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

betanodavirus是引起海鱼病毒性神经坏死的病原体，其基因组为双部正义rna。betanoda病毒具有明显的宿主特异性，尽管在betanoda病毒中病毒rna和编码蛋白的初级结构是相似的。然而，目前还没有关于宿主特异性的机制报道。我们首先构建了条带斑马鱼神经坏死病毒（SJNNV）和七条带石斑鱼神经坏死病毒（SGNNV）的cdna介导的感染性RNA转录系统。为了构建SJNNV基因组RNA1和RNA2的全长cDNA克隆，采用RACE方法确定了这两个片段的完整的5‘和3’非编码序列。根据获得的末端序列，我们进行了RT-PCR，并构建了含有这两种RNA全长cDNA拷贝的质粒克隆，定位在T7 RNA聚合酶启动子的下游。这些质粒在每个SJNNV序列的3'端下游的一个独特的限制性位点被切割，并在体外转录成具有类似帽结构的RNA。将转录本混合物转染到鱼细胞系E-11中。用抗sjnnv血清间接免疫荧光法，在转染细胞中特异性观察到荧光细胞，该培养上清液对斑jack幼虫具有致病性。这是首次报道从倍他奈达病毒cDNA克隆中可以转录感染性基因组rna。然后，我们测试了SJNNV和SGNNV之间的两个重组体对原宿主鱼的传染性。用由SGNNV RNA1和SGNNV RNA2组成的重组病毒对条纹斑马鱼和七带石斑鱼幼虫进行浸毒处理后，七带石斑鱼完全死亡，与接种SGNNV相似。相反，接种由SGNNV RNA1和SJNNV RNA2组成的重组病毒可杀死条纹斑马鱼，但对七带石斑鱼没有影响。使用抗sjnnv多克隆抗体的免疫荧光显微镜研究显示，这两种重组体在受感染鱼的大脑、脊髓和视网膜上繁殖，与亲本病毒接种感染相似。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。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