Fig. 2

PEDV inhibits dsRNA-mediated IRF-3 activation, but not NF-κB. a IECs were infected with PEDV at an MOI of 1 for 12, 24, 36 and 48 h, or treated with poly (I:C) for 12 h. To detect the activation of IRF-3 and p65 after PEDV infection, the cell extracts were prepared at the indicated times and subjected to western blot analysis with antibodies specific for IRF-3, p65, p-IRF-3, p-p65 and PEDV N McAb. Anti-β-actin was included as a control for sample loading. These experiments were performed in duplicate. b and c IECs were infected or mock-infected with PEDV at an MOI of 1 for 12 h, and then cells were cotransfected with (PRDIII-I) 4-Luc (b) or pNF-κB-Luc (c) and phRL-TK for additional 24 h. Cells were retransfected with poly (I:C) for 12 h, harvested, and then subjected to a dual-luciferase assay. All data are expressed as means ± SD of 3 independent experiments. **p < 0.01 as compared with poly (I:C). d IRF3-GFP fusion protein transfected with IECs and then infected with PEDV at an MOI of 1 and mock-infected cells served as negative controls. 24 h later, cells were retransfected with poly (I:C) (positive control) (c and d) or untransfected (a and b) for 12 h. Cells were fixed with 4 % paraformaldehyde, permeabilized with 0.1 % Triton X-100, and stained by DAPI (blue). Cells were incubated with anti-PEDV rabbit polyclonal antibody (red) and TRITC-labeled goat anti-rabbit secondary antibody, then analyzed for fluorescence by confocal microscopy. Magnification, ×20 (Leica, Wetzlar, Germany)