title: Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC).
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.
authors.affiliation: Institut für Virologie, Freie Universität Berlin; Philippstrasse 13, Haus 18; 10115 Berlin, Germany. no.34@fu-berlin.de.
authors.forename: Swaantje J
authors.initials: SJ
authors.lastname: Roth
authors.forename: Dirk
authors.initials: D
authors.lastname: Höper
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.forename: Silke
authors.initials: S
authors.lastname: Feineis
authors.forename: B Karsten
authors.initials: BK
authors.lastname: Tischer
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Osterrieder
mesh_terms: D000818:Animals; D002642:Chick Embryo; D002645:Chickens; D002522:Chlorocebus aethiops; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D015608:Cowpox virus / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli; D016679:Genome, Viral; D059765:Homologous Recombination; D008969:Molecular Sequence Data; D013937:Thymidine Kinase / Q000378:metabolism; D014709:Vero Cells
chemical_list: D013937:Thymidine Kinase