Nolden T, Pfaff F, Nemitz S, Freuling C M, Höper D, Müller T, Finke Stefan
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Molecular Virology and Cell Biology, D-17493 Greifswald - Insel Riems, Germany.
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Diagnostic Virology, D-17493 Greifswald - Insel Riems, Germany.
Sci Rep. 2016 Apr 5;6:23887. doi: 10.1038/srep23887.
Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.
反向遗传学方法是病毒复制和发病机制概念验证中不可或缺的工具。对于负链RNA病毒（NSV）而言，传染性cDNA克隆数量有限是一个瓶颈，因为这些克隆通常是从适应细胞培养或减毒的病毒中产生的，用于发病机制研究的潜力有限。我们开发了一种系统，通过线性到线性的RedE/T重组将完整NSV基因组的cDNA拷贝直接克隆到反向遗传学载体中。快速克隆多个狂犬病病毒（RABV）全长基因组并鉴定与野外病毒共有序列相同的克隆，证实了该方法的可靠性。从野外病毒cDNA克隆中拯救出重组病毒。亲代病毒和重组病毒具有相似的生长动力学，在细胞类型特异性复制中保留了野外病毒特征，并在小鼠模型中证实了其毒力。插入报告基因后滴度降低表明野外病毒的低水平复制受基因插入的影响。通过克隆正布尼亚病毒L基因组片段的多个拷贝证明了该策略的灵活性。反向遗传学技术发展中的这一重要步骤为分析病毒变异性以及在克隆水平上对重组病毒进行表型特征分析开辟了新途径。
