INFECTIOUS JAPANESE ENCEPHALITIS-VIRUS RNA CAN BE SYNTHESIZED FROM INVITRO-LIGATED CDNA TEMPLATES
INFECTIOUS JAPANESE ENCEPHALITIS-VIRUS RNA CAN BE SYNTHESIZED FROM INVITRO-LIGATED CDNA TEMPLATES
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DOI：
10.1128/jvi.66.9.5425-5431.1992
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发表时间：
1992-09-01
影响因子：
5.4
通讯作者：
TRENT, DW
中科院分区：
文献类型：
--
作者：
SUMIYOSHI, H;HOKE, CH;TRENT, DW
日本脑炎病毒（Japanese encephalitis virus，JEV）是黄病毒科（Flaviviridae）的一种正链RNA病毒。基因组JEV RNA长约11 kb，编码10种蛋白质，3种结构蛋白和7种非结构蛋白。JEV基因组的全长cDNA拷贝是通过体外连接编码基因组5 ′（核苷酸位置1至5576）和3 ′（核苷酸位置5577至10976）的两个cDNA片段构建的。T7 RNA聚合酶转录的连接全长cDNA模板的感染时，转染到BHK-21细胞。为了鉴定重组病毒，将沉默突变引入编码基因组的3'半的克隆中，其消除了核苷酸位置9131处的XbaI位点。通过用转录本转染回收的病毒含有这种沉默突变，证实了其身份。重组病毒和亲本病毒在BHK-21细胞中的生长和空斑产生、C6/36细胞中的包膜蛋白表达以及小鼠中的神经毒力和免疫原性方面相同。通过从克隆到质粒载体中的全长JEV基因组cDNA模板转录来获得感染性RNA的反复尝试是不成功的。从体外连接的JEV cDNA模板合成感染性JEV RNA将有助于黄病毒复制和毒力的分子和遗传学研究。
Japanese encephalitis virus (JEV) is a positive-stranded enveloped RNA virus that belongs to the family Flaviviridae. Genomic JEV RNA is approximately 11 kb long and encodes 10 proteins, 3 structural and 7 nonstructural. A full-length cDNA copy of the JEV genome was constructed by in vitro ligation of two cDNA fragments which encode the 5' (nucleotide positions 1 to 5576) and 3' (nucleotide positions 5577 to 10976) halves of the genome. T7 RNA polymerase transcripts of the ligated full-length cDNA template were infectious when transfected into BHK-21 cells. To identify the recombinant virus, a silent mutation was introduced into the clone encoding the 3' half of the genome, which abolished an XbaI site at nucleotide position 9131. Virus recovered by transfection with the transcripts contained this silent mutation, confirming its identity. Recombinant and parent viruses were identical with respect to growth and plaque production in BHK-21 cells, envelope protein expression in C6/36 cells, and neurovirulence and immunogenicity in mice. Repeated attempts to obtain infectious RNA by transcription from full-length JEV genome cDNA templates cloned into plasmid vectors were unsuccessful. Synthesis of infectious JEV RNA from in vitro-ligated JEV cDNA templates will be useful for molecular and genetic studies of flavivirus replication and virulence.