Establishment of a Rescue System for Canine Distemper Virus

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 3′ 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 5′ trailer region. The nonstructural proteins V and C are encoded within the P gene. V is expressed by cotranscriptional RNA editing, and C is expressed from an overlapping reading frame (8, 12). The genome of paramyxoviruses does not consist of a naked RNA molecule. In many members of the subfamily Paramyxovirinae, 6 nt are likely to be tightly associated with N protein (9, 30). The exact structure of these ribonucleoprotein complexes is not known but it has recently been suggested for vesicular stomatitis virus that the N protein binds to the sugar-phosphate backbone of the RNA, exposing the bases to the outside. As a consequence, the RNA of the Mononegavirales may be transcribed without dissociation from the nucleoprotein (27). The phosphoprotein and the RNA-dependent RNA polymerase are also associated with the RNP. Paramyxovirus genomic or antigenomic RNA as for all Mononegavirales cannot function as mRNA. Thus, to initiate an infectious cycle by introducing genome analogues into the cell, all viral proteins involved in transcription and replication have to be provided in trans. Since the generation of infectious rabies virus from a cDNA clone in 1994 (40), several other negative-stranded RNA viruses have been rescued using a number of different approaches. To date two morbilliviruses, namely MV (35) and Rinderpest virus (3), have been recovered from cDNA. These viruses are closely related and distinct from CDV and phocine distemper virus (PDV) in terms of genome length and sequence homology (6, 17, 24, 34, 41). In this study, we have established a rescue system for CDV. We generated a full-length cDNA clone of the CDV strain Onderstepoort [large plaque-forming variant (OND-LP)] and the helper plasmids encoding N, P, and L proteins. Recombinant virus (rCDV) was recovered from cell cultures transfected with all four plasmids. Immunofluorescence and a genetic tag identified rCDV. The growth characteristics of rCDV were compared with the original CDV strain.