靶向经典猪瘟病毒基因组 RNA 内部核糖体进入位点的短发夹 RNA 表达载体的构建。

Development of short hairpin RNA expression vectors targeting the internal ribosomal entry site of the classical swine fever virus genomic RNA.

机构信息

Transboundary Animal Disease Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, 890-0065, Japan.

Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, 060-0818, Japan.

出版信息

BMC Biotechnol. 2023 Sep 8;23(1):37. doi: 10.1186/s12896-023-00805-6.

DOI:10.1186/s12896-023-00805-6

PMID:37684601

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10492304/

Abstract

BACKGROUND

Classical swine fever (CSF) is a fatal contagious disease affecting pigs caused by classical swine fever virus (CSFV). The disease can be transmitted by pigs and wild boars, and it is difficult to prevent and control. To obtain necessary information to establish the CSFV resistant animals in a future study, we designed lentiviral vector-delivered short hairpin RNAs (shRNAs) targeting the conserved domain III of the internal ribosomal entry site (IRES) of the CSFV genomic RNA.

RESULTS

First, we confirmed the effects of siRNAs on CSFV-IRES activity. We observed significant inhibition of CSFV-IRES activity by si42 (domain IIIa), si107 (domain IIIc), and si198 (domain IIIf) in SK-L cells and si56 (domain IIIb), si142 (domain IIId) and si198 in HEK293 cells without affecting the amount of luciferase RNA. Next, we constructed lentiviral vectors expressing shRNA based on siRNA sequences. Treatment with shRNA-expressing lentivirus was examined at 7 and 14 days post infection in SK-L cells and HEK293 cells, and CSFV-IRES was significantly suppressed at 14 days (sh42) post infection in HEK293 cells without significant cytotoxicity. Next, we examined the silencing effect of siRNA on CSFV replicon RNA and observed a significant effect by si198 after 2 days of treatment and by shRNA-expressing lentivirus (sh56, sh142, and sh198) infection after 14 days of treatment. Treatment of sh198-expressing lentivirus significantly suppressed CSFV infection at 3 days after infection.

CONCLUSION

The IRES targeting sh198 expressing lentivirus vector can be a candidate tool for CSFV infection control.

摘要

背景

经典猪瘟（CSF）是一种致命的传染病，可感染猪，由经典猪瘟病毒（CSFV）引起。该疾病可通过猪和野猪传播，难以预防和控制。为了在未来的研究中获得建立 CSFV 抗性动物的必要信息，我们设计了靶向 CSFV 基因组 RNA 内部核糖体进入位点（IRES）保守结构域 III 的慢病毒载体递送短发夹 RNA（shRNA）。

结果

首先，我们证实了 siRNA 对 CSFV-IRES 活性的影响。我们观察到 si42（结构域 IIIa）、si107（结构域 IIIc）和 si198（结构域 IIIf）在 SK-L 细胞中以及 si56（结构域 IIIb）、si142（结构域 IIId）和 si198 在 HEK293 细胞中显著抑制 CSFV-IRES 活性，而不影响荧光素酶 RNA 的量。接下来，我们构建了基于 siRNA 序列表达 shRNA 的慢病毒载体。在 SK-L 细胞和 HEK293 细胞中，在感染后 7 天和 14 天检测表达 shRNA 的慢病毒处理，在 HEK293 细胞中，在感染后 14 天（sh42）显著抑制 CSFV-IRES，而没有明显的细胞毒性。接下来，我们研究了 siRNA 对 CSFV 复制子 RNA 的沉默作用，并在处理后 2 天观察到 si198 有显著效果，在处理后 14 天观察到表达 shRNA 的慢病毒（sh56、sh142 和 sh198）感染有显著效果。表达 sh198 的慢病毒处理显著抑制了感染后 3 天的 CSFV 感染。