*2.5. Plaque Assay*

Viral titers in cell culture supernatants were assessed as described in our previous study [25]. Briefly, virus-infected Vero and C6/36 cells were treated with dec-RVKR-cmk and DMSO. After different time points, as indicated in the results section, virus-containing cell culture supernatants were removed, serially diluted in DMEM, and adsorbed on a Vero (ZIKV) or BHK-21 (JEV) monolayer for 1 h. Afterwards, unbound viral particles were washed and overlaid with 2% carboxymethyl cellulose (CMC). After 5 days of incubation, cells were fixed with 10% formaldehyde for 12 h and then stained with 0.1% crystal violet for 6 h. The visible plaques were counted and viral loads were measured as plaque-forming unit (PFU) per ml of supernatant. All data are expressed as the means of triplicate samples.

#### *2.6. Time-of-Drug Addition Assay*

ZIKV- (0.2 MOI) and JEV- (0.2 MOI) infected Vero cells were treated with dec-RVKR-cmk under the following conditions: 1 h prior to infection, at the time of infection, or 1, 6, and 12 h post infection (hpi). Regardless of treatment time, cells were infected for 1 h. After 1 h, infectious media were replaced with fresh media and dec-RVKR-cmk added at the above time points. Supernatants were collected at 36 hpi to determine viral titer by plaque assay while cells were used to quantify viral genome copies by qRT-PCR.
