Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
PLoS One. 2012;7(3):e32857. doi: 10.1371/journal.pone.0032857. Epub 2012 Mar 7.
Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.
严重急性呼吸系统综合症（SARS）是一种由 SARS 相关冠状病毒（SARS-CoV）引起的人畜共患病，于 2002 年出现并成为全球关注的健康问题。虽然最初的爆发通过经典的公共卫生措施得到了控制，但确实存在另一种 SARS-CoV 可能从其自然宿主中重新出现的风险，无论是以原始形式还是更具毒性或致病性的菌株形式出现；在这种情况下，如果没有任何有效的抗病毒药物或疫苗，病毒将难以控制。我们使用研究充分的 SARS-CoV 分离株 HKU-39849，开发了一种基于牛痘病毒的 SARS-CoV 反向遗传系统，该系统既稳健又生物安全。SARS-CoV 基因组被克隆在单独的牛痘病毒载体（vSARS-CoV-5prime 和 vSARS-CoV-3prime）中，作为两个 cDNA，随后连接在一起，创建一个全长 SARS-CoV cDNA 模板，用于体外转录 SARS-CoV 感染性 RNA 转录本。将 RNA 转录本转染到允许的细胞中导致恢复感染性病毒（recSARS-CoV）。对 recSARS-CoV 产生的蚀斑进行的表征表明，它们的大小与亲本 SARS-CoV 分离株 HKU-39849 相似，但比 SARS-CoV 分离株 Frankfurt-1 小。复制动力学的比较分析表明，recSARS-CoV 的复制动力学与 SARS-CoV Frankfurt-1 相似，尽管释放到培养上清液中的病毒滴度约低 10 倍。最后，反向遗传系统用于生成表达 Renilla 荧光素酶的 recSARS-CoV 报告病毒，以方便分析 SARS-CoV 在人树突状细胞（hDCs）中的基因表达。同时，也将 Renilla 荧光素酶基因插入到人类冠状病毒 229E（HCoV-229E）的基因组中。通过这种方法，我们证明与 HCoV-229E 不同，SARS-CoV 不能在 hDCs 中有效介导异源基因表达。
