[Construction of genomic full-length cDNA of SARS coronavirus BJ01 strain and identification of its biological characteristics].
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.
Han
Jian-feng
JF
State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
Jiang
Tao
T
Chen
Shui-ping
SP
Yu
Man
M
Qin
E-de
ED
Zhao
Zhuo
Z
Li
Xiao-feng
XF
Qin
Cheng-feng
CF
Deng
Yong-qiang
YQ
Zhao
Hui
H
Zha
Hai-long
HL
Li
Xiao-yu
XY
D000818:Animals; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics*; D016679:Genome, Viral; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D014162:Transfection; D014709:Vero Cells
D018076:DNA, Complementary