Artificial microRNA (amiRNA)-mediated inhibition of viral replication has recently gained importance as a strategy for antiviral therapy. In this study, we evaluated the benefit of using the amiRNA vector against Japanese encephalitis virus (JEV). We designed three single amiRNA sequences against the consensus sequence of 3' untranslated region (3'UTR) of JEV and tested their efficacy against cell culture-grown JEV Vellore strain (P20778) in neuronal cells. The binding ability of three amiRNAs on 3'UTR region was tested in vitro in HEK293T cells using a JEV 3'UTR tagged with luciferase reporter vector. Transient transfection of amiRNAs was nontoxic to cells as evident from the MTT assay and caused minimal induction in interferon-stimulated gene expression. Furthermore, our result suggested that transient expression of two amiRNAs (amiRNA #1 and amiRNA #2) significantly reduced intracellular viral RNA and nonstructural 1 (NS1) protein, as well as diminished infectious viral particle release up to 95% in the culture supernatant as evident from viral plaque reduction assay. Overall, our results indicated that RNA interference based on amiRNAs targeting viral conserved regions at 3'UTR was a useful approach for improvements of nucleic acid inhibitors against JEV.

中文翻译：

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人工MicroRNA介导的对日本脑炎病毒在神经元细胞中复制的抑制。

作为抗病毒治疗的策​​略，人工microRNA（amiRNA）介导的病毒复制抑制作用最近变得越来越重要。在这项研究中，我们评估了使用amiRNA载体对抗日本脑炎病毒（JEV）的益处。我们针对JEV的3'非翻译区（3'UTR）的共有序列设计了三个单amiRNA序列，并测试了它们对神经元细胞中培养的JEV Vellore菌株（P20778）的功效。使用荧光素酶报告载体标记的JEV 3'UTR，在HEK293T细胞中体外测试了三种amiRNA在3'UTR区的结合能力。从MTT分析中可以明显看出，amiRNA的瞬时转染对细胞无毒，并且在干扰素刺激的基因表达中几乎没有引起任何诱导。此外，我们的结果表明，两个amiRNA（amiRNA＃1和amiRNA＃2）的瞬时表达显着降低了细胞内病毒RNA和非结构性1（NS1）蛋白，并减少了培养上清液中高达95％的传染性病毒颗粒释放，从病毒斑减少测定。总体而言，我们的结果表明，基于靶向3'UTR病毒保守区的amiRNA的RNA干扰是改善针对JEV的核酸抑制剂的有用方法。