Figure 4.

ZIKV crosses endothelial cell monolayer, without increasing permeability. (A) HBMECs were cultured onto transwell plates and the cells were mock-treated or infected with ZIKV_PE243 or ZIKV_MR766. TEER was measured at 24 and 48 h post infection. (B) HBMECs were infected as in (A); as controls, the cells were cultured with staurosporin (STS). After 72 hpi, cells were incubated with FITC-BSA for 30 min, the amount of extravasated albumin was measured by spectrophotometry, and the permeability coefficient (Pd) was calculated and normalized in relation to cells cultured in medium only. (C) Virus RNA was measured in the luminal (upper) and abluminal (lower) chambers of the transwell plates by qRT-PCR. Insert numbers indicate the percentage of RNA copies in relation to the corresponding upper chamber. (D) Conditioned media harvested from the lower transwell chamber of ZIKV_PE243 infected cells were inoculated into Vero cells. After 48 h, virus RNA present in the cell lysates and supernatant from Vero cells were measured by qRT-PCR. (E) HBMECs and Vero cells were cultured in the upper and lower chamber of a transwell plates, respectively. HBMECs were infected from the apical side, as described. After 72 hpi, Vero cells were harvested, and ZIKV negative strand RNA was measured by qRT-PCR. As positive and negative controls, Vero cells were mock-treated or directly infected with ZIKV_PE243 (no membrane); nd, not detected. (F) HBMECs were cultured as in (A). After 48 hpi, the cells were stained with anti-Flavivirus (4G2 antibody), followed with anti-mouse IgG-AlexaFluor488; and with anti-β-catenin, followed by anti-rabbit IgG-AlexaFluor 594; and with DAPI. ZIKV infection and β-catenin expression were then analyzed by immunofluorescence. Data are represented as mean ± SD of four independent experiments.