肠道病毒 71 型感染性 cDNA 克隆的构建：确保实验成功的因素分析。
Construction of an infectious cDNA clone of Enterovirus 71: insights into the factors ensuring experimental success.
机构信息
Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, John Street, Hawthorn, Victoria 3122, Australia.
Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, John Street, Hawthorn, Victoria 3122, Australia.
出版信息
J Virol Methods. 2014 Mar;197:67-76. doi: 10.1016/j.jviromet.2013.12.005. Epub 2013 Dec 20.
Enterovirus 71 (EV 71) is a causative agent of mild Hand Foot and Mouth Disease but is capable of causing severe complications in the CNS in young children. Reverse genetics technology is currently widely used to study the pathogenesis of the virus. The aim of this work was to determine and evaluate the factors which can contribute to infectivity of EV 71 RNA transcripts in vitro. Two strategies, overlapping RT-PCR and long distance RT-PCR, were employed to obtain the full-length genome cDNA clones of the virus. The length of the poly(A) tail and the presence of non-viral 3'-terminal sequences were studied in regard to their effects on infectivity of the in vitro RNA transcripts of EV 71 in cell culture. The data revealed that only cDNA clones obtained after long distance RT-PCR were infectious. No differences were observed in virus titres after transfection with in vitro RNA harbouring a poly(A) tail of 18 or 30 adenines in length, irrespective of the non-viral sequences at the 3'-terminus.
肠道病毒 71 型（EV 71）是一种引起手足口病的病原体，但它能够在儿童的中枢神经系统中引起严重的并发症。反向遗传学技术目前被广泛用于研究病毒的发病机制。本研究旨在确定和评估可导致 EV 71 RNA 转录本在体外感染性的因素。采用重叠 RT-PCR 和长距离 RT-PCR 两种策略获得病毒全长基因组 cDNA 克隆。研究了多聚（A）尾的长度和非病毒 3'-末端序列的存在对 EV 71 体外 RNA 转录本在细胞培养中的感染性的影响。数据显示，只有经过长距离 RT-PCR 获得的 cDNA 克隆才具有感染性。用体外 RNA 转染后，无论 3'-末端是否存在非病毒序列，带有 18 或 30 个腺嘌呤的多聚（A）尾的病毒滴度均无差异。
Zotero 插件