Biological Characterization Of Recombinant Rabies Virus Expressing H Protein Of Canine Parvovirus

Rabies is a zoonosis caused by rabies virus(RABV). The infectious disease is epidemic in the world and one of the highest mortality rate. Once symptoms appear, almost 100% death happen. Canine Distemper(CD) caused by canine distemper virus(CDV) is a highly contagious disease, which is popular in the global scope and the mortality rate reaches as high as 80%. The disease caused serious harm to dog industry and fur animal breeding industry. Vaccination is the main way to control rabies and canine distemper. Attenuated rabies vaccine has the advantages of low cost, but there are security risks of virulence residues. The import killed vaccines have the features of high safety, good immune effect, but the price is expensive, and thus it is difficult to be widely used. The development of safe, effective, low cost of animal rabies vaccine still has great significance. The CDV vaccine in market widely exists many problems including the poor thermal stability and easily affected by maternal antibody. In this study H gene of canine distemper virus was inserted between G and Lof rabies virus HEP-d G genome used the rabies virus reverse technology platform and the full-length c DNA plasmid p HEP-d G(H) was constructed and the recombinant rabies virus carrying double G and H genes was rescued.Through the study on the biological characteristics of the recombinant virus, the result indicated that the recombinant rabies virus HEP-d G(H) can multiply stablly in BHK cells and the virus titer reached higher. This RNA extracted from the 2,5,10 generation virus was used to amplify H and G, and it suggested the genes did not occur mutation. Fluorescence quantitative PCR and Western blot showed that the expression of H and G genes. The growth curves of HEP-d G(H) virus and parental virus HEP-d G strain maintained similar and the titers of two virus reached the peak at 96 h. But the titer of recombinant virus HEP-d G(H) at each time point is slightly lower than one of HEP-d G. Spread and infection experiments showed when MOI was low, proliferation ability of HEP-d G(H) was lower than HEP-d G strains,.The pathogenicity experiment was carried out. HEP-d G(H) and HEP-d G is not lethal to 6-7 weeks adult mice, and HEP-d G(H) elicited weight less than HEP-d G. The immunogenicity experiment showed that HEP-d G(H) and HEP-d G were able to induce mice to produce antibody. On 7th day post infection antibody reached the level of protection(0.5EU), and the recombinant virus HEP-d G(H) can induce neutralizing antibody of CDV. Virus challenge test showed that antibody induced by HEP-d G(H) and HEP-d G can resist the attack of CVS-24 after immunization for three weeks. These results suggest that the recombinant rabies virus can be used as a potential candidate vaccine strain.In summary, the recombinant rabies virus HEP-d G(H) has good immunogenicity, would be as a potential new genetic engineering vaccine, this study laid the foundation for the development of novel multivalent genetic engineering vaccine of canine.