A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus)
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 more ..
BioActive Compounds: 2
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
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.
VEEV is a member of the alphavirus family, and there is a possibility that compounds that effectively inhibit VEEV may have broader activity against other alphaviruses. Southern Research's Specialized Biocontainment Screening Center (SRSBSC) has screened compounds that were effective against VEEV in a 96-well microplate assay for broader activity against an additional alphavirus (Chikungunya (CHIKV) virus).
SRSBSC has developed a 96-well cell-based assay that measures CPE induced in Vero 76 cells by CHIKV infection, using a luminescent-based detection system for signal endpoint. The overall goal of this project is to discover novel compounds with broad VEEV and CHIKV inhibition and minimal cytotoxicity in vitro.
Cell Culture: Vero 76 cells obtained from ATCC (CRL-1587) were cultured and maintained in MEM-E (Invitrogen, 10370-088) with 10% Hi-FBS (Invitrogen 16000), 1% Penicillin/Streptomycin/L-glutamine (Invitrogen 10378-024) and 1% HEPES (Invitrogen 15630-080). The cells are maintained at 37C, 5.0% CO2 to 100% confluence being passaged 1:4 every 3-4 days. For cell plating, cells were detached from flask bottom by using Trypsin-EDTA solution and then re-suspended in a growth media. Cells were passaged no more than ten times after being thawed.
Compound Dosing/Plating: No positive control. The compounds were diluted in complete growth medium to 6X concentrated dosing solution which was dispensed into 96-well black clear-bottom tissue culture treated plates (25 uL volume).
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 100 uM and extending to 0.78 uM over a 8-dose 1:2 serial dilution pattern. DMSO and compounds were diluted in assay media to 4x and 25uL was dispensed to assay plates. The final DMSO in the assay for all screening concentrations was 0.25%.
Virus Addition: CHIKV stock was diluted in the culture media to 100 TCID50s/25 uL, and 25 uL were added to each test well.
Cell Plating and virus addition: 6,000 cells/well (120,000 cells/ml) were plated in 50 uL using a Matrix WellMate. All additions were done using a Matrix WellMate housed in a class II Biosafety Cabinet within the BSL-2 laboratory. The plates were incubated overnight in an actively humidified incubator with 5.0% CO2 at 37C for 18h and 95% humidity. Compounds (25 uL) were added after the cells had adhered to the plate, and CHIKV virus was added immediately after compound addition. The plates were incubated for 72 h in an actively humidified incubator with 5.0% CO2 at 37C for 72h and 95% humidity, and then endpoint reagent was added.
Endpoint Read: The assay plates were equilibrated to room temperature for 30 minutes and then 100 uL 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.
Data was analyzed using ActivityBase software (IDBS, Inc, Guilford, UK). Eight control wells containing cells only and eight wells containing cells and virus were included on each assay plate and used to calculate S/B values 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).
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.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: viability reporter:atp content
BAO: assay format: cell based format
BAO: bioassay specification: assay biosafety level: BSL3
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay stage: secondary: alternate confirmatory
BAO: detection technology: luminescence: chemiluminescence
BAO: meta target detail: binding reporter specification: interaction: protein:small molecule
BAO: meta target: biological process target: viral reproduction
BAO: meta target: molecular target: protein target: enzyme: transferase: kinase
BAO: version: 1.4b1090
Experiment Date: 26-Mar-12
Assay Format: Cell-based
Assay Cell Type: VERO 76
* Activity Concentration. ** Test Concentration.
Data Table (Concise)