The Research Of New Vaccine Candidate Strains With The Double-hemagglutinin Of Canine Distemper Virus

The non-segments negative-strand RNA viruses(NSNSV), which including paramyxovirus, rhabdovirus, Borna virus, and filamentous viruses, are most important infectious pathogens for animals and human. Reverse genetics of NSNSV is developed by operating on viral genome cDNA and finally rescuing the virus. Reverse genetics technology plays an important effect of techniques and tools in molecular virology. Reverse genetics system allowed us purposely to mutate, miss or insert exogenous gene in the virus genome cDNA, finally obtained the corresponding mutation, recombinant viruses or vaccine strains, and also taken the base of understanding the viral pathogenic mechanism and disease prevention.1. Construction of new candidated vaccine with the double-hemagglutinin of Canine Distemper Virus by the reverse genetics technologyCanine Distemper Virus (CDV), a member of the genus paramyxovirus of the family measles virus, causes an acute, febrile, a highly contagious viral diseases on a variety of animals. The prevention of canine distemper is still mainly dependent on the attenuated vaccines all over the world. However, there are some drawbacks on the attenuated vaccines. Construction of the novel vaccine of canine distemper virus is seems to be imminent.The hemagglutinin envelope glycoprotein (H) protein of CDV is primary antigen protein which can induce neutralizational antibodies in host, playing the important role in anti-CDV. In this study, full-length cDNA plasmid was constructed with inserting double H gene between P and M region. Full-length cDNA plasmid and supporting plasmids were co-transfected BSR cells and the recommbinant canine distemper virus (rCDV-JP-2H) were successfully rescued. The recommbinant virus(rCDV-JP-2H) had the lower virus tites than the wlidtype virus because the additional H gene.The tites of recommbinant virus were4.0、4.25、4.3TCID50/mL in Vero Vero-SLAM、BSR respectively. The neutralization antibody titers of dogs, which were immunized with104TCID50rCDV-JP-2H, were1:48,1:160after first and second immunization. While the neutralization antibody titers were1:24,1:96for dogs immunized with104TCID50CDV-JP. The neutralization antibody which were immunized with103TCID50rCDV-JP-2H, were1:21,1:107after first and second immunization. While the neutralization antibody titers were1:7,1:27for dogs immunized with103TCID50CDV-JP. The neutralization antibody which were immunized with1O2TCID50 rCDV-JP-2H, were1:11,1:64after first and second immunization. While the neutralization antibody titers were1:5,1:21for dogs immunized with10TCID50CDV-JP. These result indicated that we can develpoped new type live vaccine by H gene doubled expression in CDV2. The lesser pandas strain canine distemper virus full genome sequences analysisIn order to understand the genetic background of CDV-LP, our study carried out whole genome sequence analysis for CDV-LP strain and genetic characteristics comparative analysis for H gene. The17pairs of specific primers were designed, and the CDV-LP strain total RNA has been used as template. The full-length gene sequences were amplified by RT-PCR. The whole sequence had been analyzed by DNASTAR. The whole gene was15690nt and there were52nt,38nt in the3’,5’noncoding region. The N, P, M, F, H, L were1683nt,1655nt,1447nt,2209nt,1946nt,6642nt respectively. The LP had the closest genetic relationship with the standard A75/17strain with the homology of96.3%, but far genetic relationships with vaccine strains-Onderstepoort with the homology of86.6%.The evolution analysis show that CDV-LP strain belongs to Asia I gene type and has eight potential glycosylation sites in H gene.3. Construction and identification of CDV-LP reverse genetic systemTo further develop a safer and more effective CDV vaccine for the animal, we constructed CDV-LP reverse genetic system. In this study,7fragments were amplified by RT-PCR using the RNA template of CDV-LP.The full-length cDNA expression plasmid (pCI-CDV-LP) were constructed by cloning the PCR products into the eukaryotic expression vector pCI. Further, three supporting plasmids (pCI-N, pCI-P and pCI-L) were accquired by cloning N, P, L gene of CDV-LP into PCI vector. Finally, the CDV-LP were rescued by co-transfected BSR cells with the pCI-CDV-LP, pCI-N, pCI-P and pCI-L plasmids. The result of electron microscope、RT-PCR、 Indirect immunofluorescence assay suggested that reverse genetics system of LP are successfully established and the rescued virus show the same characters with the parental virus. The CDV-LP reverse genetic systems were useful for the development of novel CDV vaccine with reverse genetics technology in the future.