Background

Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease of swine characterized by severe enteritis, vomiting, and watery diarrhea and results in high mortality in piglets [1]. The causative agent belonging to the family Coronaviridae is porcine epidemic diarrhea virus (PEDV), which is first reported by Pensaert and DeBouck in Belgium and the United Kingdom [2, 3]. PED is currently a source of concern in Asia countries, where outbreaks are often more acute and severe than those observed in Europe [4]. PED is one of the most important diseases incurring economic loss in many swine-raising countries, mainly due to its high prevalence, compared to the rare incidence of transmissible gastroenteritis (TGE) and the asymptomatic characteristics of the Rotavirus (RV) infections [3, 5]. PEDV is an enveloped virus possessing an approximately 28 kb, positive-sense, single-stranded RNA genome with a 5’ cap and a 3’ polyadenylated tail [4, 36].

Table 1 Sequences of primer pairs used for qRT-PCR
Full size table

Detection of NF-κB activity

To determine the alteration of NF-κB activity by GFP and GFP-E proteins in the established cell lines, the level of NF-κB activity was measured using western blot assay and the NF-κB p65 TransAM kit (Active Motif) according to the manufacturer’s instructions. Briefly, cells nuclear extraction was prepared by using the Nuclear Extract Kit (KeyGEN, Nan**g, China) and protein concentrations were measured using the BCA Protein Assay Reagent (Pierce, Rockford, IL, US). Lysates (50 μg total proteins) were incubated in ELISA wells coated with the oligo-nucleotide motif recognized by active p65, then detected using a specific antibody against p65, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. The colorimetric reaction was measured at 450 nm. This experiment was repeated three times.

Enzyme-linked immunosorbent assay (ELISA)

The stable PEDV E gene expressing cells and the control cells were seeded in 24-well plates at a density of 1 × 105 cells/ml in DMEM with 10% new born calf serum (NCS) and cultured for 48 h. In some experiments, MG132 previously found to block IL-8 expression was added after 24 h [37]. The culture medium was then collected and centrifuged in a microcentrifuge at 1, 000 × g for 5 min to remove debris, the supernatants were then frozen at −80°C until analysed. The concentrations of IL-8 were measured using a swine IL-8 ELISA kit according to the manufacturer’s instructions (Invitrogen, USA).

Statistical analysis

Data are shown as the means ± SD of three independent experiments done in triplicate. For each assay, student’s t-test was used for statistical comparison. A value of P < 0.05 was considered significant.

Authors’ information

Dr. De-Wen Tong, professor of College of Veterinary Medicine, Northwest A&F University, Vice Dean of College of Veterinary Medicine, Northwest A&F University. Dr. Hung-Jen Liu, professor of Institute of Molecular Biology, National Chung Hsing University. Dr. **n-Gang Xu and Dr. Yong Huang are associate professors of College of Veterinary Medicine, Northwest A&F University. Honglei Zhang, graduate students of College of Veterinary Medicine, Northwest A&F University.