NiV infection and permeability of primary endothelial cells. Primary porcine brain microvascular endothelial cells (PBMEC) were cultured on fibronectin-coated filter supports for 6 days. Then, cells were infected with NiV at a m.o.i. of 0.5. (A) At 24 h p.i., cells were fixed with 4% PFA for 48 h. Subsequently, cells were stained with an NiV-specific guinea pig antiserum and AlexaFluor 568-conjugated secondary antibodies. After permeabilization with 0.1% TX-100, cell junctions were visualized with a monoclonal antibody directed against VE-cadherin and AlexaFluor 488-conjugated secondary antibodies. Magnification, 400×. (B) Effect of NiV infection on the permeability of endothelial monolayers. HRP (5 μg/ml) was added to the apical filter chamber of a filter insert with uninfected PBMEC (mock cells), or to filter inserts with NiV-infected PBMEC at 6 or 24 h p.i. (NiV 6 h p.i. or NiV 24 h p.i.). Apical-to-basolateral HRP passage was quantified by measurement of the HRP activity in the medium of the basal filter chamber every 10 min, and is given as means of 3 independent experiments normalized to the HRP concentration in mock-infected control wells.