OBJECTIVESequence and analysis the complete nucleotide of the Japanese encephalitis virus (JEV) newly strain SD08-10, isolated in 2008 in Shandong, China in order to understand the characterization of the virus.

METHODSOverlapping primers were designed according to the full-length genomes in GenBank. RT-PCR was used to amplify the fragments and the full-length genome was obtained by sequencing and splicing. Using the computer software to analysis the nucleic acid data, deduced amino acid sequence and phylogenetic tree, including Clustal X (1.8), DNASTAR, GENEDOC (3.2).

RESULTSThe result of sequence analysis shows that the genome of SD08-10 strain was 10 965 nucleotides long. An open reading frame from 96 to 10 392 including 10 296 nucleotides is capable of coding for a 3432 amino acid polyprotein. Compared with the live attenuated vaccine strain SA-14-14-2 in China, there was 1253 nucleotide difference and 82 amino acid divergence. Comparison of the complete genome sequences with 59 different JEV isolates showed a 0.7%-18.9% nucleotide sequence divergence among them, which resulted in 0.1%-5.2% amino acid sequence divergence. Phylogenetic analysis of full-length genome showed that the SD08-10 strain was belonging to genotype I.

CONCLUSIONAnalysis based on the complete genome sequences of different JEV isolates showed that the SD08-10 strain isolated in 2008 in Shandong was belonging to genotype I and close to SH17M-07 isolated in 2007 in China.

China ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Encephalitis, Japanese ; virology ; Genome, Viral ; Molecular Sequence Data ; Phylogeny

OBJECTIVETo investigate the molecular characteristics and genotype of Japanese encephalitis virus (JEV) found in vector mosquitoes in Zhejiang province from 1982 to 1983.

METHODSA total of 3188 mosquitoes were collected in Dinghai district and Yiwu city in Zhejiang province, during year 1982 and 1983. The virus was isolated by C6/36 cell, and then identified by hemagglutination inhibition test. The isolated strains were activated in year 2011, and plaque forming unit (PFU) were applied to test the virus titer. The suckling rats were tested under intracranial inoculation, where PrM and E genes were amplified and sequenced. Their nucleotide and amino acid sequences were analyzed and compared with the JEV vaccine strain SA14-14-2 and the JEV isolated in Zhejiang province during 2007 and 2010; and phylogenetic tree were constructed by bioinformatic software.

RESULTSFrom the 3188 mosquitoes captured, eleven virus strains were isolated and found to be able to cause cytopathogenic effect (CPE) in C6/36 cells within 72 hours. Virus titer ranged from 2.5 to 6.47 lg PFU/ml. The suckling rats would die within 72 hours since the inoculation. The phylogenetic analysis with the PrM and E genes showed that the JEV isolated in Zhejiang during 1982 and 1983 belonged to genotype III; while the JEV isolated in Zhejiang during 2007 and 2010 belonged to genotype I. The analysis of E genes from 5 isolated strains found that the homology of nucleotide sequence was over 98.9%, and the homology of amino acid sequence was over 99.8%. The compare between the 5 virus strains and the vaccine strain SA14-14-2 found 10 common amino acid variation sites, and showed that the homology of nucleotide sequence was over 97.7%, and the homology of amino acid sequence was 99.2%. The compare between the 5 virus strains and the JEV isolated in Zhejiang during 2007 and 2010 found a 87.7% - 87.9% nucleotide homology and an over 98.8% amino acid homology.

CONCLUSIONThe JEV isolated from the mosquitoes in Dinghai district and Yiwu city between year 1982 and 1983, were genotype III.

Amino Acid Sequence ; Animals ; China ; Culicidae ; virology ; Encephalitis Virus, Japanese ; classification ; isolation & purification ; Encephalitis, Japanese ; virology ; Genotype ; Phylogeny ; Rats

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

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

OBJECTIVETo investigate the distribution patterns of mosquito and mosquito-borne viruses in Dehong prefecture, Yunnan province, China.

METHODSMosquito samples were collected using the mosquito traps from five counties of Dehong prefecture on July, 2007 and 2010. Mosquito were cell cultured for viral isolation, and positive isolates were identified using RT-PCR and sequence analysis.

RESULTSA total of 43 634 mosquito comprised of 29 species representing six genera were collected. Culex tritaeniorhynchus and Anopheles sinensis comprised 78.69% and 14.77% of the total. Six strains of viruses were isolated from the mosquito pools. RT-PCR and phylogenetic analysis revealed three strains from Cx. tritaeniorhynchus, identified as genotype I Japanese encephalitis virus (JEV). One strain was identified from Cx. tritaeniorhynchus, as Getah virus (GETV). Two strains isolated from Cx. tritaeniorhynchus and Anopheles vagus were identified as Culex pipiens pallens Densovirus (CppDNV).

