Abstract
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.
Highlights
Cowpox virus (CPXV) belongs to the family Poxviridae, a family of large double- stranded DNA viruses replicating in the cytoplasm
We report an infectious clone in which bacterial sequences are lost via homologous recombination events in eukaryotic cells and present a Bacterial artificial chromosomes (BAC) that, upon reconstitution in cell culture, does not contain any vector or marker sequences and is genetically indistinguishable from the parental virus
For generation of the infectious clone, the thymidine kinase locus was chosen based on data from other orthopox viruses, which had shown the nonessential nature of the gene when virus is propagated in cultured cells [20]
Summary
Cowpox virus (CPXV) belongs to the family Poxviridae, a family of large double- stranded DNA viruses replicating in the cytoplasm. It is endemic in Eurasia and reportedly transmitted from rodents as the reservoir host to other mammals. CPXV causes disease in felids, zoo animals, man and, despite its name, only very rarely in cattle. Recent outbreaks in humans and its close relationship to other important members of the genus such as variola virus have drawn attention to this virus as a model, especially regarding its impressive abilities to evade host immunity [1,2,3].
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