Virucidal Action of (p-BthTX-I)₂K Against CHIKV and ZIKV

The investigation of the virucidal action of (p-BthTX-I)₂K against CHIKV was performed following previously described methods [27 (link),30 (link)] with modifications. For ZIKV, this assay was adapted based on a protocol described by Carneiro and collaborators [32 (link)]. For CHIKV, 1 × 10⁴ BHK-21 cells were seeded in the wells of 96-well white culture plates, and for ZIKV, 2 × 10⁴ Vero cells were seeded in the wells of 24-well culture plates (TPP, Trasadingen, Switzerland). The virions of CHIKV-NLuc at an amount to achieve infection with an MOI of 5 or those of ZIKV at an amount to achieve infection with an MOI of 0.1 were incubated with the (p-BthTX-I)₂K peptide at the MNTC at 37 °C (CHIKV) or room temperature (ZIKV) for 1 h. Then, the viral inoculum was added to cells. After 1 h of incubation, the supernatant was removed, the cells were washed twice with PBS, and DMEM (Cultilab, Campinas, SP, BR) supplemented with 2% FBS (Gibco—Thermo Fisher Scientific, Waltham, MA, USA) was added to each well. The cells were incubated at 37 °C for the specific period previously determined for each virus. Sixteen hours post-infection (h.p.i.), CHIKV replication was quantified by measuring NLuc activity. For ZIKV, the viral RNA in the supernatant of the cells was quantified via qRT–PCR 48 h.p.i.