Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
PLoS One. 2013 Aug 1;8(8):e71047. doi: 10.1371/journal.pone.0071047. Print 2013.
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. The lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and CHIKV-host interactions. Infectious cDNA clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. Existing CHIKV cDNA clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. To obtain a virus expected to have the general characteristics of the recent E1-226V CHIKV isolates, we have constructed a new CHIKV full-length cDNA clone, CHIKV LS3, based on the consensus sequence of their aligned genomes. Here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (CHIKV LS3-GFP). Their characteristics were compared to those of natural strain ITA07-RA1, which was isolated during the 2007 outbreak in Italy. In cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. Compared to ITA07-RA1 and clinical isolate NL10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of CHIKV replication. Cyclosporin A had no effect on CHIKV replication, suggesting that cyclophilins -opposite to what was found for other +RNA viruses- do not play an essential role in CHIKV replication. The characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that CHIKV LS3 and LS3-GFP are suitable and representative tools to study CHIKV-host interactions, screen for antiviral compounds and unravel their mode of action.
基孔肯雅病毒（CHIKV）是一种蚊媒甲病毒，于 2004 年再次出现，并在近年来引发了大规模疫情。由于缺乏许可疫苗或治疗方法，因此需要更深入地了解病毒的生命周期和 CHIKV 与宿主的相互作用。感染性 cDNA 克隆是进行此类研究以及研究抗病毒化合物作用机制的重要工具。现有的 CHIKV cDNA 克隆基于单个临床分离株的单个基因组，预计该基因组会针对宿主环境发生特定的进化，并且在随后的细胞培养传代过程中也可能发生进化。为了获得预期具有近期 E1-226V CHIKV 分离株一般特征的病毒，我们基于其对齐基因组的共识序列构建了新的 CHIKV 全长 cDNA 克隆 CHIKV LS3。在这里，我们报告了该合成病毒及其表达绿色荧光蛋白的变体（CHIKV LS3-GFP）的特性。我们将它们的特征与在意大利 2007 年爆发期间分离的天然株 ITA07-RA1 进行了比较。在细胞培养中，合成病毒表现出与天然分离株相当的表型，在小鼠模型中，它们引起的致死性感染与天然株引起的感染无法区分。与 ITA07-RA1 和临床分离株 NL10/152 相比，合成病毒对几种抗病毒化合物的敏感性相似。3-脱氮腺苷被鉴定为 CHIKV 复制的新抑制剂。环孢菌素 A 对 CHIKV 复制没有影响，这表明亲环素 -与其他+RNA 病毒的发现相反-在 CHIKV 复制中不起关键作用。基于共识序列的合成病毒的特性及其与天然分离株的比较表明，CHIKV LS3 和 LS3-GFP 是研究 CHIKV-宿主相互作用、筛选抗病毒化合物及其作用机制的合适且有代表性的工具。
