*3.5. Dec-RVKR-cmk Inhibits ZIKV and JEV Propagation at Various Time Points*

We next studied the anti-viral efficacy of dec-RVKR-cmk against both ZIKV (0.2 MOI) and JEV (0.2 MOI) in Vero cells at different time points, i.e., 24, 36, and 48 hpi, using the 100 μM concentration of dec-RVKR-cmk. Cell culture supernatant was used to determine the virus progeny titer by plaque assay. Figure 5a indicates that significant inhibition of ZIKV progeny titer was seen at 24, 36, and 48 hpi (1.6 log10, 2.25 log10, and 2.23 log10, respectively). Similarly, in the case of JEV, significant inhibition was observed at 24, 36, and 48 hpi (1.08 log10, 2.37 log10, and 2.72 log10, respectively) (Figure 5b). For IFA, infected Vero cells were fixed from both the dec-RVKR-cmk-treated and DMSO-treated control groups to see the extent of infection by counting immunoreactive cells. No significant difference in fluorescence signal of ZIKV-NS5 (Figure 5c) and JEV-NS5 (Figure 5d) were observed at 24 hpi between the dec-RVKR-cmk-treated and DMSO-treated control groups. Significant ZIKV (Figure 5c,e) and JEV (Figure 5d,f) inhibition was seen at 36 and 48 hpi, in terms of reduction in the percentage of immunoreactive cells, in the dec-RVKR-cmk-treated group as compared to the control group for ZIKV (14% and 52% inhibition, respectively) and JEV (70% and 23% inhibition, respectively). Thus, the data suggest that the maximum effects of dec-RVKR-cmk were observed at 36 and 48 hpi in terms of reduction of virus progeny titer and inhibition of ZIKV and JEV spreading to neighboring bystander cells.

**Figure 5.** *Cont.*

**Figure 5.** Dec-RVKR-cmk inhibited ZIKV and JEV infection at various time points. Vero cells were infected with ZIKV-0.2 MOI and JEV-0.2 MOI, followed by dec-RVKR-cmk treatment using the 100 μM concentration. Cell supernatant used to determine (**a**) ZIKV and (**b**) JEV viral titer by plaque assay at indicated time points. While immunofluorescence images of (**c**) ZIKV- and (**d**) JEV-infected Vero cells were acquired at different time points in both control (virus + DMSO) and treated (virus + CMK) Vero cells and quantified (**e**) ZIKV and (**f**) JEV immunoreactive positive cells to see the extent of infection. Data are presented as the mean ± SEM from three independent experiments.

We conducted an experiment to assess virus replication in more detail with the one-step growth cycle. We treated the cells using a 100 μM concentration of dec-RVKR-cmk or DMSO 1 hpi. Vero cells were infected with ZIKV at an MOI of 5 for 1 h and media were replaced with dec-RVKR-cmk or DMSO after washing the cells. The culture medium and cells were collected immediately following infection (0 h) and then every 3 h through 21 hpi. Virus titer was determined in both supernatants and cells by plaque assay on Vero cells. The data indicated that infectious viral particles began to produce between 9 and 12 h post infection in DMSO-treated cells (Figure 6a). Following initial amplification, the subsequent rise was observed at 15, 18, and 21 hpi. Similar findings were observed in the extracellular compartment (Figure 6b). In dec-RVKR-cmk-treated cells, a significant reduction in both intracellular (>1 log10 at 15, 18, and 21 hpi) and extracellular (>2 log10 at 15, 18, and 21 hpi) virus titers were observed. Taken together, the data suggests that dec-RVKR-cmk inhibits ZIKV and JEV propagation in the one-step growth cycle. Furthermore, the greater reduction in extracellular virus titer than that in intracellular virus titer indicates that dec-RVKR-cmk might inhibit virus release rather than replication.
