*2.4. ZIKV Infection Assay*

Cell cultures (mosquito C6/36, human monocytes, or endothelial vascular cells) were seeded in 12-well plate culture (Corning) until confluence. They were then infected with ZIKV at a multiplicity of infection (MOI) of 1, as previously described [31–33] and incubated for 2 h at 37 ◦C in 5% CO2. After removal of the viral inoculum, cells were maintained in media supplemented with 5% EV-depleted FBS and incubated for 24, 48, 72, 96, and 120 h. Cytopathic effect formation was observed by light field microscopy (Olympus IX71 inverted microscope; Olympus Corp. Miami, FL, USA). Images were taken with a digital camera (Olympus DP72) attached to the microscope and analyzed with ImageJ software version 1.50i (Wayne Rasband, National Institutes of Health, Bethesda, MA, USA). The cell infection was evaluated by ZIKV envelope (E) protein detection as described below.

#### *2.5. ZIKV Envelope (E) Protein Detection in ZIKV-Infected Cells by Cytofluorometry (FACS)*

The ZIKV E protein detection at the cell membrane's surface was performed as follows: The different cells were removed from the cell culture plates (C6/36, THP-1, or HMEC-1 were scrapped and homogenized by vigorous pipetting) in sterile 1.5 mL microcentrifuge tubes (Labcon, Petaluma, CA, USA), centrifuged (Eppendorf Centrifuge 5415 R; Eppendorf International, Hamburg, Germany) at 550× *g* for 10 min at 4 ◦C, and separated from the medium. The cell pellets were fixed with 2% paraformaldehyde (Sigma) for 5 min at 4 ◦C, blocked for nonspecific binding sites with 2% bovine serum albumin (BSA; Biowest) for 30 min at RT, and washed with 0.5% BSA. The samples were stained with mouse anti-ZIKV E protein clone 1,413,267 antibody (Catalog #CABT-B8528, CD Creative Diagnostics, New York, NY, USA) at a 1:300 dilution in 0.5% BSA and incubated overnight at 4 ◦C with constant 1000 rpm agitation (Vibrax VXR basic; IKA, Wilmington, NC, USA). After washing with 0.5% BSA and centrifugation, the Alexa Fluor 555-conjugated anti-mouse IgG (H + L), a highly cross-adsorbed secondary antibody (Catalog #A-31570, Thermo Fisher Scientific), was added at a 1:500 dilution in 0.5% BSA, incubated for 2 h at room temperature (RT) with constant 1000 rpm agitation, washed with 0.5% BSA, and centrifugated. The samples were suspended in 300 μL of 0.5% BSA and analyzed by the FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with CellQuest software. The mock cells were treated in the same way as the infected cells (the mock FACS average values were rested from the infected FACS values).

#### *2.6. ZIKV Envelope (E) Protein Detection in ZIKV-Infected Cells by Immunofluorescence (IF)*

To confirm the ZIKV E protein presence at the cell membrane surface level, an IF assay was performed as follows: The mosquito C6/36 cells were seeded on an 8-well separation chamber slide system (Lab-Tek II; Thermo Fisher Scientific), incubated until confluence, and infected as described above. The cells were fixed with 2% paraformaldehyde for 5 min at 4 ◦C, blocked for nonspecific binding sites with 2% BSA for 30 min at RT, and washed with 0.5% BSA. The cells were stained with mouse anti-ZIKV E protein antibody at a 1:300 dilution in 0.5% BSA and incubated overnight at 4 ◦C with constant 1000 rpm agitation. After washing with 0.5% BSA, the Alexa Fluor 555-conjugated anti-mouse IgG was added at a 1:500 dilution in 0.5% BSA, incubated for 2 h at RT, and washed with 0.5% BSA. The separation chamber was then withdrawn, the slide was covered with mounting medium with DAPI (FluoroQuest; AAT Bioquest, Sunnyvale, CA, USA), and a coverslip (Corning) was placed. The samples were observed via fluorescence microscopy (Olympus IX71 inverted microscope; Olympus Corp.), and the images were analyzed with ImageJ software.

