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

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.