Department of Genetics, Faculty of Science, Shahrekord University, Rahbar Boulevard, Postal Box: 115, Shahrekord, Iran.
Biotechnology Research Institute, Shahrekord University, Shahrekord, Iran.
BMC Biotechnol. 2020 Mar 23;20(1):18. doi: 10.1186/s12896-020-00611-4.
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.
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.
Therefore, it can be stated that RNAi is capable of being used as a potent therapeutically option against viruses like ORFV.
传染性脓疱性皮炎，又称羊口疮，是一种人畜共患病，尽管已经采取了一些控制措施，但在世界范围内仍很流行。RNAi，特别是短发夹 RNA(shRNA)，为我们提供了一种通过令人鼓舞的新方法来应对这一障碍的机会。本研究表明，设计和实验第三代慢病毒包装系统递送 shRNA 以抑制口疮病毒(ORFV)复制和感染。鉴于 DNA-聚合酶基因在病毒复制中的重要性，在这项研究中，针对该基因设计并克隆了三个 shRNA 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-聚合酶的慢病毒后，将合成的 shRNA 应用于下调病毒复制和基因表达。通过 TCID50 试验和实时 RT-PCR 评估病毒滴度和 RNA 的减少。然后将结果与对照组进行比较进行分析。
与对照组（感染 ORFV 的细胞和感染 ORFV 及乱序载体的细胞）相比，设计的 shRNA 通过 TCID50 试验分别使病毒产量降低了约 90%至 97%和 96.8%至 99.4%。实时 RT-PCR 显示病毒 RNA 的表达显著降低，与感染 ORFV 的细胞相比约为 99%，与感染 ORFV 及乱序载体的细胞相比分别为 92.6%至 99%。
因此，可以说 RNAi 有潜力作为针对 ORFV 等病毒的有效治疗选择。
