Deciphering the Pharmacological Constituents of Cow Urine Distillate Responsible for Antiviral Activity Against Chikungunya Virus

Objective: This study aims to investigate the antiviral properties of Cow-urine distillate (CUD) and identify potential compounds responsible for its antiviral activity, thereby exploring its therapeutic potential against Chikungunya virus (CHIKV). Methods: The study assessed the antiviral activity of crude CUD using in vitro cell-based assays performed in Vero cells. The characterization of CUD was performed using GC-MS to identify potential metabolites with antiviral properties. Computational docking studies elucidated these natural compounds' binding affinities and critical interactions with CHIKV nsP2 protease (nsP2Pro). The identified metabolites were evaluated for antiviral activity against CHIKV nsP2Pro via in silico, biochemical, and cell culture-based antiviral assays. The study further investigated the synergistic antiviral effect of CUD with natural compounds such as thymoquinone (TQ) and piperine (PIP) against CHIKV replication. Results: Treatment of CHIKV with CUD resulted in 90% reduction in plaques with 2% v/v of CUD (EC50 ~ 0.423%). Furthermore, four of the compounds identified from GC-MS demonstrated substantial inhibition at non-toxic doses, as validated by FRET assay and CHIKV plaque-reduction assay. The combination of 20 µM TQ + 20 µM PIP + 2% v/v GA was found to be very effective against CHIKV, which exhibited ~4 log or 99.99% virus reduction. Conclusion: The present findings establish the antiviral potential of CUD and provide an effective natural remedy against CHIKV infection.