*2.2. ZIKV Propagation and Titration*

The ZIKV MR766 strain with passage (P) number 3 was propagated in C6/36 cells and titrated in confluent Vero monolayers and was used in all experiments. The ZIKV titer was determined by the lytic plaque assay as follows: Vero cells were seeded into a 24-well culture plate (Corning, Corning, NY, USA) and incubated at 37 ◦C in 5% CO2 until confluence. Each culture well was inoculated with 400 μL of diluted ZIKV (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 at 37 ◦C in 5% CO2. The viral inoculum was removed, and each well was washed with Phosphate Buffer Saline (PBS) 1× (pH 7.4). The monolayers were overlaid with 1 mL of DMEM medium containing 1% carboxymethylcellulose (Sigma, USA) and 2.5% FBS and afterwards incubated at 37 ◦C in 5% CO2. The plaque formation began to be observed at the 7th day post-infection (PI). Cells were fixed with 96% methanol (J.T.Baker, Fisher Scientific, Allentown, PA, USA) on the 14th day PI and stained with 1% crystal violet (Sigma) for 15 min. The titer was calculated and expressed as plaque-forming units (PFU) per milliliter (mL), according to the following formula: PFU/mL = N/(*V* × *D*), where *N* corresponds to the average number of plaques counted, *V* is the inoculated volume of the viral dilution, and *D* is the less concentrated dilution from which the plaques were counted.

#### *2.3. Preparation of Fetal Bovine Serum Depleted of Extracellular Vesicles (EVs)*

The FBS was collected in sterile conical tubes (Corning) and centrifuged (GH3.8 rotor, Beckman GPR Centrifuge; Beckman Coulter, Inc., Brea, CA, USA) at 900× *g* for 10 min and filtered using a 0.22 μm pore (Millipore, Burlington, MA, USA). The samples were then centrifuged (SW28 rotor, Beckman XL-90 Ultracentrifuge, Beckman Coulter, Inc.) at 120,000× *g* for 18 h at 4 ◦C [22] and stored at 2 ◦C until use.
