Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (strain Trinidad donkey) (03)
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 ..
BioActive Compounds: 4
Depositor Specified Assays
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 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 population (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.
Biosafety and Biosecurity: All experiments with VEEV Trinidad donkey 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: Trinidad Donkey (TrD) 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 titer was 2.0XE10 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 37oC 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 200 uL 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 25 uL 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 100 uL 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). Compounds treatment were 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).
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) at 5 uM, 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.
Phenotypic Screen: Yes
Screening Concentration: 10
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
Data Table (Concise)