*2.8. Transmission Electron Microscopy (TEM)*

The placental tissues of control and ZIKV-infected women were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h at room temperature (RT). Then, the tissue was incubated for 20 h at 4 ◦C in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, washed thrice with 0.1 M sodium cacodylate buffer, and incubated for 1 h at RT with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer containing 0.5 mg/mL of ruthenium red, a marker of the paracellular pathway. Samples were then processed for TEM, as previously described [27].

## *2.9. Measurement of Transepithelial Electrical Resistance (TER)*

The transepithelial electrical resistance of BeWo cells plated at confluence on Transwell filters (12-well plate, 0.4 μm, Cat. 3460, Corning, Kennebunk, ME) was continuously measured from each insert using the automated cell monitoring system, cellZscope (nanoAnalytics GmbH, Munster, Germany). TER values were obtained using the cellZscope software version 4.3.1. Twenty-four hours after plating, when the monolayers had achieved a steady state value of TER, cells were infected with ZIKV (strain PRVABC-59-Asian, ATCC® VR-1843™) at a MOI of 1.
