*2.16. RNA Extraction and Purification*

The RNA extraction (from ZIKV stock, C6/36 EVs isolates, or cells [C6/36, THP-1, HMEC-1] pellets) was performed with the centrifugation protocol using the QIAamp RNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Briefly, the samples were first lysed under highly denaturing conditions using the lysis bu ffer. Next, 70% ethanol was added. Preparations were mixed by pulse/vortexing (Lab-Line Vortex Mixer, Alpha Multiservices, Conroe, TX, USA) for 15 s and incubated for 15 min at RT. The entire volume of the preparations was loaded onto the QIAamp Mini spin columns placed in separation tubes to promote the RNA binding to the columns' membranes. The tubes were centrifuged (Eppendorf 5415 C Centrifuge) at 6000× *g* for 2 min at RT at every step. The contaminants were washed in two steps using the Absorber Waste 1 (AW1) and AW2 bu ffers that were added onto the columns. The tubes were centrifuged at 6000× *g* for 2 min at RT at each step. Finally, eluent bu ffer AVE (RNase-free water with 0.4% sodium azide) was added to the columns, and they were centrifugated at 16,000× *g* for 2 min at RT. The RNA filtrates were collected at 4 ◦C. The RNA was quantified using a NanoDrop ND1000 Spectrophotometer (Thermo Fisher Scientific) with ND-1000 software version 3.5.2. The RT-PCR protocols were performed immediately, as described below, or samples were stored at −72 ◦C.

#### *2.17. ZIKV Inactivation on Viral Stock Samples and ZIKV-Infected C6*/*36 EVs Isolates*

To inactivate the ZIKV virions, viral stock samples were irradiated at 1200 μJ (× 100) in three consecutive cycles on a UV Stratalinker 1800 (Stratagene, San Diego, CA, USA), and genomic RNA was degraded by RNase A activity assays (Figure S4A). Briefly, the total RNA in the samples was quantified, and a concentration of 10 μg/mL of RNAse A (DNase and Protease-free; Thermo Fisher Scientific) was added. The mixtures were then incubated for 1 h, 30 min, and 15 min at 37 ◦C with 5% CO2. A 1:1 proportion of RiboLock RNase Inhibitor (40 U/μL; Thermo Fisher Scientific) was used for 15 min at 37 ◦C with 5% CO2 to inhibit RNase A activity. The RNA degradation pattern was visualized on 2% ethidium bromide-stained (Sigma) 1.2% agarose gel (Invitrogen) using a Typhoon FLA 9500 scanner (GE Healthcare, Chicago, IL, USA) with GE control software version 1.0. The images were analyzed with ImageJ software. The inactivated ZIKV (iZIKV) was evaluated by the lytic plaque assay. The mosquito ZIKV-infected C6/36 EV isolates were irradiated at 1200 μJ (× 100) in three consecutive cycles and treated with RNase A at the best condition of incubation (Figure S4B). The samples were used immediately or held at 4 ◦C. These treatments were identified as EVs (lEVs or sEVs) ZIKV C6/36 (RNase + UV) and were quantified by NTA, the RNA extraction was performed for ZIKV RNA detection by RT-PCR (as describe below), and the lytic plaque assay was performed to evaluate their plaque formation ability in Vero cells (as described above).

#### *2.18. ZIKV RNA Detection by Polymerase Chain Reaction with Reverse Transcriptase (RT-PCR)*

The ZIKV RNA detection in a single step by the RT-PCR reaction has been previously described [39,40]. The master mix was prepared according to the specifications given by the OneStep RT-PCR kit (Qiagen). The primers ZIKV FW [5-GCTGGDGCRGACACHGGRACT-3 ] (Mfg. ID 275853243, Integrated DNA Technologies [IDT], San Diego, CA, USA)] and ZIKV RV [5-RTCYACYGCCATYTGGRCTG-3] (Mfg. ID 275853246, IDT, USA)] developed by Faye et al. [40] were used. The reaction was performed on a GeneAmp PCR System 2400 (Applied Biosystems, Foster City, CA, USA) with the following conditions: pre-PCR at 50 ◦C for 40 min and 95 ◦C for 15 min; 35 cycles at 94 ◦C for 30 s, 45 ◦C for 30 s, and 72 ◦C for 1 min; final elongation at 72 ◦C for 7 min. The amplified cDNA (amplicon of 364 bp) corresponding to the more specific genome region for the ZIKV E protein, which has no cross reaction with other *Flavivirus* [40], was visualized in 2% ethidium bromide-stained 1.2% agarose gel using a Typhoon FLA 9500 scanner with GE control software. The images were analyzed with ImageJ software.

