Development of a homogeneous time-resolved fluorescence assay for detection of viral double-stranded RNA

Literature buzz Life Science

A new HTS compatible assay to screen antivirals targeting dsRNA viruses

Expert opinion

Bacteria, fungi, parasites, and viruses… the main pathogens that affect humans and trigger severe diseases. Significant research effort is dedicated to understanding infection routes and identifying solutions for treating infected patients.
Like the now well-established classification systems in place for all cellular organisms, virus classification is based mainly on phenotypic characteristics. This includes the size and shape of their protein coat, nucleic acid type (DNA or RNA), and replication mode in their host organisms. All these criteria need to be considered when setting up the most appropriate assay to screen new viral inhibitors.
A range of assays is available for virologists, such as plaque assays to measure virus infectivity, cytopathic effect (CPE) assay to assess morphological changes of infected cells, quantitative PCR (qPCR), and ELISA. However, none of these options are suitable for large-scale screening of new antiviral agents.
In this case study on Human rhinovirus (HRV), a double-stranded RNA virus, Motomichi Fujita developed a rapid, cost-effective, and HTS-compatible assay to screen compounds. She tested the robustness of her assay on several cell lines (MRC-5 and H1-Hela) infected at different MOI with different dsRNA viruses (HRV, CV, EV and MNV). Using this assay in dose-response experiments, the inhibitory effect of Rupintrivir (AG7088) was confirmed with consistent EC50 values.
All the landmarks of robust HTS are present: Z’ Factor >0.7, signal-to-background (S/B) ratio >60. The first universal high-throughput assay focused on dsRNA virus replication is at your fingertips!

Abstract

The group of positive-sense single-stranded RNA ((+) ssRNA) viruses includes many important human pathogens. However, specific antiviral agents are not currently available for many RNA viruses. For screening of antiviral agents, methods that are simple, rapid, and compatible with high-throughput are required. Here, we describe a novel method for measurement of double-stranded RNA using a homogeneous time-resolved fluorescence assay. This method allowed detection of human rhinovirus (HRV), enterovirus, coxsackievirus, and murine norovirus. Furthermore, this method detected antiviral activity of an HRV 3C protease inhibitor. The assay may be useful for discovery of antiviral agents against (+) ssRNA viruses.

Details

Analytical Biochemistry, 2019 Feb 1;566:46-49.

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