Effective inhibition of porcine transmissible gastroenteritis virus replication in ST cells by shRNAs targeting RNA-dependent RNA polymerase gene

Abstract

Transmissible gastroenteritis virus (TGEV) is identified as one of the most important pathogenic agents during swine enteric infection, leading to high mortality in neonatal pigs and severe annual economic loss in swine-producing areas. Up to date, various vaccines developed against TGEV still need to be improved. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, two shRNA-expressing plasmids (pEGFP-U6/P1 and pEGFP-U6/P2) targeting the RNA-dependent RNA polymerase (RdRp) gene of TGEV were constructed and transfected into swine testicular (ST) cells. The cytopathic effect (CPE) and MTS assays demonstrated that both shRNAs were capable of protecting cells against TGEV invasion with very high specificity and efficiency. A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the two plasmids were reduced by 95.2% and up to 100%, respectively. Our results suggest that RNAi might be a promising new strategy against TGEV infection.

Fig. 1

Effect of shRNAs on TGEV-induced CPE in ST cells. ST cells were transfected with different plasmids and then infected with TGEV at 200 CCID₅₀. CPE was examined from 20 to 40 h post-infection, and cell images were captured in the end. (A1–C1) and (A2–C2) showed the images in the middle and brim of the same well, respectively. (A) ST cells neither transfected with plasmids nor infected with virus. (E) Cells only treated with virus. From (B) to (D), cells were transfected with pEGFP-U6/P1 (B), pEGFP-U6/P2 (C) and pEGFP-U6/T (D), respectively. (A, D and E) also served as a normal, negative and mock control in the experiment, respectively. The experiment was performed in triplicate and repeated three times.

Fig. 2

Protection of ST cells from TGEV destruction by shRNAs. ST cells were transfected with plasmids as indicated and then infected with TGEV at 200 CCID₅₀. At 40 h post-infection including 4 h incubation along with MTS, viable cell numbers were evaluated by a MTS assay. OD values shown are the mean ± S.D. of three separate experiments performed in triplicate.

Fig. 3

Dose-dependent inhibition of TGEV replication. ST cells in each well were transfected with pEGFP-U6/P2 at the indicated doses, and then challenged with TGEV at 200 CCID₅₀. At 40 h post-infection, viral titers in these wells were measured and shown by log CCID₅₀/0.2 ml. Data shown are a mean of two independent experiments, and error bars represent the standard deviation.

Fig. 4

Inhibition of TGEV RNA replication by shRNAs. ST cells were transfected with plasmids as indicated and then infected with TGEV. At 40 h after infection, cell cultures in the repeated wells were collected together for total RNAs extraction, and a real-time quantitative RT-PCR analysis was performed. The amount of viral RNAs in each sample was normalized to β-actin in the same sample, relative to the mock control. The mean of three independent transfections performed in triplicate is shown, and error bars represent the standard deviation.