In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68
体外实验评估候选的抗病毒化合物对肠道病毒D68的疗效
发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2183 浏览次数: 9574
评审: Yannick DebingKristin ShinglerCristina SuárezNoam Vardi
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参见作者原研究论文
The authors used this protocol in:
Dec 2015
Abstract
In 2014 enterovirus D68 (EV-D68) caused the largest outbreak in the United States since the discovery of the virus. Distinct from before, the 2014 infections were associated with more severe respiratory disease and occasional neurological complications. So far, there are no available vaccines or antivirals for the prophylaxis or treatment of EV-D68 infections. In order to evaluate the antiviral activity of potential inhibitors of EV-D68 replication, a cell-based cytopathic effect (CPE) reduction assay was developed (Sun et al., 2015).
Keywords: Enterovirus D68 (肠道病毒D68)
Antiviral assay (抗病毒测定)
CPE (CPE)
MTS/PMS solution (MTS/PMS溶液)
Cell culture (细胞培养)
Background
As a re-emerging pathogen, antiviral compounds targeting EV-D68 were rarely reported before. It is urgently needed to establish and develop antiviral methods to combat the potential EV-D68 epidemic. Here, we report a detailed protocol which can be used to identify selective anti-EV-D68 compounds. To screen and identify potential antiviral compounds, the MTS-based CPE reduction assay is reproducible, easy-to-use and time-saving method which is widely used. This method relies on the ability of a compound to inhibit EV-D68-induced reduction of CPE. When a compound actively inhibits the replication of EV-D68, the virus-induced CPE will be reduced or absent. As a consequence, the metabolically active cells are able to convert a yellow tetrazolium salt substrate (MTS/PMS) to a brown-colored formazan product. When a compound does not have antiviral effects, the host cells die of virus-induced CPE, which will lack metabolic activity, and the yellow substrate remains un-metabolized. The colorimetric conversion is quantitative, as an EC50 can be calculated from the data generated by this assay.
Materials and Reagents
- Centrifuge tube, 14 ml (Corning, Falcon®, catalog number: 352059 )
- 1.5 ml Eppendorf Snap-Cap microcentrifuge tubes (VWR, catalog number: 21008-959 )
- 4 ml Screw neck vials, amber glass (VWR, catalog number: 548-0052 )
- Transparent 96-well (flat-bottom) tissue culture plates (Corning, Falcon®, catalog number: 353072 )
- Tissue culture flasks, 150 cm2 (TPP, catalog number: 90856 )
- Pipette tips (10 µl, 100 µl, 1,000 µl)
- Disposable serological pipets (5 ml, 10 ml and 25 ml)
- Reagent reservoir, 25 ml (Biotix, catalog number: SR-0025-5SWM )
- Nalgene rapid-flow sterile disposable filter units with SFCA membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 156-4020 )
- Hela Rh cell line (a Hela subclone, highly susceptible and permissible to rhinovirus-induced CPE, a gift from Janssen Pharmaceutica, Belgium) (Lacroix et al., 2014)
- All EV-68 strains were cultivated on HeLa Rh cells and stored at -80 °C
- EV-D68 strains (742, 947, 2042, 1348, 2284 and 670) were isolated from the Netherlands (a kind of gift from Adam Meijer, Diagnostics and Screening National Institute for Public Health and the Environment [RIVM], Bilthoven)
- EV-D68 strains (US/KY/14-18953, US/IL/14-18952 and US/MO14-18947) – these strains were originally obtained from BEI Resources
- EV-D68 strains (742, 947, 2042, 1348, 2284 and 670) were isolated from the Netherlands (a kind of gift from Adam Meijer, Diagnostics and Screening National Institute for Public Health and the Environment [RIVM], Bilthoven)
- Dulbecco’s phosphate-buffered saline (DPBS), no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
- 0.05% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
- Minimum essential media (MEM) Rega-3 (Thermo Fisher Scientific, GibcoTM, catalog number: 19993013 )
- Potential antiviral compounds
- SG85*
- Pleconaril*
- Vapendavir*
- Pirodavir*
- Rupintrivir*
- Enviroxime*
- Favipiravir*
- SG85*
- Fetal bovine serum (GE Healthcare, HycloneTM, catalog number: SH30084.03 )
- L-glutamine 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
- Sodium bicarbonate 7.5% solution (Thermo Fisher Scientific, GibcoTM, catalog number: 25080060 )
- Minimum essential media (MEM), no glutamine, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 51200038 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
- CellTiter 96® AQueous MTS reagent powder (Promega, catalog number: G1111 )
- HCl
- Dimethyl sulfoxide (VWR, catalog number: 67-68-5 )
- Growth culture medium (see Recipes)
- Assay medium (see Recipes)
- MTS/PMS solution (see Recipes)
Equipment
- Pipette controller, PIPETBOY acu2 (VWR, catalog number: 612-0927 )
- Multichannel pipette, 10-100 µl (Eppendorf)
- Incubator at 37 °C (and 35 °C ) and 5% CO2 (Binder)
- Mid bench centrifuge (Sigma Laborzentrifugen, mode: 4K15C )
- MoxiTM Z Mini Automated Cell Counter (ORFLO Technologies, catalog number: MXZ001 )
- SpectraMax 190 microplate reader (Molecular Devices, model: SpectraMax 190 )
- pH bench meter (Xylem Analytics, WTW, model: inoLab® 9310 IDS )
- Inverted microscope (Motic, model: AE21 )
- Safety cabinet (Telstar)
Software
- GraphPad Prism
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Sun, L., Delang, L., Mirabelli, C. and Neyts, J. (2017). In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68. Bio-protocol 7(6): e2183. DOI: 10.21769/BioProtoc.2183.
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分类
微生物学 > 抗微生物试验 > 抗病毒试验
细胞生物学 > 基于细胞的分析方法 > 病毒性感染
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