The Effect Of Structural Protein PrM/E On The Neurovirulence And Neuroinvasiveness Of Japanese Encephalitis Virus

Objective: To construct the infectious clone of recombinant Japanese encephalitis virus wild type strain SA14(rJEV SA14),chimeric virus of vaccine strain SA14-14-2 and wild type strain(JEV SA14-14-2/SA14),and to rescue virus,respectively,and to compare the neurovirulence and neuroinvasiveness in Kunming mouse of the two rescued virus,and to evaluate the effect on neurovirulence and neuroinvasiveness of prM/E.Methods: The infectious clone(full-length cDNA plasmid)of rJEV SA14,chimeric virus JEV SA14-14-2/SA14 were constructed with molecular clone technique,and then used as the template for transcription of RNA in vitro.RNA was transfected into BHK21 cells for virus rescue.The rescued virus were identified with plaque assay,indirect immune-fluorescence and sequencing.The growth characteristic of the rescued virus in BHK21 cells was evaluated,and the neurovirulence and neuroinvasiveness of rJEV SA14 and JEV SA14-14-2/SA14 were compared in Kunming mouse,respectively.Results: The identification of enzyme digestion showed that the infectious clone was successfully constructed,and the expression of envelope protein of rescued virus was detected by immune-fluorescence.The plaque formation experiment showed that the diameter of the plaque of rJEV SA14 was the largest,followed by JEV SA14-14-2/SA14,and vaccine strain JEV SA14-14-2.The sequencing results showed that the recovery virus were the virus of designed.The growth curve showed that JEV SA14-14-2/SA14 decreased rapidly after reaching the peak of 48 h,while JEV SA14-14-2 and rJEV SA14 reached the peak at 60 h.The titer of rJEV SA14 was higher than that of JEV SA14-14-2/SA14 and JEV SA14-14-2 during the whole observation period.The animal study showed that neurovirulence(50% lethal dose)and neuroinvasiveness(50% lethal dose)of rJEV SA14 were 0.085 PFU/0.03 mL and 2.13 PFU/0.1mL respectively,while those of JEV SA14-14-2/SA14 were 4.72 PFU/0.03 mL and 39.5 PFU/0.1mL,respectively.Conclusion: The infectious clone of rJEV SA14 and JEV SA14-14-2/SA14 were constructed successfully and the viruses were rescued successfully.The fact that neurovirulence and neuroinvasiveness of JEV SA14-14-2/SA14 was slightly lower than that of rJEV SA14,but far higher than that of vaccine strains JEV SA14-14-2 showed that prM/E plays an important role in neurovirulence and neuroinvasiveness controlling of Japanese encephalitis virus,but the nonstructural protein may also play a less role.The reverse genetics research platform for Japanese encephalitis virus was built,it laid the foundation for further research on the pathogenesis of JEV and the attenuated mechanism of JEV vaccine.