Primary cytotrophoblast cells from (A) term placentas and (C) HTR8 cells were plated for 14 days with supernatant of JFH-1 infected Huh7.5.1 at an MOI=10. Cells were stained for F-actin (red) and NS5A [green (A), blue (C)] (magnification 40X). Cells were visualized using confocal microscopy as described in the Methods. Bar represents 10 μm. Densitometry analysis confirmed significantly higher expression of NS5A protein in JFH-1-infected (B) primary syncytialized cytotrophoblasts and (D) HTR8 cells compared to non-infected control cells. Bars represent mean plus SEM, t test (n=3), * p<0.05, ** p<0.01. (E) HTR8 infected for 14 days with supernatant of JFH-1 infected Huh7.5.1 were stained for NS5A (green), early endosomal antigen 1 (EEA1, red) and actin (blue). Co-localization of NS5A with EEA1 suggests that endocytosis is involved in HCV uptake by human trophoblast cells.