#### *2.7. Mosquito C6*/*36 EVs Isolation from the Cell Culture Medium by Ultracentrifugation*

Mosquito C6/36 cells were seeded in cell culture flasks T75 (Corning). The EV isolation from the culture media of the mock and ZIKV-infected cell culture flasks were performed by ultracentrifugation (Figure S1). Briefly, C6/36 cells culture media (50 mL) were collected in sterile conical tubes and centrifuged (GH3.8 rotor, Beckman GPR Centrifuge, Beckman Coulter, Inc.) at 900× *g* for 10 min at 4 ◦C. The viable cell pellet was discarded. The supernatant was transferred to sterile conical tubes and centrifuged at 2000× *g* for 10 min at 4 ◦C. The debris pellet was discarded. The supernatant was transferred to 25 × 89 mm centrifuge tubes (Beckman Coulter, Inc.) and centrifuged (SW28 rotor, Beckman XL-90 Centrifuge) at 10,000× *g* for 35 min at 4 ◦C. The lEVs pellet was suspended in 1 mL of PBS at 4 ◦C and used immediately or stored at −72 ◦C (Revco ULT1786, Thermo Fisher Scientific). The supernatant was transferred to centrifuge tubes and centrifuged at 120,000× *g* for 70 min at 4 ◦C. The supernatant was then discarded. The sEVs and contaminant protein pellet was washed in 5 mL of PBS at 4 ◦C and incubated for 30 min at RT with constant 100 rpm agitation. The suspension was filtered with a 0.22 μm pore filter, and 25 mL of PBS was added. The samples were transferred to centrifuge tubes and centrifuged at 120,000× *g* for 70 min at 4 ◦C. The last supernatant obtained in this process was separated and identified as non-EV ZIKV SNT (used as a control in the EV stimulation assays) and stored at −72 ◦C. The sEVs pellet was suspended in 1 mL of PBS at 4 ◦C and used immediately or stored at −72 ◦C [22,34].

#### *2.8. Characterization of EVs from Mosquito C6*/*36 Cells by Nanoparticle Tracking Analysis (NTA)*

The characterization of EVs was performed by the detection of reflected light emitted by the Brownian motion of nanoparticles suspended in solution by nanoparticle tracking analysis (NTA) with the help of the NanoSight NS300 equipment and Malvern Instruments software. The optimal detection conditions were previously established (Figure S2A), and quantitative controls with 100 and 200 nm polystyrene microspheres (NTA4088 and NTA4089, Malvern Panalytical Products, Mexico City, Mexico) were used (Figure S2B). The nanoparticle concentration values (particles/mL) and the size (nm) were determined for each measurement. The ZIKV virions were detected (Figure S2C) to identify their presence in the EV samples from ZIKV-infected mosquito cells. In a parallel assay, the nanoparticles present in PBS and in FBS-EV-depleted were quantified to rest the number of nanoparticles obtained in the mock and ZIKV-infected C6/36 EV isolates.

#### *2.9. C6*/*36 lEVs Phosphatidylserine (PS)*+ *Detection by an Annexin-V Binding Assay*

Phosphatidylserine (PS) is located on the cytoplasmic surface of the cell plasmatic membrane, and, during the lEVs biogenesis, PS is translocated from the inner to the outer leaflet of the membrane, exposing the PS that can be detected by the Annexin-V binding assay [35]. The lEVs samples (50 μL) from C6/36 cells were suspended in 200 μL of the Annexin-V binding buffer 1× (Catalog #556454, BD Pharmingen, BD Biosciences, San Jose, CA, USA) that contained fluorescein isothiocyanate (FITC)-conjugated Annexin-V (Catalog #640906, BioLegend, San Diego, CA, USA) at a 1:200 dilution and were incubated for 20 min at RT with constant 100 rpm agitation. After a wash with 250 μL of PBS and centrifugation at 10,000× *g* for 35 min at 4 ◦C, samples were suspended in 300 μL of PBS. Polystyrene microspheres of a 1 μm diameter size (Polysciences, Inc., Warrington, PA, USA) were used as a FACS calibration control. The samples were analyzed by a FACSCalibur flow cytometer.

