Biological Characteristics Of Recombinant Rabies Virus Co-Expressing Canine Interleukin 12 And Interleukin 6

Rabies is caused by the rabies virus(RABV)which leads to fatal and highly neurotropic zoonotic infectious diseases.Once infection,almost 100% death happen.There is no effective therapy for this disease and prophylactic immunization is the primary countermeasure.Therefore,it is critical to develop the affordable and efficient vaccines.IL-6 is recognized to be a highly versatile cytokine,with pleiotropic actions not only in immune cells,but also in other cell types,such as cells of the central nervous system(CNS).IL-12 is a cytokine that promotes cell-mediated immunity by promoting Th1-type cytokine responses,enhancing the lytic activity of NK/LAK cells,augmenting specific CTL responses,and inducing the production of IFN-gamma.In order to explore the effects of IL-6 and IL-12 on the biological characteristics of the virus in rabies virus,This research use the recombinant viruses rHEP-dG,rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 to study.Then study on the biological characteristics of the recombinant virus rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 was performed.The results indicated that two strain viruses can multiply stablly in NA cells and the virus titer reached higher than parental virus rHEP-dG.The RNAs extracted from the 11 generation of virus rHEP-dG-CaIL12 and 7 generation of virus rHEP-dG-CaIL6-12 were used to amplify IL12 and IL6,and it suggested the genes mutation did not occur.The result of the growth curves indicated that two recombinant rabies virus(rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12)and rHEP-dG maintained similar at MOI of 0.01.The titer of recombinant virus rHEP-dG-CaIL6-12 at each time is obvious higher than rHEP-dG.The spread and activity of the virus on cells experiments showed that proliferation ability ofrHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 were higher than rHEP-dG from 48 hours,but the activity of the rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 on cells were lower than rHEP-dG at the fourth day.The apoptosis induced by rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 was inhibited in NA cell.Western bloting showed that the expression of M protein and G protein of rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 was lower than rHEP-dG in the early stage,but later on,rHEP-dG-CaIL12 expressed more M and G protein than rHEP-dG.Fluorescence quantitative PCR showed that the genomic RNA level of rHEP-dG-CaIL12 was higher than rHEP-dG.The transcription level of each gene was dynamic at MOI of 0.01,but at MOI=1 the transcription level of each gene of rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 was not high in the early stage and higher than rHEP-dG at the middle and later periods.The pathogenicity study indicated that rHEP-dG,rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 are not lethal to 6-7 weeks adult mice and there was no significant difference in body weight variation.The pathogenicity of virus rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 were lower than rHEP-dG in three day suckling mice.The three virus strains successfully induced against antibody in mice and the protection rate was100% post-immunization 21 days and 80% after 60 days.In summary,the recombinant rabies virus rHEP-dG-CaIL12 and rHEP-dG-CaIL6-12 indicated increasing virus titer and different variation replication ability,transcription level and structural protein expression,and lower pathogenicity than parent strain,this study laid the foundation for the development of novel genetic engineering rabies vaccine for canine.