Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene
Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene
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DOI：
10.1016/j.virusres.2014.10.010
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发表时间：
2015-01-02
期刊：
影响因子：
5
通讯作者：
Masuda, Michiaki
中科院分区：
文献类型：
--
作者：
Ishikawa, Tomohiro;Abe, Makoto;Masuda, Michiaki
日本脑炎病毒（JEV）基因型V最初于1952年在马来西亚分离出来，长期以来一直局限于该地区。2009年，中国和韩国突然出现了V基因型，表明其地理分布范围扩大。虽然对V基因型的研究越来越重要，但由于缺乏感染性分子克隆，其研究受到限制。本研究构建了一个在T7启动子下游含有JEV Muar株全基因组cDNA的质粒，该株属于V型。通过T7 RNA聚合酶（T7RNAP）体外转录的病毒RNA从质粒的电穿孔导致在哺乳动物和蚊子细胞中产生子代病毒。此外，证明将感染性克隆质粒转染到瞬时或稳定表达T7RNAP的哺乳动物细胞中产生感染性后代。当病毒结构蛋白基因从全长cDNA中部分缺失时，从修饰的质粒体外转录的亚基因组RNA显示出作为复制子在哺乳动物细胞中复制自身。携带萤火虫荧光素酶基因代替缺失的结构蛋白基因的复制子也显示出有效地复制自身并在哺乳动物细胞中表达荧光素酶。与来自JEV基因型III（中山株）的复制子相比，来自基因型V的复制子似乎对外源基因的导入更耐受。本研究中构建的感染性克隆和复制子可作为表征JEV基因型V的有用工具。（C）2014 Elsevier B.V.保留所有权利。
Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V. (C) 2014 Elsevier B.V. All rights reserved.