed 96 hr post infection by using CellTiter Glow to evaluate infection in each well and TCID50 was calculated followed by Reed & Muench method.
Compounds Screened:. The 55 compounds were selected through conference between SRSBSC and Burnham Chemistry center resupplied for a dose-response confirmatory assay. These compounds were tested in duplicate in a 4 points dose response in concentrations diluted 1:2 beginning at 10 uM and ending at 1.25 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.
Cell Plating: Vero E6 cells were seeded at 70% confluence in a 12-well plate in a volume of 1mL and incubated for overnight at 37oC with 5% CO2. Plates were incubated overnight at 37C, 5.0% CO2 and high humidity. Then the plates were transferred to a BSL-3 containment laboratory.
Control and Drug Preparation: Carrier Control consisted of DMSO diluted in assay media to 0.2% and 1000uL was dispensed to both cell and virus control wells of 12- well tissue culture treated plates. Test compounds were diluted in media to be at target concentration with a DMSO concentration of 0.2%.
Virus Addition: WNV stock was diluted in the culture media to 50,000 pfu/ml and 100 uL was added to the test wells and the virus control wells (viral MOI of 0.01). Media only (mock virus) was added to the cell control wells. All additions were done sed 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 48 h.
Titration of Progeny viruses
Titer of progeny viruses produced from the cell was measured by TCID50 assay in 384-well plate format with 6 wells per dilution of virus. 5 uL of 10-fold serial dilutions of progeny virus containing medium from respective samples (drug treated or untreated) were transferred to infect fresh Vero E6 cells in a 384-well format. The cell plates were incubated further 96 hours in a CO2 incubator. CellTiter Glow assay was employed to determine whether a well is infected or not. 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. A well showing luminescence signal less than mean of non infected control signal minus 5 times of standard deviation of the control was regarded as positive in infection.
Of 55 compounds selected for dose response testing, 12 compounds that showed more than 10 fold reduction in the progeny titer (<-1 of log reduction) for at least one concentration were defined as Active. If titer reduction at all doses was less than 10 fold, the compound was defined as Inactive (43 compounds).
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In this confirmatory/secondary screen, active compounds were scored on a scale of 41-80 based an inverse linear correlation of the 10 uM (log) titer reduction values. Resynthesized and purified compounds are ranked on the highest score of 81-100. Compounds that did not confirm activity are assigned the score of 0.
Possible artifacts in this assay include, but are not limited to, compounds that precipitate or are cytotoxic.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)