The Rescue And Identification Of Rabies Virus Glycoprotein 83 And 367 Mutant Strains

Rabies is an acute infectious disease caused by Rabies virus?RABV?that infects the central nervous system of human and a variety of animals.The mortality rate is almost 100%after infection.Our previous study has found that the 83,367 amino acids sites on the glycoprotein?G?of rabies virus is important for cell adaptability and mice pathogenicity.In this study,three full-length recombinant plasmids of K83R-rSAD,P367S-rSAD and K83R-P367S-rSAD were constructed from the full-length plasmid of SAD strain using overlap extension PCR and point mutation kits.The SAD parent strain and the three above recombinant viruses were successfully rescued.The recombinant virus was continuously passed on the cell for5 generations,and the multi-step growth curve of the F6 generation virus was measured.The virus titer reached to the top from 72h to 96h,and the virus titer of K83R-rSAD,P367S-rSAD,K83R-P367S-rSAD and SAD were 10^8.5FFU,10^7.5FFU,10^8.5FFU and 10^7.0FFU,respectively.The results showed that the rabies virus titer was significantly increased after the mutation of the K83R on G.The sequencing results of the recombinant virus G gene showed that K83R and P367S were stable for five serial passage.Western Blot analysis showed that the expression of G protein was significantly increased after the G83 mutation.All four strains of the virus were inoculated into Kunming white Suckling mice with brain injection,and all the suckling mice died.C57BL/6 mice were challenged with intramuscular injection,and the mice developed the symptoms and died.All mice challenged with the parental virus SAD died,and the survival rate of the mice after immunized with the recombinant viruses K83R-rSAD,P367S-rSAD and K83R-P367S-rSAD were 72%,42.6%and 63.6%,respectively.The results showed that rabies virus G83 and G367mutation can reduce the pathogenicity of C57BL/6 mice.In summary,the changes in amino acids of G83 and G367 can reduce the pathogenicity of the virus,and the mutation of G83 enhance the expression of the G.The recombinant rabies virus with less virulence was successfully constructed in this study,which provided a foundation for the development of rabies virus vaccine in the future.