#### *2.10. Mosquito C6*/*36 Cell Tetraspanin CD63-Like Protein Detection by FACS*

Tetraspanin-like proteins in arthropod cells, including mosquito C6/36 cells, have been described previously [16,30,36–38]. Briefly, the C6/36 cells (mock and ZIKV-infected cells) were collected from the cell culture plates in sterile 1.5 mL microcentrifuge tubes, centrifuged at 550× *g* for 10 min at 4 ◦C, and separated from the medium. The cell pellets were fixed with 2% paraformaldehyde for 5 min at 4 ◦C, permeabilized with 0.1% Triton X-100 (Sigma) for 5 min at 4 ◦C, blocked for nonspecific binding sites with 2% BSA for 30 min at RT, and washed with 0.5% BSA. The cells were stained with the phycoerythrin (PE)-conjugated mouse anti-human CD63 antibody (Catalog #557305, BD Pharmingen) at a 1:20 dilution in 0.5% BSA and incubated for 1 h at RT with constant 1000 rpm agitation. The samples were suspended in 300 μL of 0.5% BSA and analyzed by using the FACSCalibur flow cytometer. The mouse IgG1 kappa (P3.6.2.8.1) antibody (Catalog #14-4714-82, eBioscience, San Diego, CA, USA) was used as an isotype control. Isotype FACS average values were rested from the mock and the infected cells FACS values.

#### *2.11. Mosquito C6*/*36 Cells Tetraspanin CD63-Like Protein Detection by Immunofluorescence Assay*

To confirm the CD63-like protein presence at the cells membrane's surface and in the cytosol, the immunofluorescence (IF) assay was performed as follows: The C6/36 cells were seeded on an 8-well separation chamber slide system, incubated until confluence, and infected as described above. The cells were fixed with 2% paraformaldehyde for 5 min at 4 ◦C, permeabilized with 0.1% Triton X-100 (Sigma) for 5 min at 4 ◦C, blocked for nonspecific binding sites with 2% BSA for 30 min at RT, and washed with 0.5% BSA. The cells were stained with the PE-conjugated mouse anti-human CD63 antibody at a 1:20 dilution in 0.5% BSA and incubated for 1 h at RT. The mouse IgG1 kappa antibody was used as an isotype control. Finally, the mock and the ZIKV-infected cells were stained with mouse anti-ZIKV E protein antibody at a 1:300 dilution in 0.5% BSA and incubated overnight at 4 ◦C with constant 1000 rpm agitation. After a wash with 0.5% BSA, the FITC-conjugated anti-mouse IgG (H + L), a highly cross-adsorbed secondary antibody (Catalog #AP308F, Merck, Kennersburg, NJ, USA), was added at a 1:500 dilution in 0.5% BSA, incubated for 2 h at RT with constant 100 rpm agitation, and washed with 0.5% BSA. The separation chamber was withdrawn, the slide was covered with mounting medium with DAPI, and a coverslip was placed. The samples were observed by fluorescence microscopy, and the images were analyzed with ImageJ software.

#### *2.12. C6*/*36 sEVs CD63*+ *Detection (FACS) by Coupling to Anti-CD63-Coated Paramagnetic Nanobeads*

To identify the presence of the tetraspanin CD63 (sEVs marker) on the sEVs membrane's surface, the C6/36 sEVs isolates were coupled with anti-CD63-coated paramagnetic nanobeads (Catalog #10606D; Invitrogen, Thermo Fisher Scientific) to be detected by cytofluorometry (Figure S3). Briefly, in sterile round-bottommicrocentrifuge tubes (Labcon), 100μL of the sEVs suspension and 20μL of the paramagnetic nanobeads were added. The samples were incubated for 24 h at 4 ◦C with constant 1000 rpm agitation. After a wash with 300 μL with PBS, magnetic separation of the bead-coupled sEVs from the matrix suspension was performed using a DynaMag-2 magne<sup>t</sup> (Life Technologies, Thermo Fisher Scientific), and the supernatant was discarded. The bead-coupled sEVs were suspended in 300 μL of 0.5% BSA. For CD63 detection by FACS, the PE-conjugated mouse anti-human CD63 antibody was used as described above. The samples were analyzed using a FACSCalibur flow cytometer. The paramagnetic nanobeads were treated in the same way as the bead-coupled sEVs (the paramagnetic bead FACS average values of the bead-coupled sEVs were rested from mock and ZIKV-infected cells FACS values).

