Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus
Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus
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DOI：
10.1016/j.virol.2009.08.030
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发表时间：
2009-11-25
期刊：
影响因子：
3.7
通讯作者：
Chen, Wei-June
中科院分区：
文献类型：
--
作者：
Chuang, Ching-Kai;Chen, Wei-June
由于基因组RNA缺乏校对功能和错误修复能力，已知累积突变是驱动黄病毒属病毒进化的一种力量，包括日本脑炎（JE）病毒。基于测序数据，RNA重组最近被认为是与这些病毒基因组变异相关的另一个因素。本文利用分别从本地毒株T1P1和CJN获得的两个本地纯克隆（T1P1- s1和CJN- s1）提供实验证据，证明乙脑病毒存在RNA重组。根据含有868个核苷酸（nt 10-877）片段的C/preM连接的限制性片段长度多态性（RFLP）测定结果，重组子代病毒主要在与本研究中使用的两个克隆共感染的BHK-21细胞中形成。20种重组乙脑病毒中有9种在nt 123-323地区有交叉。从这些重组中获得的测序数据显示，该区域没有发生核苷酸缺失或插入，有利于交叉，准确地表明。不出意外地，同源重组参与其中。通过定点诱变，三个茎环二级结构在序列上被破坏和重新稳定，导致重组频率的变化。这表明二级结构的构象，而不是自由能，在调节病毒的RNA重组中是重要的。结论是，由于RNA重组在乙脑病毒中产生遗传多样性，因此在病毒进化、流行病学和可能的疫苗安全性研究中必须特别考虑到这一点。(C) 2009爱思唯尔公司版权所有。
Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely. not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety. (C) 2009 Elsevier Inc. All rights reserved.