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

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

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 compare the biological characteristics of West Nile virus (WNV) and Japanese encephalitis virus (JEV), including cells sensitivity, pathogenicity, viral morphology, as well as the results of immunological and molecular biological detection.

METHODSCytopathic effect (CPE) and pathogenicity were observed in C6/36 cells and in suckling mice inoculated intracerebrally with the WNV or JEV, respectively. The sliced tissue samples for electron microscopic examination were prepared for the morphologic observation of the viruses. Serum antibody to WNV or JEV was detected using indirect immunofluorescence assay (IFA), and the viral RNA was analyzed by RT-PCR method.

RESULTSWNV or JEV-caused CPE was characterized by cell fusion and cell shedding, respectively. There was no significant difference in the pathogenicity to suckling mice between WNV and JEV. The morphologic observation showed that the shape and size of the two virions were similar. WNV and JEV were found to have antigenic cross-reactivity. The viral RNA could be detected from both WNV and JEV samples with universal primer set, but only nucleoside fragments of corresponding virus could be amplified when specific primers were used.

CONCLUSIONCPE in C6/36 cell and detection of the viral RNA should be useful in discrimination of WNV and JEV, and simultaneously examining the titers of serum antibodies against WNV and JEV may be helpful to diagnosis of infection with these agents.

Animals ; Brain ; virology ; Cell Line ; Diagnosis, Differential ; Encephalitis Virus, Japanese ; immunology ; isolation & purification ; Encephalitis, Japanese ; diagnosis ; virology ; Flavivirus Infections ; diagnosis ; virology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; West Nile virus ; immunology ; isolation & purification

The Japanese encephalitis virus (JEV) is one of causative agents of reproductive failure in pregnant sows. An indirect enzyme-linked immunosorbent assay (I-ELISA) was examined for its potential use in the rapid monitoring of the JEV, and the results were compared with those from the hemagglutination inhibition (HI) and serum neutralization (SN) tests. The comparative analysis showed that the results of I-ELISA showed a significant correlation with the conventional HI (r = 0.867) and SN tests (r = 0.804), respectively. When the I-ELISA results were compared with the traditional diagnostic assays, the sensitivity of the I-ELISA was 94.3% with the HI test and 93.7% with the SN test, respectively. The specificity was found to be 81.4% and 80.0% with the HI and SN tests, respectively. To determine the applicability of I-ELISA in the field, the serum samples from 720 pigs were collected from 4 regions in Korea between July and August 2004. The results indicated that 21.7% of screened pigs were seropositive for the JEV. The seropositive rates of JEV in the 4 provinces were 12.6% in Gyeonggi, 45.0% in Gyeongnam, 16.7% in Jeonbuk, and 12.2% in Jeju. The I-ELISA methodology developed in this study was shown to have considerable sensitivity and specificity through a comparison with HI and the SN tests. Therefore, it might be one of convenient methods for screening a large number of samples in various fields.
Animals ; Antibodies, Viral/blood ; Antigens, Viral/immunology ; Encephalitis Virus, Japanese/immunology/*isolation&purification ; Encephalitis, Japanese/blood/immunology/*veterinary/virology ; Enzyme-Linked Immunosorbent Assay/methods/*veterinary ; Female ; Hemagglutination Inhibition Tests/veterinary ; Korea ; Neutralization Tests/veterinary ; Swine ; Swine Diseases/blood/immunology/*virology

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

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

OBJECTIVETo study the clinical and laboratory characteristics of adult Japanese encephalitis (JE) patients in a JE outbreak in Yuncheng, Shanxi Province in 2006.

METHODAll the clinical data from the Second People's Hospital in Yuncheng city were analyzed, part of patients' sera and cerebrospinal fluid were tested by serology and RT-PCR.

RESULTSThe majority of patients were middle-aged and elderly, 77.8% (35/45) of the total cases were more than 40 years old. Severe and fulminating type cases accounted for 60.0% (27/45). Most patients had underlying diseases. IgM antibody to JE virus (JEV) in serum was positive in each of the 45 patients analyzed and 4-fold or greater rise in sera neutralization antibody titer were found in convalescent serum. JEV nucleic acid was positive in part of cerebrospinal fluid specimens.

CONCLUSIONViral encephalitis emerged in Yuncheng city, Shanxi Province was Japanese encephalitis B, and most of the cases belonged to elderly group.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Viral ; blood ; Child ; Child, Preschool ; China ; epidemiology ; Disease Outbreaks ; Encephalitis Virus, Japanese ; genetics ; immunology ; isolation & purification ; Encephalitis, Japanese ; blood ; cerebrospinal fluid ; epidemiology ; Female ; Humans ; Immunoglobulin M ; blood ; Male ; Middle Aged ; Neutralization Tests ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

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