Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in vero cells
Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in vero cells
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DOI：
10.1128/jvi.72.8.6437-6441.1998
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发表时间：
1998-08-01
影响因子：
5.4
通讯作者：
Muster, T
中科院分区：
文献类型：
--
作者：
Egorov, A;Brandt, S;Muster, T
我们建立了一个针对甲型流感病毒非结构基因片段的反向遗传学系统。该系统是基于使用温度敏感（fs）reflectant病毒25 A-1。25 A-1病毒含有来自流感A/列宁格勒/134/57病毒的NS基因和来自A/波多黎各（PR）/8/34病毒的其余基因片段。这种特殊的基因组合被认为是ts表型的原因。对于NS基因的反向遗传学，将来自流感A/PR/8/34病毒的质粒衍生的NS基因核糖核蛋白转染到先前用25 A-1病毒感染的细胞中。转染上清液在40 ℃下连续传代两次，选择含有源自A/PR/8/34病毒的转染NS基因的病毒。选择过程的高效率允许拯救具有NS 1蛋白的C-末端部分的大缺失的转染子病毒。获得了含有NS 1蛋白的N-末端124、80或38个氨基酸的活的转染病毒。而所有的缺失突变体在Vero细胞中生长到高滴度，Madin-Darby犬肾（MDCK）细胞上的生长和小鼠中的复制随着缺失长度的增加而降低。在Vero细胞中，缺失突变体的病毒蛋白表达水平与野生型相似。相反，在MDCK细胞中，缺失突变体的M1蛋白水平显著降低。
We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (fs) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40 degrees C selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.