BACKGROUND Contagious ecthyma or Orf is known as a zoonotic disease remains prevalently worldwide despite the application of some control strategies against it. RNAi particularly shRNA provides us with the chance to tackle this obstacle by an encouraging new approach. The current study indicates the design and experiment of third-generation lentivirus packaging systems delivering shRNAs to inhibit Orf virus (ORFV) replication and infection. Given the importance of DNA-pol gene in virus replication, in this study, three shRNAs against this gene were designed and cloned into lentiviral vectors to stabilize the expression of shRNAs. After producing lentivectors expressing ORFV-DNA- pol in HEK293T cells, the synthesized shRNAs were applied to downregulate viral replication and gene expression. The reduction in viral titer and RNA was evaluated by TCID50 test as well as real-time RT-PCR. The results were then analyzed in comparison with the control group. RESULTS Designed shRNAs significantly reduced virus yield approximately 90 to 97% and 96.8 to 99.4%, respectively compared to the control groups (cells infected with ORFV and infected with ORFV and scrambled vector) by TCID50 test. Real-time RT-PCR revealed a dramatic reduction in the expression of viral RNA approximately 99% compared to cells infected with ORFV and from 92.6 to 99%, respectively compared to cells infected with ORFV and scrambled vector. CONCLUSIONS Therefore, it can be stated that RNAi is capable of being used as a potent therapeutically option against viruses like ORFV.

中文翻译：

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表达shRNA的慢病毒通过靶向DNA聚合酶基因抑制传染性臁疮病毒的复制。


背景技术传染性臁疮或Orf被称为人畜共患疾病，尽管应用了一些针对它的控制策略，但在世界范围内仍然普遍存在。 RNAi 特别是 shRNA 为我们提供了通过令人鼓舞的新方法解决这一障碍的机会。目前的研究表明了第三代慢病毒包装系统的设计和实验，该系统传递shRNA以抑制奥尔夫病毒（ORFV）的复制和感染。鉴于DNA-pol基因在病毒复制中的重要性，本研究设计了3种针对该基因的shRNA，并将其克隆到慢病毒载体中，以稳定shRNA的表达。在 HEK293T 细胞中产生表达 ORFV-DNA-pol 的慢病毒载体后，合成的 shRNA 用于下调病毒复制和基因表达。通过 TCID50 测试以及实时 RT-PCR 评估病毒滴度和 RNA 的降低。然后将结果与对照组进行比较分析。结果 通过 TCID50 测试，与对照组（感染 ORFV 的细胞以及感染 ORFV 和乱序载体的细胞）相比，设计的 shRNA 显着降低了病毒产量，分别约为 90% 至 97% 和 96.8% 至 99.4%。实时 RT-PCR 显示，与感染 ORFV 的细胞相比，病毒 RNA 的表达显着降低约 99%，与感染 ORFV 和乱序载体的细胞相比，病毒 RNA 的表达分别显着降低 92.6% 至 99%。结论 因此，可以说 RNAi 能够作为针对 ORFV 等病毒的有效治疗选择。