Entry Mechanism And Targeting Inhibitor Of Japanese Encephalitis Virus

Japanese encephalitis, known as epidemic encephalitis B,is an acute infection disease with central nervous system damage caused by Japanese Encephalitis Virus (JEV).The disease has low fatality for pigs, but can cause abortion,stillbirth or mummified in pregnant sows and orchitis without fertility in boars.JEV has a zoonotic transmission cycle between birds and mosquitoes, with swine serving as intermediate amplifier hosts and the virus can spread from swine to humans via mosquito bites.It not only seriously jeopardizes the healthy and stable development of swine industry, but also is a serious threat to public health security of humans. Although at present there is a great progress in the research of etiology,molecular biology and immunological characteristics of JEV, and the application of the vaccine also decreased the incidence of JE, but no effective antiviral therapy is available for survivors with serious sequelaes.And the molecular mechanism of pathogenicity of JEV is still not entirely clear.therefore, the pathogenic mechanism and antiviral research become the key point and difficulty of current research for JEV.Attachment to the surface of the host cell is the first step for viral entry and the critical step in the life cycle of the virus,which is one of the key factors affecting the host range, tissue tropism and pathogenicity of the viruses. Thus the viral entry is a promising target for antiviral therapy and antiviral drug design.The evelope glucoprotein(E) of JEV is considered to be its virus attachment protein (VAP), mediating virus binding to receptor and membrance fusion in host cells.Studies suggested that there are three structural domains in the E protein of flavivirus: domainⅠ,domainⅡand domainⅢ.The domainⅢis known as the main domain of receptor binding,and single amino acid mutation of a conserved RGD motif (Arg387-Gly387-Asp389, RGD) in the domain may affect the receptor recognition and virulence.In the present study, virus attachment protein and cellular receptors were investigated to clarify the entry mechanisms of JEV and inhibiting molecules targeting virus entry were screened and their antiviral activities were assessed.The main contents are as follows: 1. Soluble expression and polyclonal antibody preparation of the receptor binding domain of Japanese encephalitis virus (JEV) envelope proteinIn this study, prokaryotic vectors expressed Japanese encephalitis virus envelope protein domainⅢ(EDⅢ) and their conservative single amino acid RGD motif mutant (△EDⅢ, RGD mutated to RGE) were constructed using point mutation and gene recombination technology, soluble recombinant proteins were obtained through induction at low temperature(18℃).And polyclonal antibodies against EDIII was prepared in mice immunized with the purified EDⅢprotein. Western blot analysis showed that the soluble recombinant proteins EDⅢand△EDⅢwere identified by swine anti-JEV positive serum,and sera prepared in mouse specifically bound to two recombinant proteins. This study is the basis for further study.2. Analysis of E protein domainⅢand its a conserved RGD motif in mediating JEV entry into host cellsIn the study,we compared the ability to bind to the surface receptor on BHK-21 cells between recombinant E DⅢprotein or RGD peptide containing the RGD motif and their mutants recombinant△EDⅢprotein or RGE peptide. Adhesion assays demonstrated that the former was able to bind to the surface on BHK-21 cells more effectively than the latter. Recombinant EDⅢprotein or RGD peptide significantly inhibited JEV infection in BHK-21 cells pre-incubated with them,and their inhibition rates were 65% and 98%, respectively,whereas the inhibition rates were decreased to 22% and 52% when RGD mutated to RGE in recombinant△EDⅢprotein or RGE peptide. It can be implied that JEV enter into target cells through its conserved RGD motif present on envelope protein binding to the celluar receptor.These results further confirmed JEV envelope protein DⅢwas its receptor binding domain, and served as a candidate inhibitor for JEV entry.3. Screening and identification of binding peptide for evelope protein of Japanese encephalitis virusIn this study, The purified prokaryotic expression evelope protein (Trx-E) was used as the target and Trx as control to screen its binding peptides from 12-mer random phage display peptide library. After four rounds of biopanning, phage clones were identified by ELISA. The specific clones were sequenced and peptides with corresponding amino acid sequence deduced were synthesized to be further checked by Surface Plasmon Resonance (SPR) assay. ELISA results showed that 15 of 20 clones randomly selected specifically bound to the envelope protein (E) domain of the recombinant protein Trx-E,and the other 5 clones specifically bound to the Trx domain of the recombinant protein Trx-E. SPR assay further confirmed that 5 synthetic peptides to some extent bound to the envelope protein (E) domain of the recombinant protein Trx-E, one of which,named as P1 (its amino acid sequence: TPDCTRWWCPLT), had the highest affinity to the envelope protein (E) domain of the recombinant protein Trx-E.These observations provided a base for further investigation of the mechanism of JEV entry into host cells and targeting antiviral therapy for JEV.4. Antiviral activity of binding peptides for evelope protein of Japanese encephalitis virus in vivo and in vitroBased on five binding peptides for evelope protein of Japanese encephalitis virus identified in the former chapter, the activity to inhibit JEV infection was assessed further. Q-PCR assay showed that five synthetic peptides to some extent inhibit JEV infection on BHK-21 cells,one of which P1 had the highest inhibition,up to 85%.PRNT assay further confirmed that P1 inhibited JEV infection in dosage-dependent manner on BHK-21,PK15 and Vero cells.Moreover, mice challenged with JEV by intraperitoneal injection that had been incubated with peptide P1 had reduced viremia,fatality and pathology damage in the central nervous system compared with control animals. Those results provided a new thread of antiviral therapy and drug design for JEV.5. Identification of the receptors of Japanese encephalitis virus on PK15 cellsTo date,the cellular receptor for JEV on swine cells remains unknown.In the study, many proeins were identified to be JEV binding protein on the BHK-21,PK15 and Vero cells using virus overlay protein binding assay (VOPBA),and one 74kDa protein in common in three cells was reconfirmed to be heat shock cognate protein 70(Hsc 70) by Western blot analysis.And the antibody against Hsc70 was able to block JEV binding to the three cells.Indirect immunofluorescence and confocal mocroscopy analysis demonstrated localization of Hsc 70 on cell surface of the three cells. These observations indicated that Hsc70 serves as putative receptor for JEV in BHK-21,PK15 and Vero cells.In addional,the further work is the role of these proteins about 67kDa,80kDa and 105kDa in BHK-21 and Vero cells in JEV entry into cells.In summary, these results would provide novel ways for further investigation of the mechanism of JEV entry into host cells and novel antiviral drug design for JEV.