The cDNA fragment of JEV prME gene was cloned into the baculovirus shuttle vector (bacmid) to construct a recombinant baculovirus vector, defined as AcBac-prME. Then the recombinant baculovirus Ac-prME was obtained by transfecting Sf9 cells with AcBac-prME. Western blot analysis and immunofluorescence results indicated that both prM and E proteins were efficiently expressed in Sf9 cells. Electron microscopy suggested that prME was assembled into JEV-VLPs. To further evaluate the potential of JEV-VLPs as vaccine, the mice were immunized with JEV-VLPs and then challenged with lethal JEV. The results of mice survival and pathological changes demonstrated that the JEV-VLPs performed complete protection against JEV-P3 strain and relieved pathological changes in the mice brain significant. This study suggest that JEV-VLPs would be a potential vaccine for Japanese encephalitis virus.
Animals ; Antibodies, Viral ; immunology ; Encephalitis Virus, Japanese ; genetics ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; virology ; Humans ; Japanese Encephalitis Vaccines ; administration & dosage ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Sf9 Cells ; Vaccination ; Vaccines, Virus-Like Particle ; administration & dosage ; genetics ; immunology ; Viral Envelope Proteins ; administration & dosage ; genetics ; immunology

This study aimed to investigate the immune adjuvant effect and mechanism induced by chitosan nanoparticles carrying pJME/GM-CSF. In this study, plasmid DNA (pJME/GM-CSF) was encapsulated in chitosan to prepare chitosan-pJME/GM-CSF nanoparticles using a complex coacervation process. Immunohistochemistry was used to detect the type of infiltrating cells at the site of intramuscular injection. The phenotype and functional changes of splenic DCs were measured by flow cytometry after different immunogens were injected intramuscularly. The killing activity of CTLs was assessed using the lactate dehydrogenase (LDH) release assay. The preparation of chitosan-pJME/GM-CSF nanoparticles matched the expected theoretical results. Our results also found that, after pJME/GM-CSF injection, the incoming cells were a mixture of macrophages, neutrophils, and immature DCs. Meanwhile, pJME/GM-CSF increased the expression of MHC class II molecules on splenic DCs, and enhanced their Ag capture and presentation functions. Cell-mediated immunity was induced by the vaccine. Furthermore, chitosan-pJME/GM-CSF nanoparticles outperformed the administration of standard pJME/GM-CSF in terms of DC recruitment, antigen processing and presentation, and vaccine enhancement. These findings reveal that chitosan could be used as delivery vector for DNA vaccine intramuscular immunizations, and enhance pJME/GM-CSF-induced cellular immune responses.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Chitosan ; administration & dosage ; immunology ; Dendritic Cells ; immunology ; virology ; Encephalitis Virus, Japanese ; genetics ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; virology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; genetics ; immunology ; Humans ; Immunity, Cellular ; Japanese Encephalitis Vaccines ; administration & dosage ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Nanoparticles ; administration & dosage ; Spleen ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

OBJECTIVETo understand the genotypic characteristics and the neutralizing antibody levels of Japanese encephalitis virus (JEV) and Japanese encephalitis (JE) in both vector mosquitoes and in healthy people of Zhejiang province.

METHODSVirus was isolated from mosquitos sampled from the Monitoring Stations located in Xianju county during 2012 to 2013. Phylogenetic and homological studies were carried out on the E gene. A total of 1 263 blood specimens from 642 healthy people were collected before and after the seasons of JE epidemics. JEV neutralizing antibody was detected by the micro-neutralization test.

RESULTSTwenty-five JEV strains were isolated from a total of 11 650 mosquitoes. The identity of nucleotide appeared as 87.8%-99.7% both from 2012 to 2013 and from 1982 to 2010 while as 87.7%-88.0% with vaccine strain SA14-14-2, in Zhejiang. The phylogeny tree of E gene indicated that the newly isolated virus belonged to genotype I but no mutation of amino acid sequence coding conformational epitope was identified in the envelop protein. Both positive rates and the geometric mean titer (GMT) of neutralizing antibody in healthy people were 31.5%-42.0% and 1 : 2.56-1 : 3.53 in Xianju county, during 2012 and 2013, respectively. Both of the two positive rates (χ(2)≤1.76, P > 0.05) and the two GMTs (u≤0.64, P > 0.5) for antibodies pre or post the epidemic season did not show significant differences.

CONCLUSIONJEV isolated in Xianju during 2012 and 2013 belonged to genotype I. The positive rates of JEV neutralizing antibody from healthy people in Xianju were less than 42.0%, which showed no significant differendes pre or post JE epidemic season.

