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, Antiviral assay, CPE, MTS/PMS solution, Cell culture
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
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Acknowledgments
This work was supported by a fellowship to Liang Sun from China Scholarship Council (CSC) (grant 201403250056) and the Interuniversitaire attractiepolen (IUAP) BELVIR. The protocol described here was based on the following paper: Sun et al. (2015).
References
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