In order to evaluate the immune effect of the genotype Ⅰ Japanese encephalitis virus prM-E DNA vaccine and the prM-EⅢ fusion protein subunit vaccine on mice using DNA prime-protein boost strategy, the prM-E gene was inserted into the pVAX1 eukaryotic expression vector. The recombinant expression vector prM-E-pVAX1 was constructed as a DNA vaccine for initial immunity, and the recombinant prM-EⅢ fusion protein was obtained using a prokaryotic expression system as a subunit vaccine for enhanced immunity. Thirty two female BALB/c mice aged 4-6 weeks were randomly divided into four groups, and a prM-E-pVAX1 DNA vaccine group, a DNA prime-protein boost immune group, a prM-EⅢ subunit vaccine group, and a pVAX1 vector control group were set up. The specific antibody level in serum was monitored by ELISA, the neutralizing antibody titer was detected by plaque reduction neutralization, and the cellular immune responses induced by different vaccine immune groups were analyzed by cytokine expression abundance and lymphocyte proliferation experiments. The results showed that the neutralizing antibody titers induced by mice immunized with the DNA prime-protein boost strategy were close to that of the group immunized with the single prM-EⅢ subunit vaccine, but significantly higher than that of the group immunized with the single prM-E-pVAX1 DNA vaccine. DNA prime-protein boost strategies induced effective Th1/Th2 immune responses in mouse models, in particular the Th1 cell-mediated immune responses. This study provides a new immune strategy that may facilitate the prevention of Japanese encephalitis.
Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; DNA ; Disease Models, Animal ; Encephalitis Virus, Japanese/genetics* ; Female ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA/genetics* ; Vaccines, Subunit

OBJECTIVETo detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed.

METHODSBy aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.

RESULTSWith the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%.

CONCLUSIONA TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.

Animals ; Culicidae ; virology ; Encephalitis Virus, Japanese ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity

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

To explore the spatial distribution mechanism of Japanese encephalitis virus (JEV), PhyML v3.0 was used to build phylogenetic tree using JEV sequences in the dataset. PAUP v4.0 and Migrapyhla softz ware were then used to analyze the migration events. The results showed that a total of 95 migration events were observed during the dispersal of JEV throughout Asia. Further analysis revealed that Thailand, and several Chinese provinces (including Shandong, Shanghai, Sichuan and Yunnan), were the main migration sources of JEV. JEV spread from these migration sources as follows: from Thailand to Australia, Cambodia, Tibet and India; from Shanghai to eastern coastal Asian regions and Yunnan; from Shandong to Korea, Zhejiang, Hubei, Shanxi and Liaoning; from Sichuan mainly to inland regions of China, as well as Vietnam and Japan; and from Yunnan to Zhejiang. This study indicated that frequent migration events occurred during the dispersal of JEV in the Asia and Pacific regions, and that Thailand, Shandong, Shanghai, Sichuan and Yunnan were the sources of JEV dispersal.
Asia ; epidemiology ; China ; epidemiology ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; physiology ; Encephalitis, Japanese ; epidemiology ; transmission ; virology ; Phylogeny

Japanese encephalitis virus (JEV) is a pathogenic mosquito-borne flavivirus which is responsible for outbreaks of severe viral encephalitis. The cellular entry of JEV is a prerequisite for Japanese encephalitis, so the understanding of its underlying mechanisms will provide more approaches for treating such disease. In recent years, increasing research has been conducted to investigate the mechanisms of cellular entry of JEV, and the results of research on other flavivirus have expanded the research directions for JEV. More methods will be used to suppress JEV infection because of the development of E protein antibodies and the discovery of several inhibitors of the cellular entry process. This review will summarize the recent advances in the mechanisms of JEV cellular entry and membrane fusion.
Animals ; Biomedical Research ; trends ; Encephalitis Virus, Japanese ; genetics ; physiology ; Encephalitis, Japanese ; virology ; Humans ; Virus Internalization

OBJECTIVETo determine the molecular characterization of full-length genome of Japanese encephalitis virus (JEV) genotype V.

METHODSThe full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis.

RESULTSThe full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% (G I, KV1899) to 79.7% (G III, JaGAr01), for the nucleotide sequences, and from 90.0% (G I, KV1899) to 91.8% (G III, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides (encoded with a serine residue) were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3'-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V.

CONCLUSIONThe molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.

Amino Acid Sequence ; Animals ; Base Sequence ; Culex ; virology ; Encephalitis Virus, Japanese ; genetics ; Genome, Viral ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Phylogeny ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Tibet ; Young Adult

This study aimed to construct full-length cDNA clones of the Japanese encephalitis virus (JEV). SA14-14-2 strain and discuss the feasibility of constructing chimeric viruses for exogenous gene expression based on the JEV genetic skeleton. Long-fragment RT-PCR techniques were applied to amplify JEV cD-NAs, and two amplified fragments with corresponding restriction endonuclease sites at both ends were cloned into the pACYC184 vector sequentially. Using standard molecular techniques, the enhanced green fluorescent protein (EGFP) gene was inserted into the 3' non-coding region of JEV as a reporter gene. After in vitro transcription and transfection procedures, wild-type JEV and chimeric JEV that expressed the EGFP as the reporter gene were successfully rescued. The recovered viruses were characterized by RT-PCR, plaque assays, and direct fluorescence microscopy. After six serial passage generations, the stability of the recovered viruses were studied in terms of virus growth characteristics and structural gene expression. The results showed that cDNA clones of rJEV and rJEV-EGFP were successfully constructed and rescued in BHK-21 cells after in vitro transcription and transfection. Each generation of the recovered viruses was stable and the chimeric virus rJEV-EGFP could stably express EGFP. The findings of this study indicate that both rJEV and rJEV-EGFP could be constructed and rescued in BHK-21 cells, and the JEV SA14-14-2 strain could be obtained as a viral vector to express foreign genes.
Cloning, Molecular ; DNA, Complementary ; genetics ; metabolism ; Encephalitis Virus, Japanese ; genetics ; metabolism ; Encephalitis, Japanese ; virology ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Vaccines, Attenuated ; genetics ; metabolism ; Viral Vaccines ; genetics ; metabolism

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