*3.6. Dec-RVKR-cmk Inhibits ZIKV Release and Next Round Infectivity*

To elaborate on this phenomena, both ZIKV- (1 MOI) infected cells and their supernatants from treatment (dec-RVKR-cmk 100 μM) and control (DMSO) group were subjected to RT-qPCR analysis. Meanwhile, half the volume of supernatant was used to determine viral titer through plaque assay. According to the one-step growth cycle results (Figure 6), 12–21 h may reflect a single round of replication [26]. To clarify at which stage in virus life cycle dec-RVKR-cmk works, both ZIKV- and JEV-infected cells and their supernatants from the treatment (dec-RVKR-cmk 100 μM) and control (DMSO) groups were subjected to RT-qPCR analysis and plaque assay at 12, 16, and 20 hpi. At the intracellular level, there was no difference in genome copies in both the treated and control groups at different time points (Figure 7a). Extracellular viral titer, in terms of genome copies (Figure 7b) and

plaque forming unit (Figure 7c), showed a significant difference between the control and treatment groups. Afterwards, we calculated the infectivity of the virus by comparing the viral titer of ZIKV, determined by plaque assay and RT-qPCR in supernatant, for both the control and treated groups. Figure 7d indicates no significant difference in virus infectivity at 12 and 16 hpi, but at 20 hpi a 20% reduction was observed in the released virus. Taken together, these data validate the results, seen in Figure 6, which show that no significant difference in genomic RNA copies was observed in dec-RVKR-cmk-treated and untreated cells at 12, 16 and 20 hpi. Thus, dec-RVKR-cmk cannot inhibit virus replication in the first round of infection but exerts its inhibitory action on virus release and next round infectivity.

**Figure 6.** Dec-RVKR-cmk inhibits ZIKV infection in the one-step growth cycle. Vero cells were infected with ZIKV at an MOI of 5 for 1 h followed by dec-RVKR-cmk or DMSO treatment after washing the cells. At the indicated time points after infection, culture medium was collected and cells were subjected to three freeze-thaw cycles to liberate cell-associated viruses. (**a**) Intracellular and (**b**) extracellular virus titer was determined by plaque assay. Data are presented as the mean ± SEM from three independent experiments.

**Figure 7.** Dec-RVKR-cmk inhibits ZIKV release and next round infectivity. Vero cells were infected with ZIKV (1 MOI) followed by dec-RVKR-cmk treatment using the 100 μM concentration. (**a**) Infected cells were analyzed by qRT-PCR for absolute genome copies using the standard curve of in vitro transcribed ZIKV for the indicated time points. Cell supernatant was used to determine ZIKV viral titer by (**b**) qRT-PCR and also by (**c**) plaque assay. (**d**) The percentage infectivity was calculated by dividing the viral titer determined by plaque assay by the viral titer determined by qRT-PCR in supernatant, for both the control and treated groups, and then the value of the control group was normalized to 100 and compared with the treated group. Data are presented as the mean ± SEM from three independent experiments.
