| Classical swine fever (CSF) caused by classic swine fever virus (CSFV) is a highlycontagious disease of pigs，with high fever, bleeding, and immune suppression as the mainsymptoms, which causes great economic losses in the pig industry worldwide. RNA interference(RNAi) is a promising technology in development of specific antiviral therapy. Therefore,construction of RNAi-based anti-CSFV transgenic pigs is an intriguing strategy to prevent CSF.Previously, four anti-CSFV specific small interference RNA (siRNA) targeting CSFVnon-structural protein genes and corresponding single, double, and quadruple shRNA-expressingvectors were constructed and analyzed before, and then applied to generate anti-CSFV transgenicpigs via nuclear transfer technology.However, two main problems about the obtained transgenic pigs remain unresolved. First, thequantitative detection of siRNA expressed in vivo is critical for transgenic identification andassessing the antiviral effect of transgenic pigs, while a easy-to-use and reliable assay to detectsiRNA expression levels in various cells or tissues is not available. Second, it is not clear whethercytotoxicity produced by the shRNA-expressing vectors in vivo can cause high mortality ofanti-CSFV transgenic pigs，which is the bottleneck of the RNAi-based antiviral strategies.To quantify anti-CSFV specific siRNA in vivo, Taqman-based stem-loop RT-qPCR with theadvantages of high sensitivity and specificity was developed in this study. First, serial specificstem-loop primers were designed and used for reverse transcription, four of which were thenselected for RT-qPCR. Taqman-based stem-loop RT-qPCR systems were successfully developedto detect anti-CSFV specific siRNA, including siN1, siN2, siN3-2and siN5B. Then, absolutelyquantitative assay with synthetic RNA molecule siN1was established and sensitivity analysisshowed that the obtained method is capable of detecting102to108copies of siRNA moleculeswith good parallel relationship (Rsq=0.999) and high amplification efficiency (Eff.=98.2%). Inaddition, relatively quantitative assay with ssc-miR-16as internal control was established, whichcan detect expression changes of siRNA in small amounts of cells or tissues. The Taqman-basedstem-loop RT-qPCR systems were further optimized to directly use cell lysates as the template to detect siRNA, which facilitates detecting siRNA expression levels with little samples.Numerous studies have been demonstrated that the shRNA-expressing vector-based RNAistrategies cause a wide range of cytotoxicity, including unintended sequence-non-specific immuneresponses, off-target effects, oversaturation of endogenous miRNA/shRNA pathways, and evencausing animal individual lethality. Previously, it was found that11dead anti-CSFV transgenicpigs were diagnosed as negative of SFV and PRRSV, which indicated that the causative possibilityof these transgenic pigs from virus infection is low. In order to explore whether this high fatalityof anti-CSFV transgenic pigs is related to shRNA expression in vivo, porcine fetal fbroblasts(PEF)clones used as a nuclear donor cell to generate anti-CSFV transgenic pigs were subjected to detectinterferon (IFN) response and oversaturation of endogenous miRNA/shRNA pathways. Asexpected, all shRNA expression vectors can induce extensive IFN response, which is cellclone-specific, not vector-specific. miRNA expression profile analysis of quadrupleshRNA-expressing clone PEF-siRNA4showed that228Sus scrofa mature miRNA were identifiedand expression of22miRNA out of them significantly down-regulated,9significantlyup-regulated. Both number and altered expression levels of down-regulated miRNA were fargreater than that of up-regulated one, indicating that overexpression of quadruple anti-CSFVshRNA seriously affects the normal miRNA biogenesis and it may cause cytotoxicity to cells. Inaddition, miRNA is found to be involved in the early development of animals and down-regulationof miRNA at this period may cause strong toxicity to organism and even lead the animal to die.In this study, the established Taqman-based stem-loop RT-qPCR for four anti-CSFV specificsiRNA which can complete absolutely/relatively quantitative assay provide a novel technique foridentification and evaluating the antiviral effects of anti-CSFV transgenic pigs. Then,shRNA-based cytotoxicity including unintended sequence-non-specific immune responses andoversaturation of endogenous miRNA pathways were analysed. And highly lethal cause ofRNAi-based anti-CSFV strategies were explored, This concept might help to improve the successpreparation of anti-CSFV transgenic pigs. |
