Packaging Of Japanese Encephalitis Virus Like Particle

Japanese encephalitis virus(JEV)belongs to the Flavivirus in Flaviviridae family.Flavivirus members also include west Nile virus(WNV),yellow fever virus(YFV),tick-borne encephalitis virus(TBEV)and dengue virus(DENV),these are important pathogens causing human disease.JEV virus is abovirus causing central nerve damaged and the fatality rate resulting from JEV is higher as well as half of the survivors will have severe permanent neurological sequelae.Though JEV poses a major threat to human health,further reseach on JEV is limited due to potential biosafety problem of wild type virus,and there is no effective treatment for Japanese encephalitis,thus it is of great significance to package Japanese encephalitis virus like particles(VLPs)and it plays important role in the research of pathogenic mechanis,antiviral compounds screening and development of new treatment ways.Packaging JEV VLPs in this study was based on genome sequence of JEV-SA14virus.JEV-Rluc replicon and VEEV-JEV-CprME clone were previously construted in our laboratory:JEV-Rluc replicon inserted the Renilla Luciferase(Rluc)reporter gene and expression of Rluc can reflect replication of replicon sensitively;JEV structural genes were expressed effciently by a non-cytopathic Venezuelan equine encephalitis virus(VEEV)replicon with high level of replication and translation.Initially,JEV-Rluc RNA and VEEV-JEV-CprME RNA were transfected into BHK-21 cells by trasfection reagent DMRIE-C,then supernatants of different time points were obtained to infect Vero cell and luciferase activities were monitored at 48h post-infection.The results showed that JEV-Rluc replicon and VEEV-JEV-CprME could produce JEV VLPs by complementary packaging.Base on the above result,we also constructed VEEV-PAC-2A-JEV-CprME clone with a puromycin resistance gene(Puromycin Acetyltransferase,PAC)and a 2A autocleavage site of foot-and-mouth disease virus(FMDV 2A)fused to PAC genes.PAC genes could be used to select cell lines with expression of JEV structural proteins CprME and 2A gene was designed to cut and release PAC from polyproteins after translation.After construction of VEEV-PAC-2A-JEV-CprME clone,VEEV-PAC-2A-JEV-CprME RNA was obtained from vitro transcription.Similarly we utilized DMRIE-C to transfectJEV-Rluc RNA and VEEV-PAC-2A-JEV-CprME RNA into BHK-21 cells,the amount of VLPs was quantified by infecting naive Vero cells and the infected cells were then monitored for luciferase activities at 48h post-infection and Indirect immunofluorescence assay(IFA),Low luciferase signals?2.7×10~4 light units and the lower positive cell percent were obtained from the VLP-infected cells indicating inefficient VLPs production by DMRIE-C transfection.To improve the VLPs yield,we used sequential electroporations,the results demonstrated that luciferase signals increased up to 10~8 light units,and obtained higher posotive percent,indicating that electroporation could produce more VLPs.In order to achieve continuous production of VLPs,we stablished a stable BHK-21 cell line expressing JEV-CprME.In a word,this study established a new way to assebly JEV VLPs by complementary of JEV replicon with structural proteins CprME.The results demonstrated that DMRIE-C transfection could provid inefficient complementary.To some extent,sequential electroporation could improve complementary efficiency and increase VLPs production.Since Rluc reporter gene was inserted into the replicon,it providede feasibility for antiviral drugs screening.