In vitro inhibition of porcine hemagglutinating encephalomyelitis virus replication with siRNAs targeting the spike glycoprotein and replicase polyprotein genes

Abstract

Objective: The specific effect of rna interference on the replication of porcine hemagglutinating encephalomyelitis virus (phe-cov) was explored.

Methods: Four species of small interfering RNA (siRNA), targeting different regions of the PHE-CoV spike glycoprotein and replicase polyprotein genes, were prepared by in vitro transcription. After transfection of PK-15 cells with each of the siRNAs followed by infection with PHE-CoV, the cytopathic effect (CPE) was examined by phase-contrast microscope, and viral proliferation within cells was examined by indirect immunofluorescence microscopy, hemagglutination (HA) test, TCID(50) assay and real-time RT-PCR.

Results: Examination of CPE demonstrated that the four siRNAs were capable of protecting cells against PHE-CoV invasion with very high specificity and efficiency. At 48 h post-infection, only a few siRNA-treated cells were positive for viral antigen staining, whereas most untreated virus-infected cells were positive. Transfection with siRNAs also suppressed the production of infectious virus by up to 18- to 32-fold as assessed by a HA test and 93- to 494-fold as assessed by TCID(50) assay. Furthermore, treatment with siRNAs caused a 53-91% reduction in the viral genome copy number as assessed by real-time RT-PCR.

Conclusion: These results suggested that the four species of siRNAs can efficiently inhibit PHE-CoV genome replication and infectious virus production.

Fig. 1

Purity and integrity of the siRNAs transcribed in vitro: (1) SR1, (2) SR2, (3) SS1, (4) SS2, and (5) scramble siRNA.

Fig. 2

Effect of siRNAs on PHE-CoV-induced CPE in PK-15 cells. PK-15 cells were transfected with different siRNAs and then infected with PHE-CoV at 400 TCID₅₀. Images in the middle and brim of the same well, respectively, are shown. a-d Untreated cells infected with virus at 400 TCID₅₀ in 24-well plates were observed at 24, 48, 56, and 72 h post-infection, respectively. e-i Cells were transfected with SR1, SS1, SR2, SS2 and scramble siRNA, respectively, and CPE was examined 56 h post-infection. Cell images were captured at the conclusion of the study. The experiment was performed in triplicate and repeated 3 times.

Fig. 3

Protective effect of siRNA against PHE-CoV infection. The viral infection in PK-15 cells was examined by indirect immunofluorescence: a mock transfection (stained with PHE-CoV-positive serum); b scramble siRNA transfection; c mock transfection (stained with PHE-CoV-negative serum); d SR1 transfection; e SR2 transfection; f SS1 transfection, and g SS2 transfection.

Fig. 4

Inhibition of virus production in siRNA-treated cells. HA titers are the means of three repeat titrations at the time points indicated.

Fig. 5

Inhibition of virus production in siRNA-treated cells. TCID₅₀ values are the means of three repeat titrations at the time points indicated.

Fig. 6

Reduction of viral genome copy number by siRNA treatment in PK-15 cells 48 h post-infection. The PHE-CoV genome copy numbers are the means of three repeat experiments.