Complete Genome Sequences of the SARS-CoV: the BJ Group (Isolates BJ01-BJ04)

Bi, Shengli; Qin, E’de; Xu, Zuyuan; Li, Wei; Wang, Jing; Hu, Yongwu; Liu, Yong; Duan, Shumin; Hu, Jianfei; Han, Yujun; Xu, Jing; Li, Yan; Yi, Yao; Zhou, Yongdong; Lin, Wei; Wen, Jie; Xu, Hong; Li, Ruan; Zhang, Zizhang; Sun, Haiyan; Zhu, Jingui; Yu, Man; Fan, Baochang; Wu, Qingfa; Lin, Wei; Tang, Lin; Yang, Bao’an; Li, Guoqing; Peng, Wenming; Li, Wenjie; Jiang, Tao; Deng, Yajun; Liu, Bohua; Shi, Jianping; Deng, Yongqiang; Wei, Wei; Liu, Hong; Tong, Zongzhong; Zhang, Feng; Zhang, Yu; Wang, Cui’e; Li, Yuquan; Ye, Jia; Gan, Yonghua; Ji, Jia; Li, Xiaoyu; Tian, Xiangjun; Lu, Fushuang; Tan, Gang; Yang, Ruifu; Liu, Bin; Liu, Siqi; Li, Songgang; Wang, Jun; Wang, Jian; Cao, Wuchun; Yu, Jun; Dong, Xiaoping; Yang, Huanming

SARS patients were clinically diagnosed in March 2003 according to World Health Organization (WHO) guidelines (http://www.who.int/csr/sars/guidelines /en/). SARS-CoV isolates were maintained in Vero-6 cell cultures that were inoculated from autopsies and biopsies of the deceased or recovered SARS patients (Table 1 ). Viral RNA was purified from virions prepared from the cultures and subjected for cDNA syntheses. A set of primers that cover the entire viral genome were used for the reverse transcription and PCR amplification, in a product size range of 400-800 bp. PCR-amplified fragments were then cloned into amplicon-libraries and two dozens or more clones were sequenced for each PCR-amplified fragments to ensure sequence quality and to avoid errors in the procedures of the RT-PCR and cloning. Only the consensus sequences with absolute majority votes were used for the genome assembly and gene annotation, although every read was assembled and some sequence variations were clearly visible. For comparative genomic analyses, 13 other full-length SARS-CoV genome sequences were downloaded from GenBank (Table 2; http://www.ncbi.nlm.nih.gov). The nucleotide positions of Isolate BJ01 were used as the reference for analyses. Isolates BJ01 and BJ03 were derived from the autopsied lung tissue (BJ01) and liver/lymph nodes (BJ03) of the same patient who was the father of the first patient (the Index Case of Beijing) diagnosed on March 1, 2003 in Beijing. His own daughter infected him in the last week of February in Shanxi Province after she traveled to Guangdong Province during the period of February 18th to 23rd. He was 53 years old and had no detectable symptoms of hepatitis, AIDS, or heart diseases. He, as the second SARS patient hospitalized (March 5th) in Beijing, died two days after the diagnosis. His daughter, however, had recovered completely from the horrifying ordeal after medical treatments. The sequence differences between Isolates BJ01 and BJ03 are expected to reflect the sequence variations within the same patient after viral infection. Isolate BJ02 was inoculated from pooled samples of nose/throat swabs from seven patients who were all evidenced to be infected by the first patient when she (the Index Case of Beijing) was hospitalized in the first week of March at the same hospital. All of these patients were recovered after effective treatments. The sequence differences between BJ01 and BJ02 should be regarded as the variation from the first circle or round of the infection. Isolate BJ04 was inoculated from the autopsied lung tissue of a single deceased patient who had no direct contact with the Index Case of Beijing. The sequence differences between BJ04 and other BJ cases should yield variations during viral infections outside the first circle. As a whole, the group of isolates, or the BJ Group, represents the early rounds of disease transmissions in Beijing. Another relevant case concerns Isolate GD01 (previously named GZ01). It was isolated from the autopsied lung tissue of a single deceased patient who was a female of 54 years old. She was suspected being infected during her hospitalization by indirect contact with one of the "superspreaders", who stayed in the same hospital as she did. She was one of the SARS cases with known transmission path connecting to the "Index Cases" identified in Guangdong Province. Its genome sequence serves as a root or an anchor point for the BJ Group. In addition, the genome sequence of GD01 harbors