High-throughput titration of luciferase-expressing recombinant viruses.

Abstract

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

• Authors: Vanessa Garcia, Ramya Krishnan, Colin Davis, Cory Batenchuk, Fabrice Le Boeuf, Hesham Abdelbary, Jean-Simon Diallo
• Journal: Journal of visualized experiments : JoVE (J Vis Exp) Issue 91 Pg. 51890 (Sep 19 2014) ISSN: 1940-087X [Electronic] United States
• PMID: 25285536 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Video-Audio Media)

Chemical References

• Luciferases, Firefly

Topics

• Animals
• Chlorocebus aethiops
• High-Throughput Screening Assays (methods)
• Luciferases, Firefly (analysis, biosynthesis, genetics)
• Transgenes
• Vero Cells
• Vesiculovirus (enzymology, genetics)
• Virus Cultivation (methods)