Development, validation, and application of a dual-color fluorescent assay for high-throughput screening of anti-chikungunya drugs.

Chikungunya virus (CHIKV), one of the arthropod-borne viruses, has been affecting the global population for more than 70 years since it was first described with more than 620,000 cases in 2024 alone. Despite its long-standing problem, the only treatment available for chikungunya-infected patients is supportive treatment to alleviate pain. Fluorescent molecules have been used in detecting viral infection in the host cells via immunofluorescence assays because of their sensitivity. This study aimed to use this assay to rapidly screen efficacy and cytotoxicity of several compounds in a high-throughput manner. The optimized conditions were to seed Vero cells at 10,000 cells/well, and infect them with CHIKV ECSA at MOI of 0.1. These conditions resulted in a good discrimination power between infected wells and uninfected wells and minimized the cytopathic effect on host cells. Validation using two compounds with known activity against CHIKV, cycloheximide (CHX), and acyclovir (ACY), showed that the assay could properly identify active compounds and inactive compounds correctly. There was also no significant difference between the results of 3 independent rounds of compound screening, thus showing the reproducibility of the assay. Traditional primary screening were performed in parallel with the dual-color fluorescent assay for 60 unknown compounds to evaluate inhibition performance of inhibition and approximate cytotoxicity assessment. The results showed excellent performance from the analysis of the ROC curves and general agreement between two approaches from the Bland-Altman plots. Overall, the developed assay required less labor while being able to screen more compounds than the traditional assay in one round of experiment. The assay is currently being tested to screen libraries of compounds and so far, has been able to identify 22 hits for further characterization.