Rescue And Identification Of Recombinant Rabies Virus Containing N And S Gene Of Canine Coronavirus

Rabies is a zoonotic zoonosis caused by rabies virus.Once the infection has clinical symptoms,the case fatality rate is 100%.Canine Coronavirus disease is a kind of contact infectious disease caused by canine coronavirus infection.It is clinically characterized by gastroenteritis symptoms such as vomiting,diarrhea,dehydration and bloody stool.With a low mortality rate,however,the mortality rate is significantly increased when it is infected with other infectious cases such as canine parvovirus and rotavirus.In this study,the N protein of canine coronavirus was expressed by prokaryotic expression system,and polyclonal antibody was prepared after immunizing rabbits.The protein showed good reactivity and immunogenicity identified by ELISA and Western blotting.The prepared polyclonal antibody has good binding specificity with antigen.In this paper,we mainly developed the genetic engineering bivalent vaccine against rabies virus and canine coronavirus based on the reverse genetic system of rabies virus.The full-length cDNA of rabies virus with N and S genes of canine coronavirus was constructed with SAD virus as the skeleton,and then it was cotransfected with rabies virus assistant plasmids to BHK-21 cell to rescue the recombinant virus.The recombinant rabies virus BNSP-CCV-N and BNSP-CCV-S containing the target gene were screened out by immunofluorescence and qPCR tests,and then the biology Characteristics of the recombinant virus was studied.Western blotting test showed that the recombinant virus BNSP-CCV-N could express RV-G protein and CCV-N protein at the same time,and the recombinant virus BNSP-CCV-S could express RV-G protein and CCV-S protein at the same time.The growth curve of the virus showed that the titer of BNSP-CCV-N did not increase after 72 hours,and BNSP-CCV-S did not increase after 96 hours,so the optimal time of virus harvestcould be determined.The results of electron microscopy and immunofluorescence showed that the reco mbinant virus could be assembled into complete virus particles with certain morphology.At the same time,the recombinant virus was passaged in BHK-21 cells for 10 generations.The results of RT-PCR showed that the foreign gene carried by the recombinant virus could be stably inherited.Furthermore,the pathogenicity of the recombinant virus was evaluated in BALB / c mice.There were no visible clinical symptoms of mice that challenged with recombinant virus by intracerebral injection after 14 days' observation,indicating that the recombinant virus had no pathogenicity to mice.The recombinant virus was inactivated after concentrated and purified.The inactivated vaccine and attenuated vaccine were prepared in accordance with a certain percentage with GEL adjuvants,and then mice were immunized in different groups.The results showed that the inactivated vaccine and the attenuated vaccine had good safety.By comparing the antibody levels of mice immunized in different groups,the attenuated vaccine produced higher neutralizing antibody against rabies virus,and the inactivated vaccine BNSP-CCV-N+S had higher specific antibody against N protein and S protein of canine coronavirus.The result indicated that the recobminate virus have the potential to prepare bivalent genetically engineered vaccines against rabies and canine coronavirus,and further studies are needed in dog to evaluate the immune protection in the future.In this study,the recombinant rabies virus containing N and S genes of canine coronavirus was successfully rescued by reverse genetic system.The inactivated vaccine and the attenuated vaccine were used to immunize mice to iduce high neutralizing antibody against rabies virus and high specific antibodies against canine coronavirus.This study provides a certain reference value for the study of bivalent vaccine against rabies and canine coronavirus.