Institut für Virologie, Freie Universität Berlin; Philippstrasse 13, Haus 18; 10115 Berlin, Germany.
Vet Res. 2011 Jan 11;42(1):3. doi: 10.1186/1297-9716-42-3.
Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.
最近，宠物鼠和猫向人类的传播，以及猫科动物和其他动物物种的严重感染，引起了人们对牛痘病毒（CPXV）的越来越多的关注。我们报告了牛痘病毒株 Brighton Red（BR）的整个基因组作为大肠杆菌中的细菌人工染色体（BAC）的克隆，并从克隆 DNA 中恢复了传染性病毒。通过同源重组将允许在大肠杆菌中维持大的环状 DNA 的 mini-F 载体首次引入 CPXV 的胸苷激酶基因座，从而实现了全长 CPXV DNA 克隆的生成。然后将环状复制中间体电穿孔到大肠杆菌 DH10B 细胞中。成功建立感染性 BR 克隆后，我们修饰了全长克隆，使得在真核细胞中转染时可以发生重组介导的细菌序列缺失。这种 mini-F 序列内的序列重复使细菌复制子能够自我切除，从而在没有残留标记或载体序列的情况下回收重组病毒后代。从两个 BAC 克隆衍生的病毒的体外生长特性被确定，并且发现与亲本野生型 BR 几乎无法区分。最后，确定了传染性克隆的完整基因组序列，并且克隆的病毒基因组与亲本病毒相同。总之，生成的传染性克隆将极大地促进对 CPXV 个别基因和发病机制的研究。此外，现在可以使用这个新生成的工具更系统地探索 CPXV 的载体潜力。
