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Updated: Aug 14, 2026
High-throughput Titration of Luciferase-expressing Recombinant Viruses
Published on: September 19, 2014
High-throughput assays using a luciferase-expressing replicon, virus-like particles, and full-length virus for West
Francesc Puig-Basagoiti1, Tia S Deas, Ping Ren
1Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, New York 12208, USA.
Abstract:
Many flaviviruses cause significant human disease worldwide. The development of flavivirus chemotherapy requires reliable high-throughput screening (HTS) assays. Although genetic systems have been developed for many flaviviruses, their usage in antiviral HTS assays has not been well explored. Here we compare three cell-based HTS assays for West Nile virus (WNV) drug discovery: (i) an assay that uses a cell line harboring a persistently replicating subgenomic replicon (containing a deletion of viral structural genes), (ii) an assay that uses packaged virus-like particles containing replicon RNA, and (iii) an assay that uses a full-length reporting virus. A Renilla luciferase gene was engineered into the replicon or into the full-length viral genome to monitor viral replication. Potential inhibitors could be identified through suppression of luciferase signals upon compound incubation. The antiviral assays were optimized in a 96-well format, validated with known WNV inhibitors, and proved useful in identifying a new inhibitor(s) through HTS of a compound library. In addition, because each assay encompasses multiple but discrete steps of the viral life cycle, the three systems could potentially be used to discriminate the mode of action of any inhibitor among viral entry (detected by assays ii and iii but not by assay i), replication (including viral translation and RNA synthesis; detected by assays i to iii), and virion assembly (detected by assay iii but not by assays i and ii). The approaches described in this study should be applicable to the development of cell-based assays for other flaviviruses.
Insights
This study developed and compared three cell-based high-throughput screening (HTS) assays for West Nile virus (WNV) drug discovery. These assays effectively identified potential antiviral compounds and can help determine their mechanism of action.
Area of Science:
- Virology
- Drug Discovery
- Molecular Biology
Background:
- Flaviviruses cause significant global human diseases.
- Effective flavivirus chemotherapy relies on robust high-throughput screening (HTS) assays.
- The application of genetic systems in antiviral HTS for flaviviruses remains underexplored.
Purpose of the Study:
- To compare three distinct cell-based HTS assays for West Nile virus (WNV) drug discovery.
- To evaluate the utility of these assays in identifying novel antiviral inhibitors.
- To assess the potential of these assays in discriminating inhibitor mechanisms of action.
Main Methods:
- Developed and optimized three WNV cell-based HTS assays: subgenomic replicon, virus-like particle replicon, and full-length reporter virus.
- Engineered Renilla luciferase reporter into viral RNA to monitor replication.
- Validated assays with known inhibitors and screened a compound library.
Main Results:
- All three assays were optimized for 96-well format and validated.
- The assays successfully identified potential antiviral compounds through HTS.
- The distinct assay designs allow for differentiation of inhibitor action at viral entry, replication, or assembly stages.
Conclusions:
- The developed cell-based HTS assays are effective for WNV drug discovery.
- These systems can aid in elucidating the mechanism of action of antiviral compounds.
- The described approaches are adaptable for developing HTS assays for other flaviviruses.
