Rapid generation of rotavirus single-gene reassortants by means of eleven plasmid-only based reverse genetics
Rapid generation of rotavirus single-gene reassortants by means of eleven plasmid-only based reverse genetics
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DOI：
10.1099/jgv.0.001443
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发表时间：
2020-01-01
影响因子：
3.8
通讯作者：
Komoto, Satoshi
中科院分区：
文献类型：
--
作者：
Fukuda, Saori;Hatazawa, Riona;Komoto, Satoshi
重组是A组轮状病毒（RVA）进化的重要机制，产生具有新的遗传和表型性状的病毒。用于产生具有所需遗传组合的RVA抗性体的经典方法是费力且耗时的，因为分离所需抗性体需要筛选和选择过程。利用最近开发的基于仅11个克隆的编码RVA基因组的cDNA的RVA反向遗传学系统（11质粒纯系统），我们制备了一组基于猿SA 11-L2病毒的单基因表达体，每个表达体含有1个来自G1 P基因型的人KU病毒的片段[8]。结果表明，没有基因特异性的限制的重配潜力。除了这11个单基因重组子之外，还可以用11个仅质粒系统制备三基因重组子，所述三基因重组子具有来自KU的编码核心的VP 1 -3基因片段，其具有SA 11-L2遗传背景，其构成由基于KU的核心和基于SA 11-L2的中间层和外层组成的病毒体。最后，为了该系统的可能临床应用，我们有效地产生了一系列代表所有主要人类RVA G基因型（G1-4、G9和G12）的VP 7抑制剂。这些单基因抑制剂中的每一种的制备都在短短2周内完成。我们的研究结果表明，11质粒系统允许快速和可靠的RVA单基因抑制剂的产生，这将是有用的基础研究和临床应用。
Reassortment is an important mechanism in the evolution of group A rotaviruses (RVAs), yielding viruses with novel genetic and phenotypic traits. The classical methods for generating RVA reassortants with the desired genetic combinations are laborious and time-consuming because of the screening and selection processes required to isolate a desired reassortant. Taking advantage of a recently developed RVA reverse genetics system based on just 11 cloned cDNAs encoding the RVA genome (11 plasmid-only system), we prepared a panel of simian SA11-L2 virus-based single-gene reassortants, each containing 1 segment derived from human KU virus of the G1P[8] genotype. It was shown that there was no gene-specific restriction of the reassortment potential. In addition to these 11 single-gene reassortants, a triple-gene reassortant with KU-derived core-encoding VP1-3 gene segments with the SA11-L2 genetic background, which make up a virion composed of the KU-based core, and SA11-L2-based intermediate and outer layers, could also be prepared with the 11 plasmid-only system. Finally, for possible clinical application of this system, we generated a series of VP7 reassortants representing all the major human RVA G genotypes (G1-4, G9 and G12) efficiently. The preparation of each of these single-gene reassortants was achieved within just 2 weeks. Our results demonstrate that the 11 plasmid-only system allows the rapid and reliable generation of RVA single-gene reassortants, which will be useful for basic research and clinical applications.