Amino Acid Sequence ; Animals ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; China ; Culicidae ; virology ; Disease Vectors ; Encephalitis Virus, Japanese ; genetics ; immunology ; isolation & purification ; Encephalitis, Japanese ; virology ; Epitopes ; Genotype ; Humans ; Neutralization Tests ; Phylogeny

Japanese encephalitis virus(JEV)is one of the leading cause of viral encephalitis in Asia. The phenotypic and genotypic characteristics of isolated virus strains are reviewed in this paper. Studies on the biological characteristics of the isolates showed that different isolates existed apparent differences in virus plaque morphology, neuroinvasive pathogenicity in mice, protective antigenicity and hemagglutination property. In China, only genotype III JEV strains were isolated before 1977. But since 1977, both genotype I and I JEV strains were isolated and the genotype I virus, which was isolated from mosquitoes mostly, has become the dominant strain. Study on the genomic sequence indicated that there was only a few amino acid difference (< or = 43%) between the two genotype isolates. Comparison between both genotype isolates and widely used live vaccine strain SA14-14-2 revealed that there were only < or = 3% amino acid differences, most of which were the SA14-14-2 unique attenuating sites. These results indicate that the SA14-14-2 live vaccine is able to protect people against infection of the both genotype I and Ill JEV strains.
Animals ; China ; Culicidae ; virology ; Encephalitis Virus, Japanese ; classification ; genetics ; immunology ; isolation & purification ; Encephalitis, Japanese ; immunology ; prevention & control ; virology ; Genome, Viral ; genetics ; Genotype ; Humans ; Japanese Encephalitis Vaccines ; immunology ; Mice ; Phenotype ; Species Specificity ; Vaccines, Attenuated ; immunology

PrME and NS1 gene were the two main immuneprotect proteins of Japanese encephalitis virus (JEV), and they were also N-linked glycosylation proteins. To clear the effect of N-glycosylation on JEV immunity, the N-glycosylation site of prME and NS1 gene were eliminated by site-directed mutant PCR, subtituting the N to Q. And the the mutant genes were subcloned into eukaryotic expression plasmid. Four-weeks female mice were immuned with the wildtype and mutant gene by twice. The antibodies against prME were detected by ELISA and the neutralization antibodies were tested by viral neutralizing assay. The immunoprotection were determined by attack with JEV virulent strain. Compare with the wild-type gene immuned-groups, one N-glycan eliminated prME gene could induce a little higher ELISA antibody, neutralization antibody and immunoprotection, but the immunity of gene with both N-glycan absence was decreased. The similar status were observed in the wildtype and mutant NS1 groups. Thus these results show that the N-linked glycosylation in the prME and NS1 gene were correlated with the immunity, one glycan absent would enhance the immunity but both two loss would impair it.
Animals ; Antibodies, Viral ; immunology ; Encephalitis Virus, Japanese ; genetics ; immunology ; metabolism ; Encephalitis, Japanese ; immunology ; virology ; Female ; Glycosylation ; Humans ; Mice ; Mice, Inbred BALB C ; Viral Nonstructural Proteins ; genetics ; immunology ; metabolism

To express the domain III gene of Japanese encephalitis virus (JEV) and to learn the possibility of developing the Dil protein as a subunit vaccine, we amplified the JEV DIII gene by PCR and constructed the expression plasmid pET-JE DIII by inserting JEV DIII gene into the prokaryotic expression vector pET-32a(+). The domain III protein of the attenuated strain SA14-14-2 was expressed as a thioredoxin (Trx) fusion protein, which was unique in forming a large fraction of the soluble recombinant protein. We immunized the rabbits and mice with the purified protein, tested the antigenicity and immunogenicity of JEV DIII protein by ELISA, Western blotting, plaque reduction test and observed the protective efficacy on challenged weanling mice with JEV. Rabbits immunized with the purified JEV DIII protein generated 1: 7 x 10(5) anti-JEV specific antibody titers. BALB/c mice immunized with the purified JEV E DIII protein generated 1: 8.2 x 10(4) anti-JEV specific antibody titers. And the neutralized antibody titer can reach 1:256, the survival rate of the immunized weanling mice was approximately 75%. Overall, this study highlighted that recombinant JEV E DIII protein delivered in mice and rabbits can generate high antibody titers against JEV, and protect some mice challenged with JEV. These studies can provide useful information for further developing the domain III recombinant protein as subunit vaccine against JEV.
Animals ; Antibodies, Viral ; blood ; Encephalitis Virus, Japanese ; immunology ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Immunization ; Japanese Encephalitis Vaccines ; biosynthesis ; immunology ; Mice ; Mice, Inbred BALB C ; Protein Structure, Tertiary ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology

China ; Encephalitis Virus, Japanese ; genetics ; immunology ; isolation & purification ; Encephalitis, Japanese ; virology ; Humans ; Research ; trends

OBJECTIVETo prepare monoclonal antibody (mAb) against prM epitope.

