Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(؉)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(؉)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.Canine distemper virus (CDV) is an enveloped virus with a monopartite negative-stranded RNA genome. Together with Measles virus (MV) and Rinderpest virus, it belongs to the morbilliviruses which form a serologically closely related genus in the family Paramyxoviridae. CDV primarily affects dogs, but infections of other terrestrial carnivores, in both captivity and the wild, have been reported (1,2,18,21,22,25,26,29,33,36). The mortality rates associated with CDV infection vary among susceptible species and range from 0% in domestic cats to 50% in dogs and 100% in ferrets. One of the currently available vaccines (Onderstepoort strain) efficiently protects dogs, but it is insufficiently attenuated for other species, and high levels of mortality can occur due to its remaining virulence (10). Thus, there is a need to develop more attenuated vaccines for CDV to fully protect susceptible animals.A rescue system for CDV would provide the means to study attenuating effects of defined mutations and might subsequently facilitate generation and examination of new vaccines. Mutations that cause persistence of the virus could also be determined. This system would be useful in gaining a better understanding of CDV infection which can more easily be studied in cell culture and animal models by coexpression of additional reporter genes (e.g., the enhanced green fluorescent protein) from the recombinant viral genome (19,20).The CDV genome is 15,690 nucleotides (nt) in length and consists of a short 3Ј leader region and six genes encoding the N nucleocapsid (N), phospho-(P), matrix (M), fusion (F), hemagglutinin (H) and large (L) proteins (4,5,7,16,32,37,42). They are separated by intergenic regions of 3 nt and are followed by a short 5Ј trailer region. The nonstructural proteins V and C are encoded wit...