Interference of porcine reproductive and respiratory syndrome virus replication on MARC-145 cells using DNA-based short interfering RNAs.
Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit PRRSV replication on MARC-145 cells. Four short interfering RNA (siRNA) sequences (N95, N179, N218 and N294) directed against a well-conserved region of PRRSV genome ORF7 gene were selected. Sense and antisense siRNA encode sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes driven by mouse U6 promoter. Using a polymerase chain reaction (PCR)-based approach, shRNAs were generated from shRNA expression cassettes. The PCR products were cloned into pEGFP-N1 vector and shRNA expression vectors were constructed. When MARC-145 cells were transfected with shRNA expression vectors and then infected with PRRSV, N179 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by PRRSV. Western blot, indirect immunofluorescence and fluorescence quantitative PCR (FQ-PCR) confirmed that the expression of ORF7 was reduced both at protein and RNA levels comparing to controls. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of PRRS virus (approximately 681-fold reduction of viral titers) on MARC-145 cells.
He
Yun-Xia
YX
National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, PR China.
Hua
Rong-Hong
RH
Zhou
Yan-Jun
YJ
Qiu
Hua-Ji
HJ
Tong
Guang-Zhi
GZ
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005822:Genetic Vectors; D000882:Haplorhini; D016133:Polymerase Chain Reaction; D019316:Porcine respiratory and reproductive syndrome virus / Q000187:drug effects / Q000502:physiology*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics* / Q000494:pharmacology; D012367:RNA, Viral / Q000037:antagonists & inhibitors / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
D000998:Antiviral Agents; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012367:RNA, Viral; D014764:Viral Proteins