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BioAssay: AID 624286

Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (3)

Venezuelan equine encephalitis virus is one of the Alphavirus species in the family of Togaviridae characterized by a non-segmented, positive sense strand RNA virus (Griffin, D. E. 2001. Alphaviruses, p. 917-962. In D. Knipe and P. M. Howley (ed.), Fields virology, vol. 4. Lippincott, Williams, and Wilkins, Philadelphia, PA.). VEEV, endemic to North, Central and South America, is an example of more ..
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Active(6)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(8)
 
 
Active(6)
 
 
Inactive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 624286
Data Source: Southern Research Specialized Biocontainment Screening Center (VEE_VTR_03)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-06-11
Hold-until Date: 2013-05-24
Modify Date: 2013-05-24

Data Table ( Complete ):           Active    All
BioActive Compounds: 6
Depositor Specified Assays
Show more
AIDNameTypeComment
588727A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83confirmatoryPrimary and Confirmatory Screen (TC-83)
602241A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (2)confirmatoryConfirmatory Screen (TC-83)
602455A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (3)confirmatoryConfirmatory Screen (TC-83)
624063A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV TC-83 (4)confirmatoryConfirmatory Screen (TC-83)
602242A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526confirmatoryConfirmatory Screen (v3586)
602484A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (1)confirmatoryconfirmatory Trinidad Donkey
588719Vero 76 Cytoxicity Assay for VEEV CompoundsconfirmatoryCytotoxicity (Vero76) Counter Screen
602240Vero 76 Cytoxicity Assay for VEEV Compounds (2)confirmatoryCytotoxicity (Vero76) Counter Screen
602439Vero 76 Cytoxicity Assay for VEEV Compounds (3)confirmatoryCytotoxicity (Vero76) Counter Screen
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)confirmatoryCytotoxicity (Vero 76) counter screen
602307Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain)otherVirus titer reduction assay TC-83
602411Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (2) Trinidad donkey strainotherVirus titer reduction assay (WT-Trinidad Donkey)
602470A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) AssayconfirmatoryRespiratory Syncytial Virus (RSV) Counter Screen
602483HEp2 Cytoxicity Assay for VEEV Compounds (1)confirmatoryCytotoxicity (Hep-2) counter screen
623935A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus)confirmatoryChikungunya Virus Counter Screen
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)confirmatoryChikungunya VirusToxicity Screen
588723A Cell-based HTS to discover molecules that inhibit VEEV, encephalitic alphavirus - Project SummarysummarySummary AID
624450Vero 76 Cytoxicity Assay for VEEV Compounds (7)confirmatory
651735Vero 76 Cytoxicity Assay for VEEV Compounds (8)confirmatory
651884A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526 (2)confirmatory
651934A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (3)confirmatory
624449A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (6)confirmatory
651874A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (2)confirmatory
651932A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) Assay (2)confirmatory
651578Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (4)other
651734A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (7)confirmatory
651738A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (2)confirmatory
651883Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (strain Trinidad donkey) (03)other
651886Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (5)other
651917A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (8)confirmatory
651930Vero 76 Cytoxicity Assay for VEEV Compounds (9)confirmatory
Description:
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

Introduction:
Venezuelan equine encephalitis virus is one of the Alphavirus species in the family of Togaviridae characterized by a non-segmented, positive sense strand RNA virus (Griffin, D. E. 2001. Alphaviruses, p. 917-962. In D. Knipe and P. M. Howley (ed.), Fields virology, vol. 4. Lippincott, Williams, and Wilkins, Philadelphia, PA.). VEEV, endemic to North, Central and South America, is an example of an arbovirus passing from a misquito vector through rodent hosts and with epizootic subtypes causing disease in equines and humans. While there are various subtypes, human infection tends to be acute resulting in fever, headache, lymphopenia, myalgia and malaise but may increase to severe encephalitis (Bale, J.F., Jr. 1993. Viral encephalitis. Med Clin. North Am. 77:25-42.). Fatalities occur in ~1% of the (Steele, K., et al. 2007. Alphavirus encephalitides, p. 241-270. In Z. F. Dembek (ed.), Medical aspects of biological warfare. Borden Institute (U.S. Army Walter Reed), Washington, D.C.). Although recorded natural epidemics are rare, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases have classified it as a category B priority biodefense agent due to the ease at which it may be cultured, manipulated and aerosolized.
In the United States, there are no licensed human vaccines for VEEV although there is an Investigational New Drug (IND) vaccine given to at risk populations. TC-83 is a live attenuated vaccine that provides long-lasting immunity and protection. However ~25% of individuals vaccinated suffer from adverse events and ~20% of those vaccinated do not produce an immune response. C-84, derived from TC-83, is formalin-inactivated VEEV vaccine that does not produce adverse reactions but conversely does not provide protection against all VEE challenges and requires frequent boosting. Currently there is no treatment for infection with VEE beyond supportive care, so there is an intense need for potential therapeutics.
Protocol
Biosafety and Biosecurity: All experiments with TC-83 strain was done in the Regional Biocontainment Laboratory in University of Louisville. All procedures were done in compliance with Select Agent Rules.

