Experimental Research On Recombinant Pseudorabies Virus And Baculovirus Live Vaccine Expressing Protective Antigen Genes Of Rabies Virus

Rabies is an ancient zoonosis characterized by symptoms such as a fear of water, photophobia and mania. Almost all warm-blooded animals can be infected with rabies virus (RABV) and result in central lesion and lethal encephalomyelitis.In developed countries, through the strict management and prophylactic immunization of dogs, canine rabies has been basically eliminated, thus the human rabies have been effectively controlled. In China, dog is the main infection source and transmission medium for rabies virus to invade into crowd. The cases of human infection with rabies caused by dogs are about 85% to 90% of the total cases of rabies. Therefore, routine immunization of dogs and pre-exposure and post-exposure prophylaxis of human are still the main means to control canine rabies and avoid the disoperation of rabies viruses to human.Our study carried out a new explore to the new live vaccine against rabies virus. So the following researchs were explored.1. Construction, identification and immunogenicity of recombinant pseudorabies virus PRV-(TK-/gE-/2G+) expressing glycoproteins of the CTN strain and ERA strain of rabies virusPRV has a lot of advantages such as a clear genetic background, the stable genetic characteristics, broad host range, high proliferation titre, larger capacity of the inserted foreign gene (which can accommodate at least 40kb), etc. The main advantage is it can not infect humans. Therefore, this study was using pseudorabies virus TK-/gE-/LacZ+of Ea strain as a vector to construct recombinant virus PRV-(TK-/gE-/2G+) which expressing glycoproteins of the CTN strain and ERA strain of rabies virus. and the recombinant virus was identified by PCR, southern blot and western blot. Balb/c mice were immunized with recombinant virus, ELISA antibodies against glycoprotein could be detected 3 weeks after the first immunization, but ELISA antibodies detected 2 weeks after second immunization is not significantly higher. The recombinant virus can stimulate high cellular immunity, while the neutralizing antibody were negative (<0.5 IU/mL). Dogs were immunized by recombinant virus with three gradient 107.75,106,105 TCID50,Dogs were very sensitive, and all died within two weeks, indicating that this attenuated pseudorabies virus vector is with questionable safety in dogs.2. Construction. identification and immunogenicity of recombinant pseudorabies virus PRV-(Bartha-SN-SG) which expressing glycoprotein and neucleoprotein of the SRV9 strain of rabies virusBased on consideration to the safety to dogs of pre-PRV, we changed pseudorabies virus Bartha-gG-/EGFP+ strain which is natural attenuated and losed virulence factors as vector to construct recombinant pseudorabies virus PRV-(Bartha-SN-SG) expressing nucleoprotein and glycoprotein of SRV9 strain of rabies virus, and identified by PCR and western blot. Balb/c mice were immunized with recombinant virus. ELISA antibodies against glycoprotein could be detected 3 weeks after the first immunization. but ELISA antibodies detected 2 weeks after second immunization is not significantly higher. The recombinant virus can stimulate high cellular immunity, while the neutralizing antibody were negative (<0.5 IU/mL). Dogs were immunized by recombinant virus with 105 TCID50. the virus neutralizing antibody were negative (<0.5 IU/mL). while the safety to dogs did increase although the immunogenicity is not good.3. Construction, identification and immunogenicity of recombinant baculovirus BV-VSV/G-G which expressing glycoprotein of the ERA strain of rabies virus.It was found that baculovirus can not infect mammalian cells,but can access into a variety of mammalian cells, and can express foreign genes through appropriate mammalian promoter rather than replication and expression of its own genome. However the complement system in serum of mammalian can inactivate the baculovirus. It has been studied that the baculovirus particles which display a vesicular stomatitis virus glycoprotein fusion protein (VSV-G) on the membrane can have a strong tolerance against the complement system. and can utilize the fusion of the VSV-G to mediate intercellular fusion, it even can enhance the transduction efficiency into skeletal muscle cells in mice.Therefore, this study constructed a recombinant baculovirus expressing glycoprotein of ERA strain of rabies virus which display the vesicular stomatitis virus glycoprotein on the surface, and studied the immunogenicity of this candidate vaccine. ICR mice were immunized with recombinant virus. ELISA antibodies against glycoprotein and the cellular immunity were higher than pseudorabies recombinant virus. The neutralization antibodies two weeks after the second immunization were all positive (>0.5 IU/mL). The highest titre could reach to 10.26 IU/mL, and had a dose-dependent manner. Dogs immunized with recombinant virus were healthy, but the neutralization antibodies were not uniform.Comparation of the three recombinant viruses we have obtained, it indicated that the recombinant baculovirus would have better prospects as a RABV vaccine candidates.