Multiplexed multicolor antiviral assay amenable for high-throughput research

Li-Hsin Li, Winston Chiu, Yun-An Huang, Madina Rasulova, Thomas Vercruysse, Hendrik Jan Thibaut, Sebastiaan ter Horst, Joana Rocha-Pereira, Greet Vanhoof, Doortje Borrenberghs, Olivia Goethals, Suzanne J. F. Kaptein, Pieter Leyssen, Johan Neyts and Kai Dallmeier ()
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Li-Hsin Li: Laboratory of Virology and Chemotherapy
Winston Chiu: Laboratory of Virology and Chemotherapy
Yun-An Huang: Laboratory for Circuit Neuroscience
Madina Rasulova: Translational Platform Virology and Chemotherapy (TPVC)
Thomas Vercruysse: Translational Platform Virology and Chemotherapy (TPVC)
Hendrik Jan Thibaut: Translational Platform Virology and Chemotherapy (TPVC)
Sebastiaan ter Horst: Laboratory of Virology and Chemotherapy
Joana Rocha-Pereira: Laboratory of Virology and Chemotherapy
Greet Vanhoof: Janssen Pharmaceutica
Doortje Borrenberghs: Janssen Pharmaceutica
Olivia Goethals: Janssen Pharmaceutica
Suzanne J. F. Kaptein: Laboratory of Virology and Chemotherapy
Pieter Leyssen: Laboratory of Virology and Chemotherapy
Johan Neyts: Laboratory of Virology and Chemotherapy
Kai Dallmeier: Laboratory of Virology and Chemotherapy

Nature Communications, 2024, vol. 15, issue 1, 1-15

Abstract: Abstract To curb viral epidemics and pandemics, antiviral drugs are needed with activity against entire genera or families of viruses. Here, we develop a cell-based multiplex antiviral assay for high-throughput screening against multiple viruses at once, as demonstrated by using three distantly related orthoflaviviruses: dengue, Japanese encephalitis and yellow fever virus. Each virus is tagged with a distinct fluorescent protein, enabling individual monitoring in cell culture through high-content imaging. Specific antisera and small-molecule inhibitors are employed to validate that multiplexing approach yields comparable inhibition profiles to single-virus infection assays. To facilitate downstream analysis, a kernel is developed to deconvolute and reduce the multidimensional quantitative data to three cartesian coordinates. The methodology is applicable to viruses from different families as exemplified by co-infections with chikungunya, parainfluenza and Bunyamwera viruses. The multiplex approach is expected to facilitate the discovery of broader-spectrum antivirals, as shown in a pilot screen of approximately 1200 drug-like small-molecules.

Date: 2024
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DOI: 10.1038/s41467-023-44339-z

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