Thedcharoen Phathrawarin, Pewkliang Yongyut, Kiem Hoa Khanh Tran, Nuntakarn Lalana, Taengchaiyaphum Suparat, Sritunyalucksana Kallaya, Flegel Timothy W, Saksmerprome Vanvimon, Borwornpinyo Suparerk
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand; Excellent Center for Drug Discovery, Faculty of Science, Mahidol University, Bangkok, Thailand.
J Invertebr Pathol. 2020 Sep;175:107442. doi: 10.1016/j.jip.2020.107442. Epub 2020 Jul 12.
Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called "pLVX-lhRdRp", against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24-72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.
双链RNA（dsRNA）通过RNA干扰（RNAi）途径用于下调虾类病毒病原体特定基因的表达。向虾体内施用dsRNA已被证明是阻止黄头病毒（YHV）进展的有效策略。在本研究中，开发了一种载体（pLVX-AcGFP1-N1），以在巨细胞病毒（CMV）启动子下引入一个长链发夹RNA（lhRNA）沉默盒，即所谓的“pLVX-lhRdRp”，用于靶向黄头病毒的RNA依赖性RNA聚合酶（RdRp）基因。用pLVX-lhRdRp载体转染从斑节对虾分离的血细胞原代培养物，转染后12小时即可产生lhRNA转录本。在黄头病毒攻击前12小时，用pLVX-lhRdRp转染原代血细胞培养物，而对照组用pLVX-AcGFP1-N1转染或不进行转染。用pLVX-lhRdRp处理的组在黄头病毒攻击后24至72小时显著抑制了黄头病毒的复制。逆转录聚合酶链反应（RT-PCR）和免疫组织化学结果证实，pLVX-lhRdRp载体有效抑制了黄头病毒的mRNA和蛋白表达。因此，我们的血细胞培养物和具有组成型启动子的dsRNA表达质粒有潜力作为一个平台，用于测试表达针对致病性病毒感染的长链发夹RNA的DNA构建体，并作为虾类基于RNAi的DNA疫苗。
