Construction And Immunoreactivity Of Recombinant Rabies Virus Expressing Gn/Gc Protein Of SFTS Virus

Backgroud:SFTS(Severe fever with thrombocytopenia syndrome),caused by the SFTS virus(SFTSV),was first reported in China in 2009,and in Japan and South Korea since 2012,respectively.Clinical features of SFTS patients primarily include abrupt high fever and thrombocytopenia.The SFTSV appears to have a wide host range including both farm animals and wild rodents.Therefore,it is important to prevent the transmission of SFTSV from animals to humans.No therapies or vaccines are currently available.Rabies,caused by rabies virus(RABV),is a global pandemic and highly fatal zoonosis,which is transmitted mostly via bites of diseased animals and causes a fatal infection of the nervous system in both animals and humans.Currently,rabies virus is recognized as the most important lyssavirus species,given its high disease burden among humans,domestic animals and wildlife.Vaccination of domestic pets and livestock can effectively protect humans from RABV infection.Therefore,constructing a recombinant RABV expressing Gn/Gc protein of SFTSV to prevent these two zoonotic diseases is of great significance for public health.Objectives:1.The Gn and Gc genes of SFTSV were inserted into the pseudogene region of SAD strain to construct a recombinant RABV live vector strain expressing the Gn/Gc named SAD-Gn and SAD-Gc.2.To detect the biological characteristics,immunogenicity,safety and protective abilities of SAD-Gn and SAD-Gc as a dual recombinant live vector vaccine.Methods:1.The recombinant RABV named SAD-Gn and SAD-Gc expressing Gn and Gc protein were screened via reverse transcription-polymerase chain reaction(RT-PCR),indirect immunofluorescent assay(IFA)and Western blot.2.Vaccinated mice sera of 2,4,6 and 8 weeks were collected to detect virus neutralizing antibody(VNA)titers of RABV and SFTSV and evaluate the immunogenicity of SAD-Gn and SAD-Gc.3.The weight,diet and appearance of vaccinated mice were monitored daily for 21 days.The safety of recombinant virus was evaluated by change rate of body weight.4.Mice at 21 days after immunization were challenged with CVS-24.The protective abilities of SAD-Gn and SAD-Gc to prevent RABV infection were evaluated by survival rate of mice.5.Mice at 4 weeks after immunization were challenged with SFTSV.The spleens of mice were separated at 7 days to analyse pathological injury and virus load to evaluate the protective ability to prevent SFTSV infection.Results:1.Gn protein expression in SAD-Gn infected cells was successfully detected by RT-PCR,IFA and Western blot and Gc protein expression in SAD-Gc infected cells was successfully detected by RT-PCR.The molecular weights corresponded to the expected size.2.Detection of VNA titers in immunized mouse serum showed that SAD-Gc and SAD-Gn could induce strong humoral immunity.For SAD-Gn,VAN titer reached the peak at 4 weeks after immunization,RABV VAN titer was 4.13IU/ml and SFTSV VAN titer was 1:277.For SAD-Gc,VAN titer reached the peak at 6 weeks after immunization,RABV VAN titer was 4.97 IU/ml and SFTSV VAN titer was 1:256.3.Compared with mock group and SAD control group,mice in the SAD-Gn and SAD-Gc group showed slight body weight changes and showed no abnormal behavior.4.The results of challenge protection experiment showed that typical clinical signs of rabies were observed in mice of mock group,and the survival rates were 0 at the end of the observation period.RT-PCR results of brain tissue in dead mice of mock group showed dead mice died of rabies.No clinical signs of rabies were observed in mice of SAD-Gn and SAD-Gc group,and the survival rates were 100%.Conclusions:1.The recombinant RABV expressing Gn and Gc protein named SAD-Gn and SAD-Gc were successfully constructed.2.SAD-Gn and SAD-Gc induced a specific immune response against both RABV and SFTSV,which demonstrated that recombinant virus was the potential of a new bivalent vaccine for RABV and SFTSV.