Multiplexed multicolor antiviral assay amenable for high-throughput research.

Area of Science:

• Virology
• Drug Discovery
• High-Throughput Screening

Background:

• Antiviral drugs with broad-spectrum activity are crucial for combating viral epidemics and pandemics.
• Current screening methods often focus on single viruses, limiting the discovery of broad-spectrum agents.

Purpose of the Study:

• To develop and validate a cell-based multiplex antiviral assay for simultaneous high-throughput screening against multiple viruses.
• To demonstrate the assay's applicability across different viral families and co-infections.

Main Methods:

• Utilized fluorescent protein-tagged viruses (dengue, Japanese encephalitis, yellow fever) for individual monitoring via high-content imaging.
• Developed a kernel for deconvoluting multidimensional data into three Cartesian coordinates for downstream analysis.
• Validated the multiplexing approach against single-virus assays using antisera and small-molecule inhibitors.

Main Results:

• The multiplex assay successfully monitored and distinguished between multiple orthoflaviviruses (dengue, Japanese encephalitis, yellow fever).
• Inhibition profiles from multiplex screening were comparable to single-virus assays.
• The methodology proved effective for co-infections involving viruses from different families (chikungunya, parainfluenza, Bunyamwera).
• A pilot screen of ~1200 compounds demonstrated the potential for discovering broader-spectrum antivirals.

Conclusions:

• The developed cell-based multiplex antiviral assay enables efficient, simultaneous screening against diverse viruses.
• This approach facilitates the discovery of broad-spectrum antiviral drugs essential for pandemic preparedness.
• The methodology is adaptable to various viral families and co-infection scenarios.