Fig. 2.

RO8191 activity on different stages of CHIKV-nanoluc replicative cycle in BHK-21 cells. A) BHK-21 cells were treated with RO8191 for 1 h, cells were washed with PBS to remove the compound, and were infected with CHIKV-nanoluc at MOI 0.1 for 1 h. Then, the medium was removed, cells were washed to remove unbound virus, and fresh medium was added. B) BHK-21 cells were infected with CHIKV-nanoluc (MOI 0.1) and simultaneously treated with RO8191 for 1 h. Then, cells were washed to completely remove the inoculum, and a fresh medium was added. C) RO8191 and CHIKV-nanoluc at MOI 5 were incubated for 1 h (representative inoculum). Then, the inoculum was added to the cells for an additional hour. Cells were washed to remove the inoculum and fresh medium was added. D) BHK-21 cells were infected with CHIKV-nanoluc (MOI 0.1) for 1 h, cells were washed with PBS to remove unbound virus, and were treated with RO8191. All infection assays were quantified 16 h.p.i through the measurement of luminescence levels. Schematic representation of each time-based assay as indicated by BHK-21 cells (black bars), RO8191 (gray bars), CHIKV-nanoluc (orange bars), and CHIKV-RO8191 inoculum (blue tube). E) Schematic representation of the time-based BHK-CHIKV cell assay as indicated by the BHK-CHIKV-NCT cells (black bars) and RO8191 (gray bars). BHK-CHIKV-NCT cells were incubated for 72 h with the RO8191 compound at concentrations ranging from 60 to 0.46 µM. Then, cells were washed with PBS and lysed with Passive Lysis Buffer to measure the activity of Renilla. The reduction of Renilla activity in BHK-CHIKV-NCT cells is shown by 50% of the highest concentrations (EC₅₀) of RO8191 (black squares) and an effect in cell viability (CC₅₀) using MTT assay (gray circles). Mean ± SD values of a minimum of three independent experiments, each measured in triplicate. (***) P<0.001, (**) P<0.01, and (*) P <0.05 are represented. Images were generated using GraphPad Prism 8 and GIMP 2.1v.