A Genome Sequence of Novel SARS-CoV Isolates: the Genotype, GD-Ins29, Leads to a Hypothesis of Viral Transmission in South China

Qin, E’de; He, Xionglei; Tian, Wei; Liu, Yong; Li, Wei; Wen, Jie; Wang, Jingqiang; Fan, Baochang; Wu, Qingfa; Chang, Guohui; Cao, Wuchun; Xu, Zuyuan; Yang, Ruifu; Wang, Jing; Yu, Man; Li, Yan; Xu, Jing; Si, Bingyin; Hu, Yongwu; Peng, Wenming; Tang, Lin; Jiang, Tao; Shi, Jianping; Ji, Jia; Zhang, Yu; Ye, Jia; Wang, Cui’e; Han, Yujun; Zhou, Jun; Deng, Yajun; Li, Xiaoyu; Hu, Jianfei; Wang, Caiping; Yan, Chunxia; Zhang, Qingrun; Bao, Jingyue; Li, Guoqing; Chen, Weijun; Fang, Lin; Li, Changfeng; Lei, Meng; Li, Dawei; Tong, Wei; Tian, Xiangjun; Wang, Jin; Zhang, Bo; Zhang, Haiqing; Zhang, Yilin; Zhao, Hui; Zhang, Xiaowei; Li, Shuangli; Cheng, Xiaojie; Zhang, Xiuqing; Liu, Bin; Zeng, Changqing; Li, Songgang; Tan, Xuehai; Liu, Siqi; Dong, Wei; Wang, Jun; Wong, Gane Ka-Shu; Yu, Jun; Wang, Jian; Zhu, Qingyu; Yang, Huanming

After careful sequence alignment, 137 substitutions were identified in comparison with the other 16 published SARS-CoV genome sequences, yielded an overall-genome mutation rate of approximately 0.46% (Table 3). 45 substitutions were found just between GD01 and BJ01 (Supplementary Table 1). 38 out of 45 are unique to GD01. Special attention was paid to the sequences of the mutation sites and multiple clones from the amplicon-libraries constructed from multiple independent RT-PCR products were sequenced. A substantial portion (70.4%, 100/142) of the substitutions was predicted to be non-synonymous mutations in the ORFs, including 14 in the PUPs. Among the 92 substitutions detected in the R (replicase) protein, 70.7% (65/92) could lead to amino acid changes. Although the S (spike) protein has a low ratio (13/22), the ratio is higher in the other structural proteins, the M (4/4) and N proteins (3/4), as well as in BGI-PUP2 (4/5), BGI-PUP3 (2/2), and BGI-PUP (GD-Ins29, 2/2). Even though a large fraction of substitutions in the viral genome have been located in the ORF for the R protein, its substitution rate is actually the lowest among all the defined CDSs, with regard to its large size (21,222 nt). The mutation rate of SARS-CoV is quite high if we take into consideration such a short time period since it was identified from human hosts. A high mutation rate is consistent with the high error rate of RNA replication (9) and the high fraction of non-synonymous substitutions also implies the possibility that the selective pressure from the host may have worked on the virus, albeit there is very little statistical power to support the conjecture with current data set. A very important notion from our data (including tens of thousands of sequencing traces) is the obvious absence of indels (insertions and deletions), suggesting that the viral polymerase is prone to the replication errors in proofreading but remains relatively accurate in frame-moving conveyed by the RdRp (RNA-dependent RNA polymerase) activity.