Neumann G, Watanabe T, Ito H, Watanabe S, Goto H, Gao P, Hughes M, Perez D R, Donis R, Hoffmann E, Hobom G, Kawaoka Y
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin, 2015 Linden Drive West, Madison, WI 53706, USA.
Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9345-50. doi: 10.1073/pnas.96.16.9345.
We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator-together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 x 10(3) plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 x 10(4)-5 x 10(7) pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.
我们描述了一种新的反向遗传学系统，该系统能让人从克隆的互补脱氧核糖核酸（cDNA）高效生成甲型流感病毒。用人胚胎肾细胞（293T）转染八个质粒，每个质粒编码A/WSN/33（H1N1）或A/PR/8/34（H1N1）病毒的一种病毒核糖核酸（RNA），两侧分别是人核糖核酸聚合酶I启动子和小鼠核糖核酸聚合酶I终止子，同时转染编码病毒核蛋白以及PB2、PB1和PA病毒聚合酶的质粒。该策略在转染后48小时每毫升上清液产生>1×10³个病毒蚀斑形成单位（pfu）。添加表达所有其余病毒结构蛋白的质粒导致病毒产量大幅增加，达到3×10⁴ - 5×10⁷ pfu/ml。我们还利用反向遗传学生成了一种重配病毒，其含有A/PR/8/34病毒的PB1基因，其他所有基因均源自A/WSN/33。通过该方法产生的其他病毒在PA基因中有突变，或者在神经氨酸酶蛋白头部有一个外源表位。这个高效系统无需辅助病毒感染，在病毒诱变研究以及疫苗和基因治疗载体的生产中应会很有用。
