Generation of influenza A viruses entirely from cloned cDNAs

作     者：Neumann, G Watanabe, T Ito, H Watanabe, S Goto, H Gao, P Hughes, M Perez, DR Donis, R Hoffmann, E Hobom, G Kawaoka, Y 

作者机构：Univ Wisconsin Sch Vet Med Dept Pathobiol Sci Madison WI 53706 USA Hokkaido Univ Sch Vet Med Dept Dis Control Microbiol Lab Sapporo Hokkaido 0600818 Japan Univ Nebraska Dept Vet & Biomed Sci Lincoln NE 68583 USA Inst Mikro & Mol Biol D-35392 Giessen Germany 

出 版 物：《PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA》 (美国国家科学院汇刊)

年 卷 期：1999年第96卷第16期

页      面：9345-9350页

核心收录：

学科分类：07[理学] 08[工学] 

主　　题：细胞系 DNA 互补 DNA 病毒/遗传学 HN蛋白质/遗传学 流感病毒A型/遗传学 流感病毒A型/生理学 肾 质粒 RNA聚合酶Ⅰ/代谢 RNA 病毒/遗传学 逆转录聚合酶链反应 转染 病毒结构蛋白质类/遗传学 病毒复制 动物 鸡胚 狗 人类 小鼠 

摘      要：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.