Schematic representation of MNF gene region of wild-type MYXV, recombinant MYXV-GFPMNF and clinical signs associated. A. Mutant virus was obtained by homologous recombination between wild type MYXV and a transfer plasmid. The resulting recombinant MYXV-GFPMNF encodes GFPMNF fusion, under control of MNF promoter. R.R. encompasses the end of M149R-coding sequence and the intergenic sequence between M149R and MNF. To select recombinant virus, the Ecogpt gene, under control of P7.5 early poxviral promoter, was inserted between M149R and GFPMNF. So as not to disturb expression of M149R or MNF by the selection gene insertion, R.R. was duplicated. B. Pathogenicity of wild-type and mutant viruses in European rabbits. Eight-week-old New Zealand White rabbits were obtained from a local supplier and housed in biocontainment facilities according to the guidelines of the European Community Council on Animal Care (European Council directive 86/609/EEC, 24 November 1986). All procedures on animals were performed by staff accredited by the French Ministry of Agriculture and were designed to limit animal pain and distress. Infections were performed intradermally in the right ear with 5 × 103 FFU of either virus. Rabbits were monitored daily for clinical signs of myxomatosis. Rabbits that became moribund were sacrificed with T61 administered intravenously.