#### *2.19. Quantification of the Total Protein from C6*/*36 EVs Isolates by Micro BCA Protein Assay*

The protein quantification from the C6/36 EVs isolates was performed according to the specification given by the Micro BCA Protein Assay kit (Thermo-Fisher Scientific). The calibration curve standards were performed, in dilutions of 1:2, from a concentrated solution of 2 mg/mL of BSA (Thermo-Fisher Scientific). The blanks, standards, and C6/36 EVs samples (150 μL) were added in triplicate to flat bottom 96-well microplates (Corning) following the addition of 150 μL of the kit work reagen<sup>t</sup> mixture. The plate was covered with sealing tape and incubated at 37 ◦C for 2 h. The absorbances were measured at 562 nm on the Multiskan Ascent spectrophotometer (Thermo Labsystems) with the Ascent software version 2.6. The average of the 562 nm absorbances of the blank samples was rested from the 562 nm reading of each standard and C6/36 EVs samples. The standard curve was used to determine the protein concentration (mg/mL) of each C6/36 EVs sample.

#### *2.20. C6*/*36 EVs Stimulation Assays on Naïve Vero, C6*/*36, THP-1, and HMEC-1 Cells*

Naïve Vero cells were seeded in 24-well culture plates and incubated until confluence. The cells were inoculated with 400 μL of C6/36 EVs isolates in serial log (10-fold) dilutions (in serum-free medium with dilution factors from 10−<sup>1</sup> up to <sup>10</sup>−22) in duplicate and incubated for 2 h. The C6/36 EVs inoculum was removed, and each well was washed with PBS. The monolayers were overlaid with 1 mL of DMEM medium containing 1% carboxymethyl cellulose and 2.5% EV-depleted FBS. The rest of the lytic plaque assay methodology was performed as described above.

Naïve cells (C6/36, THP-1, or HMEC-1) were seeded in 12-well culture plates and incubated until confluence (C6/36 and HMEC-1 cells): For each condition, a strip of 4 plates were used and 2.5 × 10<sup>5</sup> cells/well were added, to collect 1.0 × 10<sup>6</sup> cells at the end of the assay (Figure S5). The following conditions were applied: 0.10 mg of protein in 250 μL/well of EV isolates from mock C6/36 cells (lEVs and sEVs, separately), 0.10 mg of protein in 250 μL/well of EV isolates from ZIKV-infected C6/36 cells (lEVs and sEVs, separately), 0.10 mg of protein in 250 μL/well of EV isolates from ZIKV-infected C6/36 cells RNase A + UV-treated (lEVs and sEVs, separately), and 0.10 mg of protein in 250 μL/well of non-EV ZIKV SNT. The mock cells and ZIKV-infected cells (MOI 1) were used as negative and positive controls, respectively. All conditions were added with 250 μL of non-supplemented media and incubated for 2 h; afterward, 1.0 mL of supplemented media with 5% EV-depleted FBS was added and incubated according to the best period of time established in the ZIKV infection assay: 48 h (C6/36 cells), 72 h (HMEC-1), or 96 h (THP-1 cells). The cell cultures for each condition (in a row of 4 plates) were collected (by scrapping and homogenization by vigorous pipetting) in sterile 1.5 mL microcentrifuge tubes (1.0 × 10<sup>6</sup> cells). For each condition, the ZIKV E protein was detected, and the cytopathic e ffects were observed via light field microscopy. The monocytes (CD11b, CD14, and CD16) and the endothelial vascular cells (CD142 [Tissue Factor, TF], PAR-1, and CD54 [ICAM-1]) were immunophenotyped. The detection of tumor necrosis factor-alpha (TNFα) mRNA expression by RT-PCR was performed as well, as described below.

