Construction And Identification Of A Chimeric Zika Vaccine Using Japanese Encephalitis Virus Vaccine As Backbone

Objective:To construct chimeric Japanese encephalitis virus and Zika virus(JE/ZIKA)infectious clone,rescue the virus,and identify biological characteristics and immunological characteristics of the chimeric virus,so as to explore whether the chimeric virus strain can become a vaccine candidate strain of Zika virus.Methods:The prM/E gene of Japanese encephalitis vaccine strain SA14-14-2 were replaced with Zika virus prM/E gene sequences with gene recombination techniuqes to construct JE/ZIKA chimeric virus infectious clone,and it was used as the template for RNA transcription in vitro.RNA was transfected into BHK21 cells to rescue JE/ZIKA chimeric virus and JE/ZIKA was identified with plaque assay,indirect immunofluorescence technique and sequencing,and chimeric virus growth characteristics,genetic stability and thermal stability were identified also.The neurovirulence of chimeric virus(median lethal dose,LD50)to cleaning Kunming mice were calculated after inoculation mice through intracerebral,subcutaneous and intraperitoneal pathway.Plaque reduction neutralization test(PRNT)was used to detect chimeric virus neutralizing antibody after immunization Kunming mice with chimeric virus.In order to evaluate the immune protection ability of chimeric virus,Kunming mice was immunized with chimeric virus with different doses of JE/ZIKA,2 weeks later,JE/ZIKA was used to attack immunized mice through intracerebral pathway and the mortality was recorded.Kunming mice were immunized with JE/ZIKA chimeric virus and JEV SA14-14-2 respectively,and immunized mice were attacked with Japanese encephalitis virus wild type strain SA14 and JE/ZIKA respectively,and the mortality was recorded and the cross immune protection between viruses was evaluated.Results:The results of enzyme digestion showed that the infectious clone of JE/ZIKA chimeric virus was successfully constructed.The chimeric virus was obtained from the supernatant of BHK21 cells by electroporation of BHK21 cells with RNA transcripted in vitro.Chimeric virus infected BHK21cells caused cytopathic effect.The plaque of chimeric virus was smaller than that produced by JEV SA14-14-2.The results of virus sequencing confirmed that the chimeric virus was successfully rescued and no other mutations occurred except for 1343 mutations.Indirect immunofluorescence detected the expression of E protein in Vero cells infected with chimeric virus.BHK21cells were infected with different multiplicity of infection(m.o.i was 0.1,0.01,and 0.001)got similar growth curve and when m.o.i.was 0.001,the titer of virus could reach 6.8 logPFU/ml at 108 hours of infection.The thermal stability test showed that the titer of chimeric virus decreased rapidly in the condition without liquid protected agent,and virus was deactivated at 37?for 24 hours.The chimeric virus JE/ZIKA was inoculated subcutaneously and intraperitoneally to Kunming mice,all the experimental groups were healthy in the observation period,while JE/ZIKA chimeric virus had a severe nerve toxicity(LD50 2.4 PFU/0.03ml)in Kunming mice inoculated intracerebrally.After immunization of Kunming mice with JE/ZIKA chimeric virus,the positive conversion rate of neutralization antibody was 100%.The neutralization antibody titer reached the peak in the mice inoculated intraperitoneally at 2~ndd week after inoculation and decreased rapidly.The titer of neutralizing antibody in group immunized subcutaneously was relatively high,which was relatively stable.Kunming mice immunized with JE/ZIKA protected the the attack of JE/ZIKA with a dose effect dependant manner and the immune protection rate reached 90%in the mice immunized with 2.7*10~5PFU chimeric virus,while the protection rate of MEM immuned group was 0.Immunization with chimeric virus JE/ZIKA protected mice from attack of Japanese encephalitis virus to some extent(protection rate is 40%),and mice immunized with JEV SA14-14-2 protected mice against JE/ZIKA attack(protection rate is 90%).Conclusion:The JE/ZIKA chimeric virus infectious clone were successfully constructed,and the chimeric virus JE/ZIKA was rescued.The chimeric virus has good immunogenicity,but has strong neurotoxicity in mice,and there is cross immunity between chimeric virus JE/ZIKA and Japanese encephalitis vaccine strain SA14-14-2.