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    Improved method for the recovery of non-segmented, negative-stranded RNA viruses from cDNA
    2.
    发明公开
    Improved method for the recovery of non-segmented, negative-stranded RNA viruses from cDNA 审中-公开
    Verbittenes Verfahren zur Gewinnung nichtsegmentierter negativ-strang-RNA-Viren aus cDNA

    公开(公告)号：EP2298924A2

    公开(公告)日：2011-03-23

    申请号：EP10174369.8

    申请日：2004-06-08

    申请人： Wyeth LLC

    发明人： Parks, Christopher L. ,  Udem, Stephen A. ,  Sidhu, Modinderjit S.

    IPC分类号： C12N15/86 ,  A61K39/12 ,  C12N7/00

    CPC分类号： C07K14/005 ,  A61K39/00 ,  A61K48/00 ,  A61K2039/5254 ,  A61K2039/5256 ,  A61K2039/5258 ,  C12N7/00 ,  C12N15/86 ,  C12N2760/18022 ,  C12N2760/18422 ,  C12N2760/18451 ,  C12N2760/18452 ,  C12N2760/18551 ,  C12N2760/18552 ,  C12N2760/18622 ,  C12N2760/18651 ,  C12N2760/18652 ,  C12N2760/18662 ,  C12N2760/20243 ,  C12N2760/20251 ,  C12N2760/20252 ,  C12N2760/20262

    摘要： Methods for producing infectious, non-segmented, negative-stranded RNA viruses of the Order Mononegavirales are provided that involve coexpression of a viral cDNA along with essential viral proteins, N, P, and L in a host cell transiently transfected with an expression vector encoding an RNA polymerase. In alternate methods, after the host cell is transfected with a viral cDNA expression vector and one or more vectors encoding the RNA polymerase, N protein, P protein, and L protein, the host cell is exposed to an effective heat shock under conditions sufficient to increase recovery of the recombinant virus. In other alternate embodiments, the host cells are transferred after viral rescue begins into co-culture with a plaque expansion cell, typically a monolayer of expansion cells, and the assembled infectious, non-segmented, negative-stranded RNA virus is recovered from the co-culture. Also provided within the invention are compositions for producing infectious, non-segmented, negative-stranded RNA virus of the Order Mononegavirales, recombinant viruses produced using the foregoing methods and compositions, and immunogenic compositions and methods employing the recombinant viruses. In additional embodiments, the methods and compositions of the invention are employed to produce growth- or replication-defective non-segmented negative-stranded RNA viruses and subviral particles.

    摘要翻译： 提供了用于产生单宁属病毒感染性，非分段的负链RNA病毒的方法，其涉及病毒cDNA与基本病毒蛋白N，P和L共表达在用表达载体编码的瞬时转染的宿主细胞中 RNA聚合酶。 在替代方法中，在用病毒cDNA表达载体和编码RNA聚合酶，N蛋白，P蛋白和L蛋白的一种或多种载体转染宿主细胞后，将宿主细胞暴露于有效的热休克， 增加重组病毒的恢复。 在其他替代实施方案中，宿主细胞在病毒性拯救开始与斑块扩增细胞（通常为单层扩增细胞）共培养后被转移，并且组装的感染性，非分段的负链RNA病毒从共同 -文化。 在本发明中还提供了用于产生单宁属（Mononegavirales）的感染性，非分段的负链RNA病毒的组合物，使用前述方法和组合物产生的重组病毒，以及使用重组病毒的免疫原性组合物和方法。 在另外的实施方案中，本发明的方法和组合物用于产生生长或复制缺陷型非分段负链RNA病毒和亚病毒颗粒。