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

贝塔诺达病毒是引起海水鱼类病毒性神经坏死的病原体，其基因组中含有两段正义RNA。尽管病毒RNA和编码蛋白的一级结构在不同的病毒之间是相似的，但它具有明显的宿主特异性。然而，目前还没有关于宿主特异性的机制报道。我们首次构建了条斑杰克神经坏死病毒(SJNNV)和七带石斑鱼神经坏死病毒(SGNNV)的感染性RNA转录系统。为了构建SJNNV基因组RNA1和RNA2的全长cDNA克隆，用RACE方法测定了两个片段的5‘和3’非编码全序列。根据获得的末端序列，我们进行了RT-PCR，并构建了包含这两个RNA的全长c DNA拷贝的质粒克隆，位于T7 RNA聚合酶启动子下游。这些质粒在每个…的3‘末端下游的一个独特的限制位点上被切割更多的SJNNV序列并在体外转录成具有帽子结构的类似RNA。将该转录本混合物导入FISH细胞系E-11。用SJNNV抗血清进行间接免疫荧光检测，在转基因细胞中可观察到特异的荧光细胞，其培养上清对条纹叶枯病幼虫具有致病性。这是首次报道了可以转录出感染性基因组RNA的BetanodaVirus的cdna克隆。然后，我们测试了SJNNV和SGNNV之间的两个重组子在原始宿主鱼中的感染性。用含有SJNNV RNA1和SGNNV RNA2的重配病毒感染斑点石斑鱼幼虫时，与接种SGNNV相似，只杀死7条石斑鱼；反之，接种含有SGNNV RNA1和SJNNV RNA2的重配病毒对斑点石斑鱼有杀灭作用，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表明，这两种重组体都在受感染鱼的大脑、脊髓和视网膜中繁殖，类似于亲本病毒接种的感染。这些结果表明，病毒RNA2和/或编码的外壳蛋白控制着SJNNV和SGNNV的宿主特异性。较少

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