Construction And Biological Characterization Of Recombinant Rabies Virus Carrying RFP Gene

Rabies?Rabies?is a severe zoonotic infectious disease,caused by highly neurotrophic rabies virus?RABV?,human and all warm-blooded animals are susceptible to rabies,its mortality is almost 100%.About 55,000 people die from rabies each year,and rabies is still a serious threat to public health and safety in the world,especially in Asia and Africa.RABV enters the organism usually through wound or mucosal surface,it replicatesfirst in the infected muscle cells,and enters the nerve cells through the binding to acetylcholine receptors?AchR?and neural cell adhesion molecules?NCAM?by G protein.After RABV invades central nervous system?CNS?through the motor endplates and motor axons in an inverted axonal manner,it replicates largely and ultimately causes lethal encephalitis.In order to explain the distribution and migration of rabies virus in infected CNS,and the removal process of attenuated strains,the red fluorescence protein?RFP?was inserted into the pseudo genetic region of the viral genome using the reverse genetic system based on rHEP-Fluryto rescue the recombinant rabies virus rHEP-RFP in this study.The biological characteristics and the distribution and migration mode of the recombinant virus were studied in cell and mouse infection models.The biological characteristics of recombinant rHEP-RFP showed that rHEP-RFP could be stably proliferate on BHK-21 cells,with a slightly lower titer thanr HEP-GFP and HEP-Flury.The RNAfrom the 10th and 20th generation of the recombinant virus was extracted and the RFP gene was amplified by RT-PCR and the gene show nomutation.The growth curve showed that HEP-RFP had similar growth characteristics with r HEP-GFP and rHEP-Flury,and peaked at 72 h.The titers of rHEP-RFP at different time points were lower than those of r HEP-Flury and r HEP-GFP.Animal experiments showed that the LD₅₀ of recombinant virus r HEP-RFP on the neonatal 10^-5.428/0.03mL,intracranial vaccination 5-6weeks of adult mice,shows no fatal,effect of r HEP-RFP on the weight of mice less than the parent strain r HEP-Flury.Whole brain tissue frozen sections can be observed in the rHEP-RFP nasal infection in mice on the 7th day in the temporal lobe of the brain and hippocampus back to observe the red fluorescence,after 14 days it spread to the cerebral cortex,cerebellum and brain stem,At 21 days post infection,there is almost no fluorescence in cerebellum and brain stem,the fluorescence in cerebrum is also significantly reduced.In this study,we found that the recombinant virus rHEP-RFP decreased the cell proliferation ability and the pathogenicity to the mice compared with the parent strain.After infection with r HEP-RFP,the virus appeared earlier in the olfactory center of the brain,and then spread to the cerebellum and brain stem and other parts,at 21 days post infection virusin the cerebellum and brain stem was completely removed.The recombinant virus rHEP-RFP,which expresses red fluorescent protein,provides a convenient condition for further study on the proliferation and clearance mechanism of rabies virus in the CNS.