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, TCID₅₀ 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 TCID₅₀ 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.

Xenotransplantation panel for the detection of infectious agents in pigs

“…The assays described here can detect the coronaviruses PHEV, TGEV and the closely related virus porcine respiratory coronavirus (PRCV), as well as porcine epidemic diarrhea virus (PEDV) (22–24). Specimens tested from the animals described above were all obtained during the summer months and none of them tested positive for any of these viruses.…”

Porcine Hemagglutinating Encephalomyelitis Virus: A Review

“…In addition, PHEV infection can block the phagosome and lysosomes fusion, inducing an atypical autophagy response necessary for viral replication in neurons (99). Furthermore, PHEV genome replication in PK-15 cells and; therefore, the production of infectious virus in vitro can be inhibited through small interfering RNAs (siRNA) that target different regions of the PHEV spike glycoprotein (100) or nucleocapsid genes (101).…”

Drawing Comparisons between SARS-CoV-2 and the Animal Coronaviruses

“…Combined siRNA therapy was shown to prevent escape of siRNA-resistant FCoV mutants in vitro [ 229 ]. siRNAs restricted viral protein expression and inhibited the replication of PEDV, PDCoV, PHEV, and SADS-CoV in vitro [ 230 , 231 , 232 ]. siRNA expression inhibited virus replication, lowered inflammation, significantly decreased mortality, and improved weight gain in poultry experimentally infected with IBV [ 233 ].…”