Biacchesi S, Thoulouze M I, Béarzotti M, Yu Y X, Brémont M
Unité de Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France.
J Virol. 2000 Dec;74(23):11247-53. doi: 10.1128/jvi.74.23.11247-11253.2000.
Infectious hematopoietic necrosis virus (IHNV) is a Novirhabdovirus and is the causative agent of a devastating acute, lethal disease in wild and farmed rainbow trout. The virus is enzootic throughout western North America and has spread to Asia and Europe. A full-length cDNA of the IHNV antigenome (pIHNV-Pst) was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Recombinant IHNV (rIHNV) was recovered from fish cells at 14 degrees C, following infection with a recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) and cotransfection of pIHNV-Pst together with plasmids encoding the nucleoprotein N (pT7-N), the phosphoprotein P (pT7-P), the RNA polymerase L (pT7-L), and the nonvirion protein NV (pT7-NV). When pT7-N and pT7-NV were omitted, rIHNV was also recovered, although less efficiently. Incidental mutations introduced in pIHNV-Pst were all present in the rIHNV genome; however, a targeted mutation located in the L gene was eliminated from the recombinant genome by homologous recombination with the added pT7-L expression plasmid. To investigate the role of NV protein in virus replication, the pIHNV-Pst construct was engineered such that the entire NV open reading frame was deleted and replaced by the genes encoding green fluorescent protein or chloramphenicol acetyltransferase. The successful recovery of recombinant virus expressing foreign genes instead of the NV gene demonstrated that the NV protein was not absolutely required for viral replication in cell cultures, although its presence greatly improves virus growth. The ability to generate rIHNV from cDNA provides the basis to manipulate the genome in order to engineer new live viral vaccine strains.
传染性造血器官坏死病毒（IHNV）是一种诺达病毒，是野生和养殖虹鳟鱼中一种毁灭性急性致死疾病的病原体。该病毒在北美西部呈地方性流行，并已传播到亚洲和欧洲。从亚基因组重叠cDNA片段组装了IHNV反基因组的全长cDNA（pIHNV-Pst），并克隆到T7 RNA聚合酶启动子和自催化丁型肝炎病毒核酶之间的转录质粒中。在用表达T7 RNA聚合酶的重组痘苗病毒（vTF7-3）感染并将pIHNV-Pst与编码核蛋白N（pT7-N）、磷蛋白P（pT7-P）、RNA聚合酶L（pT7-L）和非病毒体蛋白NV（pT7-NV）的质粒共转染后，在14℃从鱼细胞中回收了重组IHNV（rIHNV）。当省略pT7-N和pT7-NV时，也回收了rIHNV，尽管效率较低。pIHNV-Pst中引入的偶然突变都存在于rIHNV基因组中；然而，通过与添加的pT7-L表达质粒进行同源重组，位于L基因中的一个靶向突变从重组基因组中消除。为了研究NV蛋白在病毒复制中的作用，对pIHNV-Pst构建体进行了改造，使得整个NV开放阅读框被删除，并被编码绿色荧光蛋白或氯霉素乙酰转移酶的基因取代。成功回收表达外源基因而非NV基因的重组病毒表明，NV蛋白在细胞培养中并非病毒复制绝对必需的，尽管其存在能极大地促进病毒生长。从cDNA产生rIHNV的能力为操纵基因组以构建新的活病毒疫苗株提供了基础。
