Porcine epidemic diarrhea virus inhibits dsRNA-induced interferon-ß production in porcine intestinal epithelial cells by blockade of the RIG-I-mediated pathway

Cao, Liyan; Ge, Xuying; Gao, Yu; Herrler, Georg; Ren, Yudong; Ren, Xiaofeng; Li, Guangxing

IECs were seeded in 24-well plates and then transfected with 1 μg of IRF3-GFP fusion expression constructs per well using Lipofectin transfection reagent (Invitrogen Corp.) when cells reached confluence. Cells were then mock-infected or infected with PEDV at a multiplicity of infection (MOI) of 1. At 24 h postinfection, cells were transfected with 1 μg of poly (I:C) or left untransfected. After 12 h, cells were removed from the culture medium and washed three times in cold phosphatebuffered saline (PBS). Next, cells were fixed in 4 % paraformaldehyde for 15 min at room temperature, quenched with 0.1 M glycine for 5 min, and then permeabilized with 0.1 % Triton X-100 for 10 min. Afterward, the cells were incubated with anti-PEDV antibody (dilution, 1:500) for 1 h followed by TRITClabeled goat anti-rabbit secondary antibody (dilution, 1:200) for 30 min at 37°C. The nuclei were stained with 4′,6-diamidino-2-phenylindole-dihydrochloride (DAPI) (Invitrogen Corp.). Cells were examined using a TCS SP2 AOBS confocal microscope (Leica Camera AG, Wetzlar, Germany).