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

Child ; China ; epidemiology ; Encephalitis Virus, Japanese ; Encephalitis, Viral ; epidemiology ; prevention & control ; Female ; Humans ; Japanese Encephalitis Vaccines ; immunology ; Male ; Vaccination

OBJECTIVETo investigate the molecular basis of pathogenicity of Japanese encephalitis virus (JEV) by sequencing of complete nucleotide sequence and analyze the characteristics of full-length genome of genotype I Japanese encephalitis virus strains (GZ56) which was isolated from the first cerebrospinal fluid (CSF) of Japanese encephalitis patients.

METHODSThe complete nucleotide sequence was obtained by RT-PCR and sequencing was performed directly. Bioinformatics was used to analyze the nucleic acid data, deduced amino acid sequence and phylogenetic trees.

RESULTSThe result of sequence analysis showed that the genome of GZ56 strains had 10 965 nucleotides, which coded for a 3432-amino acid polyprotein. Phyolngenetic analysis based on full-length genome showed that GZ56 strains and M-28 strains which were the first isolated from mosquitoes in Yunnan in 1977 were in the same evolutionary branch. GZ56 strains belongs to genotype I of Japanese encephalitis virus, the homology of genome ranged from 96.2% to 98.6% in nucleotide and from 98.2% to 99.7% in amino acid sequences respectively when compared with selected genotype I of JEV strains in GenBank. There were 11 amino acid divergences in E protein when compared with the JEV inactivated P3 strain but they are not the key virulence sites. However, there were 14 amino acid divergences in E protein when compared with the JEV live attenuated vaccine SA14-14-2 strain and 8 amino acid divergences were the key virulence sites.

CONCLUSIONThis study indicated that the full length of genome GZ56 strains had no ignificant change. It can be hypothesized from genomic level that the currently available JEV vaccines(inactivated and live attenuated) can protect against GZ56 strains infection, meanwhile, the JEV live attenuated vaccine (SA14-14-2) formulation conferred higher levels of protection.

Computational Biology ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Encephalitis, Japanese ; virology ; Enzyme-Linked Immunosorbent Assay ; Genome, Viral ; Genotype ; Japanese Encephalitis Vaccines ; immunology ; Phylogeny ; Sequence Analysis, DNA

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

OBJECTIVEUsing the advantages of Japanese encephalitis live attenuated and inactivated vaccine, to reduce the rate of immunization reaction and to increase the effect, we conducted a study on the strategy of immunization in Japanese encephalitis using live attenuated vaccine combined with inactivated vaccine.

METHODSObserving the safety and immune effects of different groups.

RESULTSData on side effect showed that the rate of moderate and severe systematic reactions of the group who were inoculated with combined vaccine was 0.73%, with local reaction 1.46% while the combined rate of moderate and severe systematic reaction of the group who were inoculated with inactivated vaccine was 2.8%. Under the detection of serum neutralizing antibody, the GMT rose from 1:1.05 - 1:3.35 before vaccination to 1:47.34 - 1:101.30 after vaccination in the different groups. Neutralizing antibody was detected in 97.67% of the combined group. There was a significant difference by comparing neutralizing antibody seroconversion rate of the combined group with the inactivated group (chi(2) = 3.89, P < 0.05), but no significant difference with attenuated group (chi(2) = 0.74, P > 0.05).

CONCLUSIONResults showed that in children who previously had been immunized with two doses of inactivated vaccine, the booster administration of live attenuated vaccine was both effective and safe.

Antibodies, Viral ; blood ; Child, Preschool ; Encephalitis Virus, Japanese ; immunology ; Humans ; Immunization ; Japanese Encephalitis Vaccines ; administration & dosage ; adverse effects ; immunology ; Vaccines, Attenuated ; immunology ; Vaccines, Inactivated ; immunology

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

BACKGROUNDTo determine if the attenuated Japanese encephalitis (JE) virus SA14-14-2 vaccine strain interacts efficiently with Culex tritaeniorhynchus and Culex pipiens quinquefasciatus, and further to acquire a new knowledge of its characteristics and safety for human beings.