Cell Culture: Vero 76 cells (ATCC; CRL1586) were cultured 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 strain TC-83 was used for screening. The VEEV TC83 stock was prepared in BHK cells using an initial stock obtained from USAMRIID. The 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 1mL 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 37C, 5% CO2 and high humidity. The supernatant was harvested and the cell debris pelleted by centrifuging at 1,000 rpm for 5 minutes at 18C. 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 1.2XE09 pfu/ml.

Cell Plating: Vero 76cells were seeded at 70% confluence in a 12-well plate in a volume of 1mL and incubated for overnight at 37C with 5% CO2 and high humidity.

Virus Addition: The cells were infected with virus by adsorption for an hour. Cell culture media in the 12-well plates were removed completely and the cells were infected with either mock virus (media only) or VEEV. VEEV stock was diluted in the culture media to 9XE03 pfu/ml and 200uL was added to the test wells and the virus control wells (final MOI of 0.1). The plates were incubated in an actively humidified incubator with 5.0% CO2 at 37C for one hour. During the incubation, plates were gently rocked every 20 min. to ensure the coverage of the cells with virus. After the adsorption, the cells were rinsed 1mL of PBS per well and then replenished with the media containing testing articles. All additions were performed in a class II Biosafety Cabinet. The plates were incubated in an actively humidified incubator with 5.0% CO2 at 37C for 48 h.

Control and Drug Preparation: Carrier Control consisted of DMSO diluted in assay media to 0.25% 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 0.25%.

Titration of Progeny viruses (Mini plaque assay)
Titer of progeny viruses produced from the cell was measured by a mini plaque assay in 96-well plate format. Fresh Vero 76 cells were seeded and grown in 96-well plates overnight. The cell culture supernatants from the 96-well plates were emptied and the cells were infected with 25uL of 10-fold serial dilutions of progeny virus containing medium from respective samples (drug treated or untreated). The plates were incubated for one hour in an incubator at 37C, 5% CO2. The cells were rinsed with 100uL per well of PBS once and then replenished with DMEM with 0.75% methyl-cellulose and 10% FBS. The cell plates were incubated at 37C, 5% CO2, and high humidity for an additional three days. Crystal violet solution with 4% paraformaldehyde was used to developed plaques in the wells. The assay plates were equilibrated to room temperature for 10 minutes and then an equal volume of the crystal violet solution was added to each well. The plates were incubated for 60 min at room temperature and stained one more time. After a wash the plates with water, the number of plaques in each wells were determined by a visual counting. Virus titers were calculated by: No. of plaques X 10E (dilution fold at the counting) * 1000 / 25 (pfu/mL). Compound treatment was done in a duplicate independently and mean from duplicates of titration was used. Log reduction of titer was calculated by: Log10(titer of Pos control) - Log10 (titer of sample).
Comment
Possible artifacts in this assay include, but are not limited to, compounds that precipitate.

Scoring: Of the compounds selected for dose response testing, those showing more than 10 fold reduction in the progeny titer (<-1 of log virus titer change), and were defined as Active. Scoring for this assay was based on the decreasing in virus titer relative to the untreated, infected control.
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. Purified and resynthesized compounds are scored on a relative scale of 0-80 based on result, in this case the log virus titer change.
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: alternate confirmatory

BAO: bioassay specification: assay biosafety level: BSL2

BAO: assay format: cell based format

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: format detail: reagent: inducer: none

BAO: meta target: molecular target: protein target: enzyme: transferase: kinase

BAO: meta target: biological process target: viral reproduction

BAO: meta target detail: binding reporter specification: interaction: protein:small molecule

BAO: assay design: viability reporter:atp content

BAO: detection technology: luminescence: chemiluminescence

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Modifier - Log virus titer change Rep 1String
2Log virus titer change @ 5 uM Rep 1Float
3Modifier - Log virus titer change Rep 2String
4Log virus titer change @ 5 uM Rep 2Float
Additional Information
Grant Number: 1 R03 MH087448-01A1

Data Table (Concise)
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