Significant Interference with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strain Replication in Small-Intestine Epithelial Cells Using an shRNA Expression Vector

Abstract

Porcine epidemic diarrhea (PED) re-emerged in China in 2010 and is now widespread. Evidence indicates that highly virulent porcine epidemic diarrhea virus (PEDV) strains belonging to genotype G2 caused a large-scale outbreak of diarrhea. Currently, vaccines derived from PEDV classical strains do not effectively prevent infection by virulent PEDV strains, and no specific drug is available to treat the disease. RNA interference (RNAi) is a novel and effective way to cure a wide range of viruses. We constructed three short hairpin RNA (shRNA)-expressing plasmids (shR-N307, shR-N463, and shR-N1071) directed against nucleocapsid (N) and determined their antiviral activities in intestine epithelial cells infected with a classical CV777 strain and LNCT2. We verified that shR-N307, shR-N463, and shR-N1071 effectively inhibited the expression of the transfected N gene in vitro, comparable to the control shRNA. We further demonstrated the shRNAs markedly reduced PEDV CV777 and LNCT2 replication upon downregulation of N production. Therefore, this study provides a new strategy for the design of antiviral methods against coronaviruses by targeting their processivity factors.

Keywords: N gene; RNA interference; intestine epithelial cells; porcine epidemic diarrhea virus; processivity factor.

Figure 1

Schematic description of the target viral genome, and small interfering RNA (siRNA)-expressing cassette. (A) Genomic structure of porcine epidemic diarrhea virus (PEDV) and position of target short hairpin RNA (shRNA) at N gene. ShR-N307, shR-N463, and shR-N1071 indicate initial shRNA target sites; (B) sequences and design map for shRNA constructs; (C) structure of predicted shRNA.

Figure 2

Phylogenetic analysis and virus-induced cytopathic effect (CPE) in intestine epithelial cells (IECs). (A) Phylogenetic trees of PEDV based on complete genomic DNA. Black squares, strains in United States of America (USA); black triangles, strains in South Korea; black rhombuses, strains in China; red circle, G1 CV777 strain from this study; red rhombus, G2 LNCT2 strain from this study. (B) Production and growth properties of PEDV CV777 and LNCT2 in IEC cells. CPE 48 h post-infection (hpi) (upper panels, bar: 400 µm) and immunofluorescence assay (IFA) 48 hpi (lower panels, bar: 200 µm) of CV777 and LNCT2 in IEC cells. IEC cells were infected with PEDV CV777 or LNCT2 at 100 50% tissue culture infective dose (TCID₅₀)/mL. CPE and IFA were examined at 48 hpi, and cell images were captured.

Figure 3

ShRNA treatment did not affect cell viability. Cell viability was detected by cell counting kit-8 (CCK-8) assays after transfection of IEC cells with shRNAs for 48 h. Absorption at 450 nm was recorded and expressed as a percentage of relative cell viability. Values are means ± SD (n = 3). Significant differences were assessed by Student’s t-test; ns, no significant difference compared to control; p > 0.05.

Figure 4

Inhibitory effects of shR-N307, shR-N463, and shR-N1071 on the PEDV N protein. (A) Western blots for effect of shR-N307, shR-N463, and shR-N1071 on N gene expression. PMyc-N was co-transfected with increasing doses of shR-N307, shR-N463, or shR-N1071 into IEC cells. After 48 h, transfected cells were lysed. Equal amounts of cell lysates were resolved by 12.5% SDS-PAGE. Reaction products were probed with anti-Myc or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Densitometric data for N/GAPDH from three independent experiments are shown as means ± SD. * p < 0.05. The p value was calculated using Student’s t-test; (B) shR-N307, shR-N463, and shR-N1071 influence on pAcGFP-N expression in cultured IEC cells. PAcGFP-N was co-transfected with increasing doses of shR-N307, shR-N463, or shR-N1071 into IEC cells. PAcGFP-N expression plasmid was the unrelated control. Images show enhanced GFP (EGFP) expression at 48 h post-transfection.

Figure 5

Dose-dependent inhibitory effects of shR-N307, shR-N463, and shR-N1071 against N on target gene expression and PEDV CV777 replication in infected IEC cells. IEC cells with shRNA (1, 2, 4 μg) for 24 h before infection with PEDV CV777 for 48 h. (A) PEDV N protein by Western blot with anti-N protein monoclonal antibody (mAb). Densitometric data for N/GAPDH from three independent experiments, shown as means ± SD. * p < 0.05. The p value was calculated using Student’s t-test; (B) PEDV CV777 titers in shRNA-transfected IEC cells. ShRNA transfection and viral infection were as in panel A. Viral titers in supernatants collected at 48 hpi were determined using the Reed–Muench method. Error bars represent standard errors of the mean from three independent experiments. * p < 0.05. The p value was calculated using Student’s t-test.

Figure 6

Dose-dependent inhibitory effects of shR-N307, shR-N463, and shR-N1071 directed against N on target gene expression and PEDV LNCT2 replication in infected IEC cells. IEC cells with shRNA (1, 2, 4 μg) for 24 h were infected with PEDV LNCT2 for 48 h. (A) PEDV N protein was detected by Western blot with anti-N protein mAb. Densitometric data for N/GAPDH from three independent experiments are shown as means ± SD. * p < 0.05. The p value was calculated using Student’s t-test; (B) PEDV LNCT2 titers in shRNA-transfected IEC cells. ShRNA transfection and viral infection were as in panel A. Viral titers in supernatants collected at 48 hpi were determined using the Reed–Muench method. Error bars represent standard errors of the mean from three independent experiments. * p < 0.05. The p value was calculated using Student’s t-test.