METHODSLaboratory colonies of the two species of mosquitoes were set up and were inoculated intrathoracically with the attenuated vaccine virus and wild JE virus (Nak), both of which were used with different dilution from 10(-1) to 10(-9). Subsequently, the virus titers in the mosquitoes were detected by the plaque assay.

RESULTSInoculated with the vaccine strain, two species of mosquitoes were infected with the titers ranged from 10(0)-10(-3), and the maximum titers in Culex tritaeniorhynchus and Culex pipiens quinquefasciatus were 4.48 logPFU/ml and 5.63 logPFU/ml, respectively. Inoculated with wild JE virus, Culex pipiens quinquefasciatus was infected with titers ranged from 10(0)-10(-5), and the maximum titer in the mosquitoes was 6.59; Culex tritaeniorhynchus was infected with titers ranged from 10(0)-10(-4) and the maximum titer was 5.74 logPFU/ml.

CONCLUSIONBy intrathoracic infection, the attenuated JE virus SA14-14-2 vaccine strain can replicate in both species of mosquitoes.

Animals ; Culex ; classification ; virology ; Encephalitis Virus, Japanese ; genetics ; growth & development ; immunology ; Encephalitis, Japanese ; virology ; Humans ; Insect Vectors ; virology ; Japanese Encephalitis Vaccines ; immunology ; Species Specificity ; Vaccines, Attenuated ; immunology ; Viral Plaque Assay

OBJECTIVETo construct infectious Japanese encephalitis virus (JEV) based on the in vitro-ligated cDNA template of the vaccine strain SA14-14-2, and identify the virus.

METHODSFull-length genomic cDNA of JEV SA14-14-2 strain was ligated and then RNA was transcribed in vitro, the infective virus was obtained by transfecting the RNA into Vero cells and identified.

RESULTSThe infective clone of JEV was constructed, the virulence was weaker than the wild virus.

CONCLUSIONIt was possible to construct infectious clone from the production strain of live attenuated Japanese B encephalitis vaccine.

Animals ; Animals, Newborn ; Base Sequence ; Cells, Cultured ; Cercopithecus aethiops ; Cloning, Molecular ; DNA, Complementary ; genetics ; Encephalitis Virus, Japanese ; genetics ; immunology ; pathogenicity ; Encephalitis, Japanese ; pathology ; virology ; Genome, Viral ; Japanese Encephalitis Vaccines ; immunology ; Mice ; RNA, Viral ; genetics ; Vaccines, Attenuated ; immunology ; Vero Cells ; Virulence

Animals ; Antibodies, Monoclonal ; immunology ; Cell Line ; Cercopithecus aethiops ; Cricetinae ; Culicidae ; Dengue Virus ; immunology ; Encephalitis Virus, Japanese ; immunology ; Encephalitis, Japanese ; immunology ; Mice ; Mice, Inbred Strains ; Vero Cells ; Viral Vaccines ; immunology

To screen the best genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens, the prMEIII gene, the polytope gene and the prMEIII-polytope fusion gene of the GenotypeⅠJapanese encephalitis virus GS strain were cloned into prokaryotic expression vector pET-30a. The recombinant proteins were obtained after the induction and purification. The prepared recombinant proteins were immunized to mice, and the immunogenicity of the subunit vaccine candidate antigens was evaluated through monitoring the humoral immune response by ELISA, detecting the neutralizing antibody titer by plaque reduction neutralization test, and testing the cell-mediated immune response by lymphocyte proliferation assay and cytokine profiling. The recombinant proteins with the molecular weights of 35 (prMEIII), 28 (polytope antigen) and 57 kDa (prMEIII-polytope) induced strong humoral and cellular immune responses in mice. Compared with prMEIII-polytope and polytope proteins, the prMEIII protein induced a significant expression of IL-2 and IFN-γ (P<0.05) and the significant lymphoproliferation of splenocytes (P<0.05). The neutralizing antibody titer induced by the prMEIII protein was close to that induced by the commercial attenuated vaccine SA14-14-2 (P>0.05). The study suggests that the prMEIII protein can be used for the development of the Japanese encephalitis virus subunit vaccine.
Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; immunology ; Encephalitis Virus, Japanese ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; Immunogenicity, Vaccine ; Mice ; Mice, Inbred BALB C ; Vaccines, Subunit ; immunology ; Viral Vaccines ; immunology