Han Jian-feng, Jiang Tao, Chen Shui-ping, Yu Man, Qin E-de, Zhao Zhuo, Li Xiao-feng, Qin Cheng-feng, Deng Yong-qiang, Zhao Hui, Zha Hai-long, Li Xiao-yu
State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
Wei Sheng Wu Xue Bao. 2006 Dec;46(6):922-7.
Severe acute respiratory syndrome (SARS) is an important emerging infectious disease which caused by SARS coronavirus (SARS-CoV), and the study of its pathogenesis is needed for the treatment and prevention of this disease. To study the pathogenesis of SARS-CoV using reverse genetics technology, by in vitro ligation using 7 contiguous cDNAs that span the entire genome of the SARS-CoV BJ01 strain, a genomic full-length cDNA was assembled, then using T7 RiboMAX Large Scale RNA Production Systems with the genomic full-length cDNA as template, the RNA transcript was attained. The typical SARS-CoV-resulted cell pathogenic effects were observed when RNA transcript was electroporated into Vero E6 cells. The results of RT-PCR and sequencing of the rescued virus showed that it originated from transcript which derived from the full-length cDNA construct. Rescued virus-infected cells were detected by indirect fluorescent antibody staining demonstrated that it can specifically reaction with SARS-CoV. By CPE method and plaque assay, the titers of the rescued virus and wild-type virus were assessed, which demonstrated there are no significant difference between the viruses and they have similar biological characteristics. Construction of the genomic full-length cDNA of SARS-CoV BJ01 stain successfully and study of the biological characteristics of the rescued virus will provide a useful tool serving for the discovery of molecular pathogenesis and development of candidate vaccines against SARS-CoV.
严重急性呼吸综合征（SARS）是一种由SARS冠状病毒（SARS-CoV）引起的重要新发传染病，对其发病机制的研究对于该疾病的治疗和预防至关重要。为了利用反向遗传学技术研究SARS-CoV的发病机制，通过体外连接跨越SARS-CoV BJ01株全基因组的7个连续cDNA，组装出一个基因组全长cDNA，然后以该基因组全长cDNA为模板，使用T7 RiboMAX大规模RNA生产系统，获得了RNA转录本。当将RNA转录本电穿孔导入Vero E6细胞时，观察到了典型的SARS-CoV引起的细胞病变效应。对拯救病毒进行RT-PCR和测序的结果表明，它源自全长cDNA构建体的转录本。通过间接荧光抗体染色检测拯救病毒感染的细胞，结果表明其能与SARS-CoV发生特异性反应。通过CPE法和蚀斑试验评估了拯救病毒和野生型病毒的滴度，结果表明两种病毒之间无显著差异，且具有相似的生物学特性。成功构建SARS-CoV BJ01株的基因组全长cDNA并对拯救病毒的生物学特性进行研究，将为发现分子发病机制和开发抗SARS-CoV候选疫苗提供有用工具。
