Development of reverse genetics systems for bluetongue virus: Recovery of infectious virus from synthetic RNA transcripts
Development of reverse genetics systems for bluetongue virus: Recovery of infectious virus from synthetic RNA transcripts
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DOI：
10.1128/jvi.00808-08
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发表时间：
2008-09-01
影响因子：
5.4
通讯作者：
Roy, Polly
中科院分区：
文献类型：
--
作者：
Boyce, Mark;Celma, Cristina C. P.;Roy, Polly
蓝舌病毒（BTV）是一种由昆虫传播的野生反刍动物和家畜的新兴病原体，在世界许多地区对农业造成了严重的经济影响。由于缺乏反向遗传系统，对蓝舌病毒复制和发病机制的研究受到了阻碍。通过用从转录核心在体外获得的10种纯化病毒mRNA的完整集合转染允许细胞，可以恢复有感染性的蓝舌病毒（M.博伊斯和P.罗伊，《病毒学杂志》81：2179 - 2186，2007年）。在此，我们报道从cDNA克隆获得的体外合成的T7转录本可以使用T7转录本和核心来源的mRNA的混合物被引入蓝舌病毒基因组。描述了基因组节段10的替换以及编码两种具有重要免疫作用的外衣壳蛋白VP2和VP5的节段2和5的同时替换。此外，我们证明了完全从T7转录本恢复有感染性的蓝舌病毒，这证明了在加帽类似物存在的情况下合成的转录本能够在蓝舌病毒复制周期的所有阶段功能性替代病毒转录本。具有完全确定基因组的蓝舌病毒的产生允许在确定的遗传背景下恢复突变。产生特定突变体的能力为研究蓝舌病毒复制周期提供了一种新工具，并且允许产生设计疫苗株，这在许多国家是非常需要的。
Bluetongue virus (BTV), an insect-vectored emerging pathogen of both wild ruminants and livestock, has had a severe economic impact in agriculture in many parts of the world. The investigation of BTV replication and pathogenesis has been hampered by the lack of a reverse genetics system. Recovery of infectious BTV is possible by the transfection of permissive cells with the complete set of 10 purified viral mRNAs derived in vitro from transcribing cores (M. Boyce and P. Roy, J. Virol. 81:2179-2186, 2007). Here, we report that in vitro synthesized T7 transcripts, derived from cDNA clones, can be introduced into the genome of BTV using a mixture of T7 transcripts and core-derived mRNAs. The replacement of genome segment 10 and the simultaneous replacement of segments 2 and 5 encoding the two immunologically important outer capsid proteins, VP2 and VP5, are described. Further, we demonstrate the recovery of infectious BTV entirely from T7 transcripts, proving that synthetic transcripts synthesized in the presence of cap analogue can functionally substitute for viral transcripts at all stages of the BTV replication cycle. The generation of BTV with a fully defined genome permits the recovery of mutations in a defined genetic background. The ability to generate specific mutants provides a new tool to investigate the BTV replication cycle as well as permitting the generation of designer vaccine strains, which are greatly needed in many countries.