Depletion of cholesterol by MβCD inhibits JEV infection. (A) PK15 cells were treated with 10 mM MβCD for 1 h and infected with JEV in the presence of inhibitor; 36 hours post-infection (hpi), the cells were fixed and stained with anti-JEV E primary and Alexa488 anti-mouse IgG secondary antibodies. The nuclei were stained with DAPI. Scale bars, 50 μm. (B) The cells were treated with MβCD as described above and then infected with JEV; 36 hpi, the cells were lysed and processed for western blot analysis of JEV E protein. β-actin was used as an internal loading control. +, control cells untreated with MβCD; -, control without JEV infection. (C) PK15 cells were untreated (control) or treated with 10 mM MβCD for 1 h at 37°C and then incubated with 10 μg/ml AF 555-labelled Cholera toxin B (CTxB) for 30 min at 37°C. Nuclei were stained with DAPI. Bar, 10 μm. (D) PK15 cells were plated on 96-well plates and then either left untreated (as a control) or treated with MβCD (10 mM) for 6 h at 37°C. After treatment, cell viability was determined with a CCK-8 kit. The results are presented as the mean ± SD of three independent experiments.