Switching species tropism: an effective way to manipulate the feline coronavirus genome
Switching species tropism: an effective way to manipulate the feline coronavirus genome
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DOI：
10.1128/jvi.77.8.4528-4538.2003
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发表时间：
2003-04-01
影响因子：
5.4
通讯作者：
Rottier, PJM
中科院分区：
文献类型：
--
作者：
Haijema, BJ;Volders, H;Rottier, PJM
猫传染性腹膜炎病毒(FIPV)是一种冠状病毒，是猫致死性感染的病原体。像其他冠状病毒一样，FIPV含有一个约30kb的超大正链RNA基因组。我们在这里描述了基于靶向RNA重组的FIPV反向遗传学策略的开发和使用，该策略类似于已经描述的小鼠肝炎病毒(MHV)(L.Kuo等人，J.Virol。74：1393-1406,2000)。在这两步过程中，我们首先通过靶向重组构建了FIPV的突变体，命名为mFIPV，其中尖峰糖蛋白的胞外区被MHV的胞外区取代。这种转变允许在小鼠细胞中选择重组病毒：mFIPV在这些细胞中生长到高滴度，但已经失去了在猫细胞中生长的能力。在第二个反向过程中，mFIPV被用作受体，FIPV尖峰的重新引入现在允许通过它们重新获得在猫细胞中生长的能力来选择候选重组体。用这种方法，我们构建了野生型重组病毒(r-wtFIPV)，并产生了非结构基因7b起始密码子已被破坏的定向突变体FIPV(FIPVDelta7b)。R-wtFIPV与其亲本病毒FIPV 79-1146不仅在组织培养上的生长特性没有区别，而且在猫身上也是如此，表现出高度致死性。FIPV Delta7b失去了其7b基因的表达，但在细胞培养中生长未受损害，证实了7b糖蛋白在体外不是必需的。我们建立了针对冠状病毒的第二个靶向RNA重组系统，为FIPV基因组的基因工程提供了有力的工具。
Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPVDelta7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPVDelta7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.