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Establishment of an infectious RNA transcription system for piscine nodaviruses and molecular analyses of the pathogenicity

鱼类诺达病毒感染性RNA转录系统的建立及其致病性分子分析

基本信息

批准号：
14360110
负责人：
NAKAI Toshihiro
金额：
$ 9.6万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14360110/
关键词：
betanodavirus viral nervous necrosis in vitro infectious RNA transcription system viral coat protein gene RNA-dependent RNA polymerase gene reassortant virus host specificity SJNNV リアソータント血清型トランスフェクション

项目摘要

Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNAs as the genomes. Betanodaviruses have marked host specificity although the primary structures of the viral RNAs and encoded proteins are similar among betanodaviruses. However, no mechanism underlying the host specificity has yet been reported. We first constructed a cDNA-mediated infectious RNA transcription system for striped jack nervous necrosis virus (SJNNV) and sevenband grouper nervous necrosis virus (SGNNV). To construct full-length cDNA clones of the SJNNV genomic RNA1 and RNA2, the complete 5'-and 3'-noncoding sequences of both segments were determined using the RACE method. Based on the terminal sequences obtained, we performed RT-PCR and constructed plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 RNA polymerase promoter. These plasmids were cleaved at a unique restriction site just downstream of the 3' terminus of each … More SJNNV sequence and transcribed in vitro into RNA with a cap structure analog. The transcript mixture was transfected into fish cell line E-11. Using indirect immunofluorescence with anti-SJNNV serum, fluorescing cells were observed specifically in these transfected cells, and this culture supernatant exhibited pathogenicity to striped jack larvae. This is the first report of betanodavirus cDNA clones from which infectious genomic RNAs can be transcribed. Then, we tested two reassortants between SJNNV and SGNNV for the infectivity in originated host fish. When striped jack and sevenband grouper larvae were bath-challenged with the reassortant virus comprising SJNNV RNA1 and SGNNV RNA2, sevenband grouper was exclusively killed similar to inoculation with SGNNV Conversely, inoculations with the reassortant virus comprising SGNNV RNA1 and SJNNV RNA2 killed striped jack but did not affect sevenband grouper. Immunofluorescent microscopic studies using anti-SJNNV polyclonal antibodies revealed that both the reassortants multiplied at the brains, spinal cords, and retinas of infected fish similar to infection with parental virus inoculations. These results indicate that viral RNA2 and/or encoded coat protein control host specificity in SJNNV and SGNNV. Less

贝塔诺达病毒（Betanodaviruses）是引起海洋鱼类病毒性神经坏死的病原体，其基因组为二分正义RNA。尽管病毒RNA和编码的蛋白质的一级结构在Betanodavirus中是相似的，但Betanodavirus具有显著的宿主特异性。然而，还没有宿主特异性的潜在机制的报告。本研究首次构建了条纹杰克神经坏死病毒（striped jack nervous necrosis virus，SJNNV）和七带石斑鱼神经坏死病毒（sevenband grouper nervous necrosis virus，SGNV）cDNA介导的感染性RNA转录系统。为了构建SJNNV基因组RNA 1和RNA 2的全长cDNA克隆，使用RACE方法确定两个片段的完整5 '和3'非编码序列。基于获得的末端序列，我们进行了RT-PCR和构建质粒克隆含有全长cDNA拷贝的两种RNA，位于下游的T7 RNA聚合酶启动子。这些质粒在每个质粒的3'末端下游的独特限制性位点处被切割。 ...更多信息 SJNNV序列并在体外转录成具有帽结构类似物的RNA。将转录物混合物转染到鱼细胞系E-11中。用抗SJNNV血清间接免疫荧光检测，在这些转染细胞中特异性地观察到荧光细胞，并且该培养上清对条纹杰克幼虫表现出致病性。这是第一次报告的β-结节病毒的cDNA克隆，从感染性基因组RNA可以转录。然后，我们测试了SJNNV和SGNV之间的两种抑制剂在起源宿主鱼中的感染性。当用包含SJNNV RNA 1和SGNV RNA 2的回复病毒对条纹鱼和七带石斑鱼幼虫进行浴攻击时，与用SGNV接种类似，七带石斑鱼被专门杀死。相反，用包含SGNV RNA 1和SJNNV RNA 2的回复病毒接种杀死条纹鱼，但不影响七带石斑鱼。使用抗SJNNV多克隆抗体的免疫荧光显微镜研究显示，这两种rerectants在受感染的鱼的大脑，脊髓和视网膜上繁殖，类似于用亲本病毒接种的感染。这些结果表明，病毒RNA 2和/或编码的外壳蛋白控制SJNNV和SGNV中的宿主特异性。少