Development of a novel single-step reverse genetics system for the generation of classical swine fever virus
Development of a novel single-step reverse genetics system for the generation of classical swine fever virus
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开发用于产生猪瘟病毒的新型单步反向遗传学系统
DOI：
10.1007/s00705-016-2851-6
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发表时间：
2016-07-01
影响因子：
2.7
通讯作者：
Pan, Zishu
中科院分区：
文献类型：
--
作者：
Li, Ling;Pang, Huining;Pan, Zishu
我们描述了一种基于猪RNA聚合酶I启动子（pSPI）介导的vRNA转录产生猪瘟病毒（CSFV）的替代反向遗传系统。构建了猪瘟石门株全长cDNA重组质粒pSPTI/SM，其5′端为猪RNA聚合酶I （pol I）启动子序列，3′端为鼠pol I终止子序列。将pSPTI/SM质粒转染到PK-15细胞中，可直接产生与亲本病毒末端相同的传染性猪瘟病毒。从这种反向遗传系统中拯救出来的猪瘟病毒与亲本猪瘟病毒相比，表现出相似的生长动力学和斑块形成。利用该反向遗传系统制备猪瘟疫苗C株时，在转染重组质粒pSPTI/C的PK-15细胞上清液中检测到传染性病毒。这种新型的反向遗传系统是研究病毒基因组结构和功能、分子致病性研究以及开发猪瘟基因工程疫苗的一种简单而有效的工具。
We describe an alternative reverse genetics system for generating classical swine fever virus (CSFV) based on swine RNA polymerase I promoter (pSPI)-mediated vRNA transcription. The recombinant plasmid pSPTI/SM harboring a full-length CSFV Shimen strain cDNA, flanked by a swine RNA polymerase I (pol I) promoter sequence at the 5′ end and a murine pol I terminator sequence at the 3′ end, was constructed. When the plasmid pSPTI/SM was introduced into PK-15 cells by transfection, an infectious CSFV with termini identical to those of the parental virus was generated directly. CSFV rescued from this reverse genetics system exhibited similar growth kinetics and plaque formation compared with the parental CSFV. When the novel reverse genetics system was used to generate the CSFV vaccine C-strain, infectious virus was detected in the supernatant of PK-15 cells transfected with the recombinant plasmid pSPTI/C. This novel reverse genetics system is a simple and efficient tool for the investigation of the structure and function of the viral genome, for molecular pathogenicity studies, and for the development of genetically engineered vaccines for CSFV.