## *2.21. Monocyte and Vascular Endothelial Cell Immunophenotyping*

The monocytes and endothelial vascular cells from EV stimulation assays were fixed and blocked for nonspecific binding sites as described above. The human monocytes were immunophenotyped, separately, with the mouse PE-conjugated anti-human CD14 antibody (Catalog #325606, BioLegend), the mouse anti-human CD16 antibody (Catalog #555404, BD Pharmingen), and the mouse PE-conjugated anti-human CD11b antibody (Catalog #301306, BioLegend). Likewise, the ECs were immunophenotyped with the mouse FITC-conjugated anti-human CD142 (TF) antibody (Catalog #13133-MM05-F, Sino Biological, USA), the mouse anti-Protease Activated Receptor (PAR-1) antibody (Catalog #sc-13503, Santa Cruz Biotechnology, USA), and the mouse FITC-conjugated anti-human CD54 (ICAM-1) antibody (Catalog # 35-0549-T025, Tonbo Biosciences, USA). For primary antibodies, the Alexa Fluor 555-conjugated secondary antibody was added at a 1:500 dilution in 0.5% BSA. The samples were analyzed by the FACS Calibur flow cytometer.

#### *2.22. Monocytes and Vascular Endothelial Cells TNF-*α *mRNA Expression by RT-PCR*

The RNA extraction was performed from cells collected after EV stimulation assays for the TNFα mRNA detection by RT-PCR. The master mix was prepared according to the specifications given by the OneStep RT-PCR kit (Qiagen), as described above. The primers TNFα FW [5-ACAAGCCTG-TAGCCCATGTT-3 (Mfg. ID 110182256, IDT, USA)], TNFα RV [5-AAAGTAGACCTGCCC-AGACT-3 (Mfg. ID 170166847, IDT, USA)], GAPDH housekeeping FW [5-CCATGTTCGTCATGG-GTGTGAACCA-3 (Mfg. ID 110179057, IDT, USA)], and GAPDH housekeeping RV [5-GCCAGT-AGAGGCAGGGATGATGTTC-3 (Mfg. ID 110179058, IDT, USA)] were used. The reaction was performed on a GeneAmp PCR System 2400 with the following conditions: pre-PCR at 45 ◦C for 60 min and 95 ◦C for 15 min; 35 cycles at 94 ◦C for 30 s, 60 ◦C for 30 s, and 72 ◦C for 30 s; final elongation at 72 ◦C for 10 min. The amplicons [TNFα: 600 bp and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: 294 bp)] were visualized on 2% ethidium bromide-stained 1.2% agarose gel using a Typhoon FLA 9500 scanner with GE control software. The images were analyzed with ImageJ software.

## *2.23. Endothelial Vascular Cells Permeability Assay*

The endothelial vascular barrier integrity after the stimulation with EVs from naïve or ZIKV-infected C6/36 cells was evaluated by a Transwell assay. Briefly, sterile polycarbonate tissue culture-treated Transwell inserts (12 mm) with a 0.4 μm microporous membrane pore size (Corning) were used. The HMEC-1 cells (3.5 × 105) were seeded in the inserts' upper chambers; meanwhile, the lower chambers were filled with MCDB-131 medium supplemented with 5% EVs-depleted FBS. The cultures were incubated for 48 h at 37 ◦C with 5% CO2 to reach confluence. The EVs samples (0.10 mg of protein in 250 μL) from mock C6/36 and ZIKV C6/36 (RNase + UV-treated or untreated), as described above, were applied in the upper chambers (Figure S6). The inserts were incubated for 2 h; afterward, 100 μL of the medium supplemented with 5% EVs-depleted FBS were added in the upper chambers and incubated for 48 h at 37 ◦C with 5% CO2. The mock HMEC-1 cells, non-EV ZIKV SNT (0.10 mg of protein in 250 μL), and ZIKV-infected HMEC-1 (MOI 1) were used as controls. To determine the cellular permeability degree, FITC (40 kDa)-Dextran (Catalog #60842-46-8, Sigma-Aldrich) was diluted to 1:60 in MCDB-131 medium supplemented with 5% EV-depleted FBS; afterward, 250 μL was added to each upper chamber. An empty insert (without cells) with FITC-Dextran was identified as the no-cell control (NCC), corresponding to 100% of permeability. The inserts were incubated for 1 h at 37 ◦C with 5% CO2. The fluorescence emitted by the FITC-Dextran solution that passes through the cell monolayer to the lower chamber was measured as follows: 100 μL of each lower chamber medium was separated and diluted to 1:50; afterward, the media were passed to a black 96-well plate (Merck) that was analyzed at 492/520 nm in a Synergy H4 hybrid multi-mode microplate reader (BioTek Instruments Inc., USA) with the Gen5 software version 2.09. The permeability percentage (p%) was calculated according to the following formula: p% = (Abs InsertX/Abs NCC) × 100, where *Abs InsertX* corresponds to the absorbance of each different EV stimulus condition or control, and *Abs NCC* corresponds to the absorbance of the no-cell control.
