Evaluating (p-BthTX-I)2K Antiviral Activity

The antiviral activity of the (p-BthTX-I)₂K peptide mediated by blocking viral entry into the cells was analyzed according to previously described methods [27 (link),30 (link),33 (link)] with modifications. BHK-21 cells (for CHIKV) and Vero cells (for ZIKV) were plated as described above. After 24 h of culture, the cells were infected with CHIKV-NLuc at an MOI of 0.1 or ZIKV^BR at an MOI of 0.1 in the presence of the (p-BthTX-I)₂K peptide at the MNTC. After 1 h of incubation at 37 °C, the supernatant was aspirated, the wells were washed twice with PBS, and DMEM (Cultilab, Campinas, SP, Brazil) with 2% FBS (Gibco—Thermo Fisher Scientific, Waltham, MA, USA) was added to the cells. The plate was incubated at 37 °C for 16 h.p.i (CHIKV) or 48 h.p.i (ZIKV). (p-BthTX-I)₂K inhibitory effects on CHIKV were evaluated via the measurement of the NLuc activity, and the effect on ZIKV was evaluated via qRT–PCR as described above.