Recovery of infectious bluetongue virus from RNA
Recovery of infectious bluetongue virus from RNA
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DOI：
10.1128/jvi.01819-06
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发表时间：
2007-03-01
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;Roy, Polly
蓝舌病毒(Bluetongue Virus，BTV)是一种以昆虫为媒介的新型反刍动物病原体，可能对欧洲农业造成毁灭性的经济影响。BTV和许多呼肠孤病毒科的其他成员仍然顽固地抵制开发从克隆核酸中拯救传染性病毒的方法(反向遗传学)。部分分解的病毒颗粒在转录上是活跃的，在受感染细胞的细胞质中合成病毒转录本，本质上是原位运送病毒核酸。为了建立BTV的反向遗传学系统，我们研究了用BTV核心颗粒体外合成的BTV转录本(单链RNA[ssRNA])导入BSR细胞恢复感染性BTV的可能性。转染后，免疫印迹检测到病毒-蛋白质合成，共聚焦检查显示，与感染细胞中看到的相似，包涵体在细胞质中呈点状分布。从转单链RNA的细胞中分离到病毒双链RNA(DsRNA)，证明单链RNA已经复制。此外，在BSR细胞中的感染性传递证明，在转基因细胞的培养液中存在传染性病毒。侵染性对单链特异性RNaseA敏感，将BTV基因组dsRNA与转录的单链单链RNA共转染后，活性成分为单链单链RNA，而不是单链双链RNA。我们的结论是，完全从单链RNA中恢复感染性BTV是可能的，这为建立呼肠孤病毒科成员的辅助病毒非依赖反向遗传学系统提供了一种方法。
Bluetongue virus (BTV) is an insect-vectored emerging pathogen of ruminants with the potential for devastating economic impact on European agriculture. BTV and many other members of the Reoviridae have remained stubbornly refractory to the development of methods for the rescue of infectious virus from cloned nucleic acid (reverse genetics). Partially disassembled virus particles are transcriptionally active, synthesizing viral transcripts in the cytoplasm of infected cells, in essence delivering viral nucleic acids in situ. With the goal of generating a reverse-genetics system for BTV, we examined the possibility of recovering infectious BTV by the transfection of BSR cells with BTV transcripts (single-stranded RNA [ssRNA]) synthesized in vitro using BTV core particles. Following transfection, viral-protein synthesis was detected by immunoblotting, and confocal examination of the cells showed a punctate cytoplasmic distribution of inclusion bodies similar to that seen in infected cells. Viral double-stranded RNA (dsRNA) was isolated from ssRNA-transfected cells, demonstrating that replication of the ssRNA had occurred. Additionally, infectious virus was present in the medium of transfected cells, as demonstrated by the passage of infectivity in BSR cells. Infectivity was sensitive to single-strand-specific RNase A, and cotransfection of genomic BTV dsRNA with transcribed ssRNA demonstrated that the ssRNA species, rather than dsRNA, were the active components. We conclude that it is possible to recover infectious BTV wholly from ssRNA, which suggests a means for establishing helper virus-independent reverse-genetics systems for members of the Reoviridae.