Construction Of Reverse Genetic System Of Canine Distemper Virus And Evaluation Of Its Potential As A Live Vaccine Vector

Canine distemper virus (CDV) is an enveloped, single-stranded, negative-sense RNA virus of the Morbillivirus genus within the family Paramyxoviridae. The size of CDV genome is15690nucleotides with a short3’leader region and six genes encoding nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H) and large polymerase (L) proteins, followed by a short5’trailer region organized as3’-N-P-M-F-H-L-5’. CDV can infect most members of the order Carnivora which is resulted in CD, a highly contagious disease that can be controlled only by vaccination.Many members of the nonsegmented negative-strand RNA viruses (NNSVs) have been developed as vaccine vectors since several features make them attractive for this purpose. NNSVs replicate only in the cytoplasm and therefore have no chance to integrate with the host genome. Their genes are nonoverlapping, each having its own transcriptional signal, and proteins are synthesized separately. This modular organization facilitates insertion of exogenous genes. Previous studies have demonstrated mat NNSVs can accommodate several foreign genes with an aggregate length of4to5kb. Vaccine vectors based on NNSVs have been shown to stably express exogenous genes for more than10passages. Therefore, CDV has properties highly advantageous to a live vaccine vector. In the present study, a rescue system was established for the attenuated canine distemper virus strain CDV-L, furthermore, we investigated whether the CDV-L has the potential as a live recombinant virus vector.Based on previously published CDV sequences in GenBank, the specific primers were designed. Six fragments of the viral genomic cDNA were prepared by reverse transcription and PCR amplification (RT/PCR) and sequenced directly. The full-length antigenome was then obtained by seamless ligation of all fragments with In-Fusion method. Finally, T7promoter, the full-length antigenome, the hepatitis8-ribozyme and the T7terminator were also ligated seamlessly using one In-Fusion reaction. Three helper vectors expressing the CDV nucleocapsid (N), phosphoprotein (P), and large protein (L) were also constructed, respectively. A BHK-21-derived cell line constitutively expressing T7RNA polymerase, named BHK-T7was generated. The recombinant virus rCDV-L was rescued by a traditional T7RNA polymerase-based system.Expression of a foreign gene EGFP by rCDV-L-EGFP recovered from the infectious cDNA clone demonstrating that CDV can be used as an expression vector. Furthermore, we generated a recombinant CDV vaccine strain, rCDV-RV-G, expressing the rabies virus glycoprotein (RV-G) and another recombinant CDV virus expressing mink enteritis virus VP2(MEV-VP2). Expression of G was identified and we firstly found out that the G protein was incorporated in rCDV-RV-G particles by immuno-electron microscopy. Immunization of mice and dogs with rCDV-RV-G could elicit protective neutralizing antibody against CDV and RV. This result demonstrate that recombinant CDV has the potential to serve as bivalent live vaccine against rabies and canine distemper. Detection of expression of VP2was failed. Another recombinant virus in which the sequence of a peptide of N terminus of MEV-VP2was ligated seamlessly with the G gene was rescued. Expression of G was identified but immunization of mice with the recombinant virus failed to elicit protective neutralizing antibody against MEV.In the study, we developed a rescue system for CDV-L and proved that recombinant CDV-L has the potential to serve as a live recombinant virus vector.