Institute of Virology, University of Bonn Medical Center, Bonn, Germany.
Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
J Gen Virol. 2012 Sep;93(Pt 9):1991-1995. doi: 10.1099/vir.0.043752-0. Epub 2012 Jun 6.
Chikungunya virus (CHIKV) has caused massive epidemics in the Indian Ocean region since 2005. It belongs to the genus Alphavirus and possesses a positive-stranded RNA genome of nearly 12 kb in size. To produce genetically modified viruses for the study of various aspects of the CHIKV life cycle, a reverse genetic system is needed. We report the generation of a T7 RNA polymerase-driven infectious cDNA clone of CHIKV. Electroporation of in vitro-transcribed RNA resulted in the recovery of a recombinant virus with growth characteristics comparable to the parental strain. Using the established cDNA clone, the red fluorescent marker gene mCherry was introduced into two different sites within the CHIKV nsP3 gene. Both constructs allowed the rescue of stable fluorescent reporter viruses with growth characteristics similar to the wild-type virus. The latter reporter viruses represent valuable tools for easy follow-up of replicating CHIKV useful in several applications of CHIKV research.
基孔肯雅病毒（CHIKV）自 2005 年以来在印度洋地区引发了大规模疫情。它属于甲病毒属，拥有一条大小约为 12kb 的正链 RNA 基因组。为了研究 CHIKV 生命周期的各个方面而产生遗传修饰病毒，需要一个反向遗传学系统。我们报告了 CHIKV 的 T7 RNA 聚合酶驱动的传染性 cDNA 克隆的生成。体外转录 RNA 的电穿孔导致了重组病毒的恢复，其生长特征与亲本株相当。利用建立的 cDNA 克隆，将红色荧光标记基因 mCherry 引入 CHIKV nsP3 基因的两个不同位置。这两个构建体都允许稳定荧光报告病毒的拯救，其生长特征与野生型病毒相似。后一种报告病毒是用于研究 CHIKV 的几种应用中复制 CHIKV 的简便后续的有用工具。
