Dulbecco modified eagle medium (dmem)

Baby hamster kidney fibroblast cells (BHK-21; ATCC CCL-10) and African green monkey kidney cells (Vero; ATCC CCL-81) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Cultilab, Campinas, SP, Brazil) supplemented with 10% fetal bovine serum (FBS, Gibco—Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin (10,000 IU/mL)/streptomycin (10 mg/mL) (P/S) (Cultilab, Campinas, SP, Brazil). The cells were maintained in a humidified incubator at 37 °C in 5% CO₂.
C6/36 cells (ATCC CRL-1660) from Aedes albopictus mosquitoes were cultured in Leibovitz L-15 medium (Cultilab, Campinas, SP, Brazil) supplemented with 10% FBS (Gibco—Thermo Fisher Scientific, Waltham, MA, USA) and 1% P/S (Cultilab, Campinas, SP, Brazil). These cells were maintained in an incubator at 28 °C.

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.

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.

Human umbilical vein endothelial cells were purchased from the Rio de Janeiro Cell Bank (UFRJ, Brazil) originated from the ATCC (American Type Culture Collection, USA). Cultivation was performed in a humidified incubator at 37°C and 5% CO₂ in bottles using modified Eagle's Dulbecco's medium (DMEM) plus 10% fetal bovine serum, amphotericin B, and L glutamine (Cultilab, Brazil).
After the cultivation step, the cells were quantified in a Neubauer chamber and seeded onto 96-well plates containing 200 μL DMEM medium at 1×10⁴ concentrations. Then, the medium was discarded, and the wells containing cells were treated with PV3 at concentrations of 10, 20, 30, and 250 µg/mL for 24 h. The negative control group was treated with extract diluent, 0.9% (w/v) sodium chloride solution. The positive control group was treated with diluent and, after 24 h, was exposed to 3% (v/v) hydrogen peroxide (H₂O₂) for one hour. All assays were performed in three independent and triplicated experiments.

The protective effect of the peptides against CHIKV infection was evaluated as previously described [55 (link)]. BHK-21 and Huh-7 cells (1 × 10⁴ per well) were seeded in 96-well white culture plates (Greiner Bio-One, Americana, SP, Brazil). After 24 h, the cells were treated with peptides at the MNTC for 1 h at 37 °C. Then, the supernatant was removed, each well of the plate was washed twice with PBS, and the virus at an MOI of 0.1 was added to the cells and incubated for 1 h at 37 °C. After that, cells were washed twice with PBS again, and DMEM (Cultilab, Campinas, SP, BR) supplemented with 2% FBS (Gibco—Thermo Fisher Scientific, Waltham, MA, USA) was added to the cells. The plate was incubated at 37 °C, and the protective effect against CHIKV infection was evaluated at 16 h.p.i. by measurement of NLuc activity.

The cytotoxicity of the compounds against HFF-1 fibroblasts and HepG2 hepatocytes was evaluated using the MTS tetrazolium assay (Promega, Madison, WI, USA) [13 (link)]. HFF-1 fibroblasts were seeded at 4x10³/well in 96-well culture plates in DMEM without phenol red and incubated overnight (37°C, 5% CO₂). HepG2 hepatocytes were seeded at 7x10³/well in 96-well culture plates in DMEM (Cultilab) with supplements (10% FCS, 1% pen/strep) and incubated overnight (37°C, 5% CO₂). The compounds were added in 2-fold serial dilutions at the same concentrations described before, and the plates were incubated as described before. Each compound concentration was evaluated in duplicate. All the plates included doxorubicin (DOX) (Sigma-Aldrich) as a positive control (10–0.01 μM) and untreated wells as a negative control (100% growth). After 72 h, 15 μL of MTS (Promega) was added, and the plates were incubated for 4 h. The absorbance was measured at 490 nm in an automated SpectraMax 384 microplate reader (Sunnyvale, CA, USA), and the data were analysed using GraphPad Prism version 8.0 for CC₅₀ value calculation. The percentage of nonviable cells was determined and compared to the negative control wells (100% growth).

RAW 264.7 macrophages (ATCC number TIB-71) and 3T3 fibroblast (ATCC CRL-1658) cell lines were cultured in DMEM (Cultilab, Campinas, SP, Brazil) supplemented with 10% (v/v) fetal serum bovine (FBS) (Cultilab, Campinas, SP, Brazil) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). The cells were incubated at 37 C in a humidified atmosphere with 5% CO₂. For maintenance of the cells, the culture medium was changed every three days, and the cells were further subcultured at 80% confluence using a cell scraper (RAW cells) or trypsin/EDTA (3T3 cells).