Yao K, Vakharia V N
Center for Agricultural Biotechnology, University of Maryland, College Park, Maryland 20742, USA.
J Virol. 1998 Nov;72(11):8913-20. doi: 10.1128/JVI.72.11.8913-8920.1998.
We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3'-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3' end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.
我们利用从克隆的互补脱氧核糖核酸（cDNA）衍生而来的正链核糖核酸（RNA）转录本，开发了一种针对传染性胰腺坏死病毒（IPNV）的反向遗传学系统，IPNV是双RNA病毒科的原型病毒。构建了包含RNA片段A和B的整个编码区和非编码区的IPNV基因组全长cDNA克隆。片段A编码一种106 kDa的前体蛋白，该蛋白被切割后产生成熟的VP2、非结构蛋白酶和VP3蛋白，而片段B编码依赖RNA的RNA聚合酶VP1。通过用T7 RNA聚合酶对线性化质粒进行体外转录，制备了两个片段的正义RNA转录本。用片段A和B的组合转录本转染奇努克鲑鱼胚胎（CHSE）细胞，在转染后10天产生了有传染性的IPNV颗粒。此外，还产生了一种含有基因标记序列的转染病毒，以证实该系统的可行性。通过用兔抗IPNV血清对感染的CHSE细胞进行免疫荧光染色以及核苷酸序列分析，确定了回收病毒的存在和特异性。此外，对回收病毒的RNA进行3'末端序列分析表明，体外转录过程中在3'末端合成的多余核苷酸在复制过程中被精确修剪或排除，因此这些核苷酸没有被纳入基因组。我们试图确定IPNV和另一种双RNA病毒传染性法氏囊病病毒（IBDV）的依赖RNA的RNA聚合酶是否能在异源组合中支持病毒拯救。因此，用来自IPNV片段A和IBDV片段B的转录本转染CHSE细胞，并用来自IBDV片段A和IPNV片段B的转录本转染非洲绿猴肾（Vero）细胞。在这两种情况下，即使在细胞培养中传代三次后，也没有产生有传染性的IPNV或IBDV颗粒，这表明病毒依赖RNA的RNA聚合酶具有种属特异性。然而，我们开发的IPNV反向遗传学系统将极大地促进对病毒复制和发病机制的研究以及新一代减毒活疫苗的设计。