METHODSThe gene encoding prM was isolated using RT-PCR from brain of JEV infected mouse and cloned into prokaryotic expression vector pET-32a. Recombinant plasmid was transformed into E.coli BL21/DE3/LysS, then the transformed cells were expressed with the induction of IPTG. The expression and purification of the prM protein was analyzed by SDS-PAGE. The BALB/c mice were immunized with purified prM protein. Hybridoma cell lines secreting monoclonal antibodies against prM were established after cell fusion of mouse splenic cell and P3-X63-Ag8.653 cells. The specificity of mAb was identified by ELISA, Western Blot and Immunohistochemistry assay.

RESULTSmAb against prM epitope of JEV was prepared successfully.

CONCLUSIONThe obtained prM specific mAb was valuable for the prevention and dignosis of Japanese encephalitis.

Animals ; Antibodies, Monoclonal ; analysis ; immunology ; isolation & purification ; Antibody Specificity ; BALB 3T3 Cells ; Cell Line ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Encephalitis Virus, Japanese ; genetics ; immunology ; Epitopes ; immunology ; Escherichia coli ; genetics ; Mice ; Plasmids ; genetics ; metabolism ; Prokaryotic Cells ; metabolism ; Sequence Analysis, DNA ; Viral Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification

BACKGROUNDTo analyze the difference of nucleotides and deduced amino acids sequences E gene between the newly isolated Japanese encephalitis (JE) virus strains from mosquitoes or patients and P3 strain.

METHODSThe E gene sequences of corresponding strains of JE virus were obtained from GenBank. Computer analyze of nucleic acid data and deduced amino acid sequence were accomplished using the Clustal X (1.8), DNASTAR, GENEDOC (3.2) programs.

RESULTSThe result showed that compared with the Fujian strains and P3 strain the nucleotide sequence homology was up to 98.3%, and the amino acid sequence homology was up to 98.2%, respectively. Compared with the Shanghai strains and P3 strain, the nucleotide sequence differences were 12%, and the amino acid sequence homology was up to 98.2%, respectively. Compared with P3 strain, there were nineteen amino acid variations in E gene of all the newly isolated strains. Between P3 and all the newly isolated JE virus strains, there are three common variations at E-129, E-222, E-366. And two common variations E-160 and E-487 were found only in Fujian strains, common variations at E-129, E-222, E-227, E-366 in Shanghai strains.

CONCLUSIONThere are some differences between P3 strain and JE viruses which were isolated from mosquitoes belonging to genotype I in Shanghai and from patients belonging to genotype III from Fujian province. But these variations are not in the important locations affecting the biological characteristic of the viruses.

Amino Acid Sequence ; Animals ; Culicidae ; virology ; Encephalitis Virus, Japanese ; genetics ; immunology ; isolation & purification ; Encephalitis, Japanese ; immunology ; virology ; Genetic Variation ; Humans ; Sequence Homology, Nucleic Acid ; Vaccines, Inactivated ; Viral Envelope Proteins ; genetics

Genes encoding for the premembrane and envelope (prME), envelope (E) and nonstructural protein (NS1) of Japanese encephalitis virus (JEV) were cloned. Each protein was expressed in baculovirus expression system. Of the three proteins expressed in baculovirus system, only prME had hemagglutination activity. The prME (72 and 54 kDa), E (54 kDa) and NS1 (46 kDa) proteins could be detected by Western blotting in the recombinant virus infected cells. Immunogenicity of the recombinant proteins obtained from infected Spodoptera frugiperda (Sf-9) cells was examined in mice. The 3 week-old ICR mice immunized intraperitoneally with three recombinant proteins three times were challenged with a lethal JEV. A survival rate was increased from about 7.7% in unimmunized mice to 92.3% in E + prME and only E groups. The complete protection was shown in prME and live vaccine inoculated groups, respectively. We also measured neutralizing antibody and three immunoglobulin subtypes of IgG1, IgG2a and IgG2b in the sera of mice before and after challenge. Titers of IgG1 antibodies were approximately two to three times higher than that of IgG2b antibodies in all the immunized groups as compared to the control group. However, IgG2a antibody level somewhat increased after challenge, indicating T-helper type 1 (Th1) cell response. The results of this study can provide useful information for developing efficacious subunit vaccine against JEV.
Animals ; Antibodies, Viral/blood ; Baculoviridae/genetics ; Blotting, Western ; Cloning, Molecular ; Encephalitis Virus, Japanese/genetics/*immunology ; Encephalitis, Japanese/*immunology/prevention&control ; Female ; Immunization ; Immunoglobulin Isotypes/blood ; Japanese Encephalitis Vaccines/*immunology/standards ; Mice ; Mice, Inbred ICR ; Microscopy, Fluorescence ; Plasmids ; Recombinant Proteins/genetics/immunology ; Viral Envelope Proteins/genetics/*immunology ; Viral Matrix Proteins/genetics/*immunology ; Viral Nonstructural Proteins/genetics/*immunology