Schematic presentation of rationale for cloning of cowpox virus infectious clone. A. Shown is a schematic of the full-length CPXV.BR genome and the TK locus in greater detail. In a first step, the viral TK was amplified by PCR, cloned into the pCRII vector, which was then used for an inverse PCR to introduce an Fse I restriction site into TK. Mini-F vector sequences containing gfp under the control of the late 4B promoter were finally inserted into the singular Fse I site to obtain transfer plasmid #7. B. Shown are the constructs and the strategy used to obtain recombinant virus BR.TK- and the full-length CPXV.BR BAC pBRf. Recombinant BR.TK- was used to infect Vero cells. Infection resulted in the formation of replication intermediates, concatemers, some of which were circularized. Viral DNA extracted from cells infected with BR.TK- was electroporated into E. coli DH10B giving rise to pBRf. The full-length CPXV BAC clone was ultimately transfected into Vero cells using FWPV as a helper. Scale bars indicate the sizes of the molecules. Abbreviations: ITR, inverted terminal repeats; cat, chloramphenicol resistance gene; gfp, green fluorescent protein gene: loxP, loxP sites; H, "head", T, "tail" orientation of the ITR present in the replicative intermediates.