*3.3. Dec-RVKR-cmk Inhibits ZIKV and JEV Infection at Di*ff*erent Times of Drug Administration*

The ZIKV and JEV life cycle has multiple vulnerable points where suitable therapeutics can potentially be developed. For this purpose, a time-of-addition assay was performed using 100 μM dec-RVKR-cmk added to ZIKV and JEV infected cells 1 h prior to infection, at the time of infection, and 1, 6, and 12 hpi, followed by virus titer determination by plaque assay and quantification of viral genome copies inside the cells by qRT-PCR at 36 hpi. The data indicated that the maximum reduction in both ZIKV and JEV viral titer (Figure 3a,b) and genome copies (Figure 3c,d) were observed when

dec-RVKR-cmk was added post infection. Interestingly, the infectious JEV and ZIKV particles released into the media from infected cells and inside the cells were still reduced even when peptidyl cmk was added at the time of infection.

**Figure 3.** Time-of-addition assay of dec-RVKR-cmk against ZIKV and JEV infection. ZIKV (0.2 MOI) and JEV (0.2 MOI) infected Vero cells were treated with dec-RVKR-cmk (100 μM) at the indicated condition. Time-of-addition study revealed the effect of dec-RVKR-cmk against (**a**,**c**) ZIKV and (**b**,**d**) JEV viral titer production and intracellular genome copies, respectively. Data are presented as the mean ± SEM from three independent experiments.
