College of Veterinary Medicine, Yangzhou University, 48 Wenhui Road, Yangzhou 225009, Jiangsu, People's Republic of China.
Arch Virol. 2013 Jan;158(1):55-61. doi: 10.1007/s00705-012-1455-z. Epub 2012 Sep 5.
A robust artificial microRNA (amiRNA) strategy against porcine reproductive and respiratory syndrome virus (PRRSV) was developed by targeting the untranslated regions (UTRs). Six candidate amiRNAs targeting the 5' or 3' UTR were used for vector construction, and four effective amiRNAs were selected for further study using a vector transfection/virus infection assay. In cell cultures stably transfected with the four amiRNA vectors, expression of the sequence-specific amiRNAs was confirmed using poly(A)-tailed RT-PCR. After infection with three different PRRSV strains, the viral RNA genome and/or transcript were inhibited by ~90 % (semi-quantitative RT-PCR), and the viral titers were decreased by more than six log CCID(50) (viral titration assay) before day 3 postinfection. The potent anti-PRRSV effects lasted for at least 5 days. Sequence analysis showed that the amiRNA antiviral activities were not compromised by the presence of one or two mismatches in their binding targets. This work constitutes a step towards developing a more effective RNAi strategy against PRRSV.
针对猪繁殖与呼吸综合征病毒（PRRSV），开发了一种稳健的人工 microRNA（amiRNA）策略，该策略针对非翻译区（UTR）。使用针对 5'或 3'UTR 的六个候选 amiRNA 进行载体构建，并使用载体转染/病毒感染测定法选择了四个有效的 amiRNA 进行进一步研究。在稳定转染四种 amiRNA 载体的细胞培养物中，使用 poly(A)-尾 RT-PCR 证实了序列特异性 amiRNA 的表达。在感染三种不同的 PRRSV 株后，通过~90%（半定量 RT-PCR）抑制病毒 RNA 基因组和/或转录物，并且在感染后第 3 天之前，病毒滴度降低超过六个对数 CCID(50)（病毒滴定测定法）。强大的抗 PRRSV 作用至少持续 5 天。序列分析表明，amiRNA 的抗病毒活性不受其结合靶标中存在一个或两个错配的影响。这项工作是朝着开发针对 PRRSV 的更有效的 RNAi 策略迈出的一步。
