Generation of influenza A viruses entirely from cloned cDNAs
Generation of influenza A viruses entirely from cloned cDNAs
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DOI：
10.1073/pnas.96.16.9345
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发表时间：
1999-08-03
影响因子：
11.1
通讯作者：
Kawaoka, Y
中科院分区：
文献类型：
--
作者：
Neumann, G;Watanabe, T;Kawaoka, Y
我们描述了一种新的反向遗传学系统，它允许人们完全从克隆的cDNA中有效地产生甲型流感病毒。用8种质粒转染人胚肾细胞（293 T），每种质粒编码一种病毒RNA。A/WSN/33（H1N1）或A/PR/8/34（H1N1）病毒，侧翼为人RNA聚合酶I启动子和小鼠RNA聚合酶I终止子-以及编码病毒核蛋白和PB 2、PB 1和PA病毒聚合酶的质粒。该策略在转染后18小时产生>1 × 10（3）噬斑形成单位（pfu）/ml上清液的病毒。添加表达所有剩余病毒结构蛋白的质粒导致病毒产量显著增加，3 x 10（4）-5 x 10（7）pfu/ml。我们还使用反向遗传学来产生包含A/PR/8/34病毒的PB 1基因的重组病毒，所有其他基因代表A/WSN/33。通过这种方法产生的其他病毒在PA基因中具有突变或在神经氨酸酶蛋白的头部具有外来表位。这种不需要辅助病毒感染的有效系统，在病毒诱变研究和疫苗和基因治疗载体的生产中应该是有用的。
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 mi of supernatant at is 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.