| Objective:To construct chimeric virus JE/ZIKV(PRVABC59)that can express ZIKA virus prM/E protein and use Japanese encephalitis virus vaccine as backbone.To analyze the growth,immunogenicity,safety and protectiveness of the virus,so as to evaluate whether the chimeric virus can be developed into a candidate ZIKA virus vaccine strain.Methods:Adapting standard reverse genetics technology,we replaced the prM/E of the Japanese encephalitis virus vaccine strain with ZIKA virus prM/E and constructed the infectious clone with Japanese encephalitis virus vaccine as backbone.RNA had been transcribed in vitro,and then the chimeric virus was saved by electrical transfection into BHK-21 cells.Afterward,the virus was identified by plaque assay,immunofluorescence and gene sequencing.The growth was shown by the growth curve.The neurovirulence of the virus is determined by intracerebral injection of it into 3-week-old Kunming mice and 3-day-old Kunming Suckling mice.The chimeric virus immunized Kunming mice.Blood was collected 1,3,and 5 days after immunization,plasma was separated,and plaque experiments were performed to detect viremia;After three weeks of injection,we collected their blood,isolated the serum,and measured the ability of virus producing neutralizing antibodies by applying 50%plaque reduction neutralization experiment.Kunming mice were inoculated with JE/ZIKV(PRVABC5 9)chimeric virus by intraperitoneal route.After 3 weeks of immunization,JE/ZIKV(MR766)was used to attack the mice to test their immunity against JE/ZIKV(MR766).Results:The result of enzyme digestion and gene sequencing showed that the infectious clone of chimeric virus was successfully constructed.Five days after the electrical transfection of the virus into BHK21 cells,the cells suffered obvious lesions.The virus had been detected via plaque assay.Virus sequencing showed that the virus was successfully rescued and no mutation occurred.Immunofluorescence confirmed the expression of prM/E protein of Zika virus and NS1 protein of Japanese encephalitis virus.The plaque test showed that the chimeric virus caused needle-like plaque,which is smaller than plaque caused Japanese encephalitis virus vaccine strain.The growth curve displayed that the proliferous rate was slower than that of the Japanese encephalitis vaccine strain.It reached a peak titer of 6.071gPFU at the 72nd hour,when M.O.I.was 0.01.Cell proliferou’s activity assay showed that cells infected with JE/ZIKV(PRVABC59)chimeric virus had higher proliferation activity than those infected with Japanese encephalitis virus vaccine strain 60h after infecting the BHK21 cells(M.O.I.=0.1).The results of animal experiments showed that neither adult mice nor suckling mice were killed by the chimeric virus,and the neurotoxicity was extremely low.Viremia results showed that the chimeric virus did not detect significant viremia in mice.The 50%PRNT showed that the positive conversion rate of neutralizing antibodies in the serum of the chimeric virus immunized mice reached 100%from the 3rd to 10th weeks,and the titer of antibodies peaked at the third week(GMT=85).Immune protection experiments demonstrated that the chimeric virus immunized mice had a 30%protection rate against the intracranial attack of JE/ZIKV(MR766).Conclusion:The infectious clone of JE/ZIKV(PRVABC59)chimeric virus was successfully constructed,and saved the chimeric virus.Chimeric virus JE/ZIKV(PRVABC59)had little neurovirulence for mice and was reliablysafe.The virus can produce effective neutralizing antibodies and had certain immunogenicity.Therefore,it has the potential to be development as a candidate zika vaccine strain. |
