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

The plasmids IFN-β-Luc for IFN-β, PRD (III-I) 4-Luc for IRF-3, and pNF-κB-Luc for NF-κB were kindly donated by Dr. Shaobo Xiao (Huazhong Agricultural University, Wuhan, Hubei Province, China) [25] . The pEF-BOS empty vector and pEF-Flag-RIG-I recombinant expression plasmid were kindly provided by T. Fujita (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) [32] . The pEF-Bos-Flag-TRIF, pCDNA3-Flag-IKKε and pCDNA3-Flag-TBK1 recombinant expression plasmids, and the pCDNA3 empty vector, and the conjugate IRF3-green fluorescence protein (GFP) expression construct were kindly provided by K. Fitzgerald (University of Massachusetts Medical School, Worcester, MA, USA) [27] . The porcine IPS-1 (NCBI accession No: EU082069.1) gene was cloned from porcine kidney cells by reverse transcription polymerase chain reaction (RT-PCR) using the specific primer pair. The IPS-1 primers were 5′-CCGGGTACCACCATGACGTTTGCCGAGGACAA-3′ and 5′-TTTCTCGAGTCACTGGGGCAGGCGCCGCC-3′. Porcine IPS-1 was inserted into pcDNA3.1 (+) using the restriction enzymes KpnI and XhoI. Cells were harvested and luciferase activity was analyzed using a dual-luciferase assay. All data are expressed as means ± SD of 3 independent experiments. **p < 0.01 compared with PEDV-infected, expression plasmids or vector-transfected control when the cells reached 70 %-80 % confluence. The cells were then infected or mock-infected with PEDV for 24 h. Cells were retransfected with or without poly (I:C) (1.0 μg) for an additional 12 h. Or IECs were infected or mock-infected with PEDV for 12 h prior to transfection the luciferase reporter plasmids alone or cotransfection the indicated expression plasmids (0.5 μg). The cell lysates were harvested and luciferase activity was analyzed using a dual-luciferase assay system and a luminometer (Turner BioSystems, Inc. Sunnyvale, CA, USA) according to the manufacturer's instructions. Data represent relative firefly luciferase activity normalized to Renilla luciferase activity. The resulting ratios were used to compare the expression of the firefly luciferase gene in PEDVinfected cells to that present in mock-infected cells.