Figure 5

PEDV enters cells through the caveolae-mediated pathway. A Vero cells and IPEC-J2 cells were incubated with a mixture of Alexa-555 labeled CTB (red) and PEDV (green) at 4 °C for 1 h, and then shifted to 37 °C for 30 min. The cells were fixed and stained for PEDV using monoclonal antibody against S protein. The cellular localizations of CTB and PEDV were observed with a confocal fluorescence microscope. Light exposure was avoided throughout this process. B Vero cells (up) and IPEC-J2 cells (down) were transfected with wild-type caveolin-1 (GFP-Cav-WT) and domain negative mutant of caveolin-1 (GFP-Cav-M), respectively, and infected them with PEDV strains at 24 h after transfection. The cells were fixed at 12 hpi and stained for confocal analysis. C, D The Vero cells and IPEC-J2 cells were transfected with siCav twice and infected with PEDV strains at 24 h after the second transfection. The cells were collected at 6 hpi and 9 hpi for qRT-PCR and Western blotting analysis, respectively. Ctrl means control. E Cells were pre-cooled at 4 °C for 15 min, incubated with PEDV strains at 4 °C for 1 h, shifted to 37 °C to initiate internalization for 10 min, and washed for three times to remove viral particles that were not internalized. The cells were fixed and stained with anti-PEDV-S (red) and anti-caveolin-1 (green) primary antibodies. Scale bars indicate 50 μm in A, 25 μm in B, and 5 μm in E. *P < 0.05; **0.05 < P < 0.01; ***0.01 < P < 0.001; ****P < 0.001.