Figure 1.

USUV and WNV differentially impact the human BBB integrity in vitro. (a) The human in vitro BBB model used in this study is composed of brain pericytes (basolateral compartment) allowing the differentiation of CD34⁺-derived endothelial cells towards hBLEC on transwell filters (apical compartment). (b) After 2 h infection of hBLECs (MOI of 0.1, inoculum represented on Y axis), cell were rinsed and refilled with fresh medium, supernatants from apical and basolateral compartments were collected at 6 and 12 h post-infection, then at 1, 2, 4, 7 and 10 dpi; USUV (blue dots) and WNV (orange dots) replication was determined using the TCID50 method. Results are expressed as mean ± SEM (n = 12, from six independent experiments). (c) At 7 dpi, mRNA of mock, USUV or WNV-infected (MOI of 0.1) hBLECs were collected. The expression of tight junction factors and transporters were normalized to housekeeping gene HPRT1 and compared to mock-infected hBLECs (CTL) in the context of USUV (blue bar chart) and WNV (orange bar chart) infection. Results are expressed as mean ± SEM (n = 6; * p < 0.05, ** p < 0.01, from three independent experiments). (d) Infected- or mock-infected hBLECs (MOI of 0.1) were fixed after 10 dpi and indirect immunofluorescence assays performed to show hBLECs structure with actin (green), viral replication with pan-flavivirus (magenta), junctions with β-catenin labelling (cyan) and nuclei with Hoescht (blue). Scale bar 10 µm. (e) The permeability coefficient (Pe) was measured using the Lucifer Yellow transport assay at 10 dpi with USUV at MOI 0.1 (blue), USUV MOI 1 (dark blue), WNV MOI 0.1 (orange), WNV MOI 1 (dark orange) and mock-infected CTL (black). Bars represent mean ± SEM (n = 6; * p < 0.05, ** p < 0.01, from three independent experiments).