Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.
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Method Article
Assays for the Identification of Novel Antivirals against Bluetongue Virus
In This Article
Summary
Abstract
To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.
Introduction
BTV is a prototype double-stranded RNA virus in the genus Orbivirus, family Reoviridae. BTV is one of the most important diseases of domestic livestock, including sheep, goat, cattle and other domestic animals, with $3 billion/year loss worldwide1,2. The exotic BTV serotype is an important animal pathogen listed in the "USDA High Consequence Livestock Pathogens." Recently, the re-emerging of BTV has caused a major outbreak of disease in cattle and sheep in several countries across Northern Europe3,4. As a result of its economic significance and as a model system, BTV has been the subject of extensive molecular, genetic a....
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Protocol
1. Cells, Virus and the Antiviral Compounds
- Maintain BSR cells, a derivative of baby hamster kidney (BHK) cells10, in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS), 100U/ml penicillin and 100 μg/ml streptomycin.
- For all three assays, plate cells in DMEM with 1% FCS, 100U/ml penicillin and 100 μg/ml streptomycin, as optimized previously5. This medium is referred as assay medium for all three assays.
- Incubate all cells in the incubator at 37 °C, with 5% CO2 and 80-95% humidity.
- Plaque-purify and propagate the type 10 BTV (BTV-10) as describe....
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Results
1. Antiviral efficacy of compound
The cell-based CPE assay was developed, optimized and validated in vitro using the luminescent-based CTG kit to identify novel antivirals against BTV as described previously2,5. The ten-dose response assay was employed to reflect the antiviral efficacy and cytotoxicity of an identified lead compound by measuring the number of metabolically viable cells in culture based on quantitation of cellular ATP presented in the living cells5,11
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Discussion
For the initial identification of antiviral hits, one of the key steps for antiviral drug discovery and development is to develop robust assays, which includes selecting a quantifiable marker, developing a simple protocol, obtaining sufficient signals and less than 10% CV. Most biochemical or cell-based screens are designed to provide a chemical starting point based upon the most robust, simple and inexpensive assay, due to the required reproducibility in the screening process and the potentially large number of molecule.......
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Disclosures
The authors declare that they have no competing financial interests.
Acknowledgements
This project was supported by grant 1R03MH08127-01 and 7R03MH08127-02 from NIH to Q. Li, and by the IMPACT funds from Department of Medicine at UAB to Q. Li. Support from the Molette Fund and Auburn University is appreciated. We also thank the technical assistances from Ms. Pulin Che and Mr. Volodymyr Musiienko during the course of the work.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| DMEM medium | Gibco | 1134218 | For cell culture |
| FBS | Gibco | 16000044 | For cell culture |
| 0.05% Trypsin-EDTA | Gibco | 1000185 | For cell culture |
| DPBS | Gbico | 1049769 | For cell culture |
| CellTiter-Glo (CTG) kit | Promega | TB288 | For cell viability measurement |
| 70% ethanol | Fisher | S25309B | Diluted from 95% |
| Antiviral huashilcompounds | NIH MLSMR and de novo synthesis | ||
| BTV-10 | ATCC | VR-187 | |
| BSR cell | Developed in house | ||
| Synergy-II multi-mode microplate reader | BioTek | For luminescent signal reading | |
| MicroFlo select dispenser | BioTek | Adding cells, virus, and reagents | |
| 384-well flat-bottom microplate | CORNING | 28908031 | For cell culture |
| Gen. 5 software | BioTek | For analysis of reading outputs from Synergy-II multi-mode microplate reader | |
| GraphPad Prism 5 | GraphPad | Version 5 | For biostatic analysis and plot |
References
- Hemati, B., et al. Bluetongue virus targets conventional dendritic cells in skin lymph. J. Virol. 83, 8789-8799 (2009).
- Gu, L., et al. Novel Virostatic Agents against Bluetongue Virus. PLoS ONE. 7, e43341(2012).
- Meiswinkel, R., et al.
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