Johnson M C, Simon B E, Kim C H, Leong J A
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA.
J Virol. 2000 Mar;74(5):2343-50. doi: 10.1128/jvi.74.5.2343-2350.2000.
Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31 degrees C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11, 550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.
乌鳢弹状病毒（SHRV）感染东南亚的温水鱼类，凭借其非病毒粒子基因（NV）属于诺达病毒属。由于SHRV在28至31摄氏度之间生长最佳，我们能够利用T7表达系统从病毒基因组的克隆cDNA拷贝中产生有活力的重组SHRV。11550个核苷酸的SHRV基因组的正链RNA拷贝与病毒核衣壳（N）、磷蛋白（P）和聚合酶（L）蛋白的表达，导致在预先感染了用于T7聚合酶表达的痘苗病毒载体的细胞中产生传染性SHRV。通过检测在NV和L基因之间的SHRV基因组中设计的一个独特限制位点，验证了重组病毒的产生。由于我们现在能够开始研究NV基因的功能，我们构建了一种重组病毒，该病毒在NV蛋白编码序列中第22个密码子处含有一个无义突变。NV基因敲除病毒在培养的鱼类细胞中的产生浓度与野生型病毒一样高，并且在电子显微镜照片中，产生的病毒粒子似乎与野生型病毒粒子相同。这些初步研究表明，NV在培养的鱼类细胞中的SHRV复制中没有关键功能。