#### *2.13. C6*/*36 EVs Morphological Characterization by Transmission Electron Microscopy (TEM)*

The C6/36 EV isolates were fixed with a 1:1 mixture of 2.5% glutaraldehyde (Electron Microscopy Sciences [EMS], Hatfield, PA, USA) and 4% paraformaldehyde (Sigma) for 2 h at RT and washed three times (5 min each) with PBS. After fixation, the samples were incubated with 2% osmium tetroxide (Alfa Aesar, Thermo Fisher Scientific) for 90 min at RT. The fixed pellets were washed three times with PBS and dehydrated in an ascending graded series of ethanol (30, 50, 70, 80, 90, and 96%), including three passes (5 min each) in absolute ethanol (J.T.Baker) at RT. Three passes (5 min each) in propylene oxide (Sigma-Aldrich) at RT were then performed. The samples were placed in a 1:1 mixture of propylene oxide/epoxy resin for 18 h at RT and embedded in pure epoxy resin (EMS) at 60 ◦C for 48 h. Ultrathin sections (40–50 nm thick) were obtained in an ultramicrotome (Leica EM UC7, Leica Microsystems, Bu ffalo Grove, IL, USA) and mounted on copper grids (EMS) covered with formvar (EMS). The sections were contrasted with uranyl acetate (Merck, Kennerworth Fort, NJ, USA) for 30 min and lead citrate (EMS) for 10 min at RT. The preparations were observed with a transmission electron microscope (JEM1010 model; JEOL, Peabody, MA, USA) operating at 80 kV. The images were captured with a CCD300-RC camera (DAGE-MTI, Michigan City, IN, USA) adapted to the microscope and analyzed with ImageJ software.

#### *2.14. ZIKV E Protein Detection in lEVs Isolates from ZIKV-Infected C6*/*36 Cells by FACS*

The ZIKV E protein detection at the lEVs membrane's surface was performed as follows: The lEVs isolates were centrifuged at 10,000× *g* for 35 min at 4 ◦C, and the supernatant was discarded. The lEVs pellets were fixed with 2% paraformaldehyde for 5 min at 4 ◦C, blocked for unspecific binding sites with 2% BSA for 30 min at RT, and washed with 0.5% BSA. The lEVs were stained with mouse anti-ZIKV E protein antibody at a 1:300 dilution in 0.5% BSA and incubated overnight at 4 ◦C with constant 1000 rpm agitation. After washing with 0.5% BSA and centrifugation, the FITC-conjugated anti-mouse IgG secondary antibody was added at a 1:500 dilution in 0.5% BSA, incubated for 2 h at RT with constant 1000 rpm agitation, washed with 0.5% BSA, and centrifugated. The samples were suspended in 300 μL of 0.5% BSA and analyzed by the FACSCalibur flow cytometer with CellQuest software. The lEVs from mock cells were treated in the same way as those from the infected cells (the lEVs mock C6/36 FACS average values were rested of the lEVs ZIKV C6/36 FACS values).

#### *2.15. ZIKV E Protein Detection in sEVs Isolates from ZIKV-Infected C6*/*36 Cells by FACS*

The ZIKV E protein detection at the sEVs membrane's surface was performed as follows: The sEVs isolates were coupled with anti-CD63-coated paramagnetic beads as described above. The bead-coupled sEVs were fixed with 2% paraformaldehyde for 5 min at 4 ◦C, blocked for nonspecific binding sites with 2% BSA for 30 min at RT, and washed with 0.5% BSA. The bead-coupled sEVs were stained with mouse anti-ZIKV E protein antibody at a 1:300 dilution in 0.5% BSA and incubated overnight at 4 ◦C with constant 1000 rpm agitation. After washing with 0.5% BSA and recovery by magnetic separation, the Alexa Fluor 555-conjugated anti-mouse IgG secondary antibody was added at a 1:500 dilution in 0.5% BSA, incubated for 2 h at RT with constant 1000 rpm agitation, washed with 0.5% BSA, and recovered by magnetic separation. The samples were suspended in 300 μL of 0.5% BSA and analyzed by the FACSCalibur flow cytometer with CellQuest software. The EVs from mock cells were treated in the same way as those from the infected cells (the sEVs mock C6/36 FACS average values were subtracted from the sEVs ZIKV C6/36 FACS values).
