Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dong Hoon Chung, University of Louisville
Award: 1 R03 MH087448-01A1

Venezuelan Equine Encephalitis Virus is a mosquito transmitted virus effecting both humans and horses. The last major outbreak in 1995, reported an estimated 70,000 - 100,000 humans infected and a similar number of known horse infections. In humans the symptoms present as fever, headache, and encephalitis. Although the mortality rate is below 1%, the neurological disease is apparent in >/= 14% of patients. There is no FDA approved vaccine or therapeutic and supportive care is limited. Thus, prophylaxis and efficacious treatments are critical to minimizing the impact of the transmissible disease on human and equines.

The US Army has been developing vaccines for VEEV as they appreciate the impact of the disease on soldiers as well as its potential use as a bioweapon. The vaccines, which are comprised of attenuated live virus, are still in the investigational new drug (IND) stage and are only available through the Special Immunization Program at United State Army Medical Research Institute of Infectious Diseases (USAMRIID) for protecting personnel working with the virus. A few other vaccine candidates are in the IND stage, such as formalized killed TC-83 vaccine and the live attenuated V3526 vaccine. Again those vaccines have not been FDA-approved due to lack of efficacy and adverse effects seen during clinical trials.

The assay provider has developed and validated a 384-well cell-based assay that measures CPE induced in Vero 76 by VEEV infection, using a luminescent-based detection system for signal endpoint. The overall goal of this project is to discover novel compounds with anti-VEEV replication efficacy and minimal cytotoxicity in vitro.
This assay is to confirm the antiviral activity of the selected compounds against a wild type strain VEEV. The primary and confirmatory assays (AID; 588727) were done with an attenuated strain, TC-83, therefore a confirmation of anti-VEEV activity against a wild type virus is critical to validate the activity. We used a dose-response format to evaluate the anti-VEEV activity against TrD in Vero 76 cells.

Biosafety and Biosecurity: All experiments with VEEV Trinidad donkey (TrD) strain was done in the Regional Biocontainment Laboratory(RBL) in University of Louisville. All procedures were done in compliance with Select Agent Rules.

Cell Culture: Vero 76 cells obtained from ATCC (CRL-1586) were cultured and maintained in in Dulbecco's Modified Eagle's Medium (DMEM) with 4500 mg/L glucose, 2 mM L-glutamine, and 10% FBS (culture media). The cells are maintained at 37C, 5.0% CO2 to 100% confluence being passaged every three to seven days. For cell plating, cells were detached from flask bottom by using 0.05% Trypsin-EDTA solution and then re-suspended in a growth media.

VEEV culture: VEEV TrD strain was used for screening. The VEEV TrD stock was prepared in Vero 76 cells using an initial stock obtained from World Reference Center for Emerging Viruses and Arboviruses (Dr. Robert Tesh). Briefly, cells were grown in two T-175 flasks to 50% confluence in a culture media. The cells were infected with 1 mL of diluted virus stock (1:10 dilution of the original stock) per T175 for 1.5 hours and then washed, and replenished with 25mL media. The cells were incubated for 2 days in an incubator at 37 oC, 5% CO2 and high humidity. The supernatant was harvested and the cell debris pelleted by centrifuging at 1,000 rpm for 5 minutes at 18 oC. The supernatant was aliquoted (1 ml per tube) and stored at -80C. These virus stocks were titrated in Vero 76 cells using an agarose overlay plaque method and the titers were 2.0XE10 pfu/mL.

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 12.5uM and extending to .09uM over 8 points 1:2 serial dilution pattern. DMSO and compounds were diluted in assay media to 3x and 30 uL was dispensed to assay plates. The final DMSO in the assay for all screening concentrations was 0.25%.


Assay Set up: Forty five ul of Vero 76 cell suspension (267,000 cells/mL) were plated in clear bottom black well 96-well plates and the plates were incubated in an incubator at 37oC in a humidified 5% CO2 atmosphere. The next day, thirty uL of drugging media (0.75% DMSO) were added to the each wells and the plates were incubated at 37oC for an hour. The plates were transferred into a BSL-3 lab in the RBL. Each well received fifteen uL of VEEV TrD virus diluted in Complete media ( 40,000 pfu/mL, final 0.05 MOI). For the cell control wells, Complete media were added instead of virus solution. Drug plating was conducted using a EVO100, 96MAC (Tecan) and virus solution was added using MicroFlo (Biotek). The assay plates were incubated for two days at 37 degrees C, 5% CO2 and 90% relative humidity.
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 Synergy4 HT multi-label reader.

Data Analysis: Eight control wells containing cells only and eight 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. 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.

Of the compounds tested, those that showed at least 50% inhibition at any tested dose were considered active.

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 the confirmatory dose response screen, active compounds were scored on a scale of 41-80 based on the IC50 result while compounds that did not confirm as actives were given the score of 0. A scale of 81-100 based on the IC50 of active compounds is used for purified/synthesized compounds to indicate a high level of confidence in the results. Inactive compounds are given a score of 0.

Phenotypic Screen: Yes

Multiplexing: No

BSL: BSL2

Screening Concentration Range Max: 12.5

Screening Concentration Range Min: 0.098

Assay Format: Cell-based

Assay Type: Viability/Toxicity

Assay Method: End-point

Assay Detection: Bio-luminescence

Used for Hit Validation?: No

Used during SAR?: Yes

Grant Number: 1 R03 MH087448-01A1