*3.4. Dec-RVKR-cmk Suppresses ZIKV and JEV Propagation in a Dose-Dependent Manner*

Next we investigated the antiviral activity of dec-RVKR-cmk against ZIKV and JEV. Vero cells were infected with ZIKV (0.2 MOI) or JEV (0.2 MOI) in the presence of increasing concentrations of dec-RVKR-cmk (1, 10, 50, 100 μM), followed by viral titer determination in supernatant by plaque assay. Dec-RVKR-cmk inhibited ZIKV and JEV in a dose-dependent manner in the viral titer reduction assay. In the case of ZIKV infection, a 1.48 log10 and 2.44 log10 decrease in virus titer was observed with 50 and 100 μM dec-RVKR-cmk treatment, respectively (Figure 4a, left panel). In the case of JEV infection, treatment with 50 μM dec-RVKR-cmk led to a 1.22 log10 decrease in virus titer, while treatment with 100 μM led to a 2.53 log10 decrease in virus titter (Figure 4b, left panel). No significant inhibition of ZIKV and JEV was observed at 1 and 10 μM concentrations of dec-RVKR-cmk. The IC50 of dec-RVKR-cmk, i.e., the concentration at which 50% virus inhibition occurred, was determined to be 18.59 and 19.91 μM against ZIKV and JEV, respectively (Figure 4a,b, right panel).

**Figure 4.** Antiviral assessment of dec-RVKR-cmk against ZIKV and JEV in a dose-dependent manner. Vero cells were infected with ZIKV or JEV at 0.2 MOI followed by dec-RVKR-cmk treatment using the indicated concentration. Right panels (**a**,**b**) indicate virus titer while left panels indicate IC50 of dec-RVKR-cmk against the indicated MOI of ZIKV and JEV. (**c**) ZIKV-0.2 MOI and (**d**) JEV-0.2 MOI infected Vero cells were treated by dec-RVKR-cmk in a dose-dependent manner and then analyzed by qRT-PCR for absolute genome copies using standard curve of in vitro transcribed ZIKV and JEV RNA at 36 hpi. Meanwhile, infected Vero cells with similar condition of RNA analysis were fixed to analyze virus spreading from infected cells to neighboring cells. Immunofluorescence images of (**e**) ZIKV- and (**f**) JEV-infected Vero cells were acquired at 36 hpi, and quantified (**g**) ZIKV and (**h**) JEV immunoreactive positive cells to visualize the inhibition of infection in a dose-dependent manner. Data are presented as the mean ± SEM from three independent experiments.

To obtain a detailed insight into the efficacy of dec-RVKR-cmk against ZIKV and JEV, RT-qPCR was performed. Reduced ZIKV (0.2 MOI) and JEV (0.2 MOI) genome copies were observed in a dose-dependent treatment in Vero cells at 36 hpi. A significant decline in the viral RNA of ZIKV (~2 log10) and JEV (2.2 log10) was observed in the 100 μM treatment of dec-RVKR-cmk. While at the 50 μM treatment, a 1 log10 decrease in ZIKV RNA and a 1.16 log10 decrease in JEV RNA were observed (Figure 4c,d). Treatment with 1 and 10 μM concentrations of dec-RVKR-cmk did not cause significant inhibition of ZIKV and JEV RNA.

Meanwhile, the effect of dec-RVKR-cmk was observed on virus spreading from infected to bystander cells by IFA. Immunofluorescence images of ZIKV- (0.2 MOI) and JEV- (0.2 MOI) infected Vero cells were taken after 36 hpi using different concentrations of dec-RVKR-cmk (Figure 4e,f). The fluorescence signals revealed that a significant ~22.67-fold inhibition of infection was found when ZIKV-infected cells were treated with 100 μM dec-RVKR-cmk and, to a lesser extent, with 50 μM (~12-fold) and with 10 μM (~1.7-fold), as compared to the control (Figure 4g). Similarly, in the case of JEV-infected cells, the counting of immunoreactive cells showed a strong decrease of viral spreading in a dose-dependent manner (Figure 4h). Taken together, the data suggest that dec-RVKR-cmk significantly reduces intracellular and extracellular virus particles along with the inhibition of ZIKV and JEV spreading from infected cells to bystanders in a dose-dependent manner.