CONCLUSIONCx. tritaeniorhynchus had been the major species of mosquito and mainly transmitting vector of mosquito-borne viruses in Dehong prefecture. Genotype I JEV, GETV and CppDNV were the vectors causing transmission of mosquito-borne diseases in this area. Data from phylogenetic analysis showed that these newly discovered isolates seemed to have had close relationship with those viruses previously circulating in Yunnan and other provinces of China.

Alphavirus ; isolation & purification ; Animals ; Arboviruses ; classification ; isolation & purification ; China ; Culicidae ; virology ; Disease Vectors ; classification ; Encephalitis Virus, Japanese ; isolation & purification

OBJECTIVETo sequence and analyze the whole genome of Japanese encephalitis virus (JEV) strain named 47 which was isolated from patient's cerebrospinal fluid sample in Heilongjiang province in 1950.

METHODSRNA was extracted from the recovery strain 47 and amplified with self-designed JEV genome sequencing primers. The differentiation analysis for nucleotides and coding amino acids and phylogenetic analysis were performed by the software of DNAStar, Modeltest, and Phylip.

RESULTSThe whole genome of strain 47 has 10,977 nucleotides. An open reading frame from 95 to 10,391 including 10,296 nucleotides is capable of coding a 3432 amino acid polyprotein. The nucleotide difference between strain 47 and 5 vaccine strains is 2.4%-4.4%, the amino acid difference between strain 47 and 5 vaccine strains is 0.3%-1.1%. The best evolution model for the whole genome is GTR + I + G. Based on the phylogenetic analysis, strain 47 belongs to the genotype III JEV.

CONCLUSIONStrain 47 is highly conserved on whole genome nucleotide and amino acid sequence. And it is belongs to the genotype III JEV.

China ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Encephalitis, Japanese ; virology ; Genome, Viral ; Humans ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; RNA, Viral ; cerebrospinal fluid ; genetics ; isolation & purification

The biological and genetic characteristics of a highly neurovirulent JE virus strain SA4 were studied. Mice were inoculated intracerebrally with strain SA4 and SA14, and observed for 14 days, respectively. On different days, mice brains were harvested for titrations of the virus content in the brains. Full-length genome of SA4 was sequenced and compared with SA14 as well as other JE virus strains in the world. The results indicated that the mice inoculated by SA4 induced sickness and death more rapidly (24 hours faster) than those induced by the SA14. The virus titers in the brains of mice infected with SA4 were 0.5-1.0 lg PFU/mL higher than that infected with SA14. The sequence comparison indicated that the nucleotide and amino acid homology between SA4 and the other 21 JE strains were 84.6%-99.0% and 95.2%-99.7% respectively. Comparison with strain SA14 revealed that there were 17 amino acid differences between the two strains, of which 5 were in the E protein region. The results demonstrate that strain SA4 is a highly neurovirulent strain. The substitutions of the 17 amino acids in the SA4 strain can be the molecular basis for the biological characteristics of high neurovirulence.
Animals ; Brain ; virology ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; pathogenicity ; Encephalitis, Japanese ; mortality ; virology ; Genotype ; Humans ; Mice ; Sequence Analysis ; Viral Envelope Proteins ; genetics ; Virulence

OBJECTIVEFor constructing the high-throughput platform of sequencing the JEV whole genome, the two systems of multiplex primers for genotype I and III should be designed and used for detected the whole genome of genotype I and III JEV in the research.

METHODSThe two systems of JEV genotype-specific primers was designed based on the reference sequence of all the available genotype I and III JEV genome sequence on GenBank, then, they were used to amply and sequence the 121 JEV strains isolated in China contains 63 GIII JEV and 58 GI JEV.

RESULTSThe self-designed genotype-specific primers for genotype I and genotype III JEV were 16 pairs and 17 pairs respectively, which were used for detecting the whole genome of 121 JEV. The average quality value for GI JEV is 40.037. The average quality value for GIII JEV is 40.857.

CONCLUSIONThe two systems of JEV genotype-specific primers could sequenced the genotype I and III JEV qualified and specific. It is the basis of the high throughput platform of sequencing the JEV whole genome.

China ; DNA Primers ; genetics ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Encephalitis, Japanese ; virology ; Genotype ; Humans ; Molecular Sequence Data ; Phylogeny ; Species Specificity

OBJECTIVETo sequence and analyze the whole genome of Japanese encephalitis virus (JEV) isolated from mosquitoes in Liaoning province in 2008.

METHODSUsing RT-PCR to amplify fragments with genome sequencing primer. The full-length genome was obtained by sequencing and splicing. The differentiation analysis for nucleotides, deduced amino acid sequence and phylogenetic tree was performed by the software of Clustal X (1.83), ATGC (V4), DNAStar, GENEDOC (3.2) and Mega (4.0).

RESULTSThe whole genome of strain LN0828 possesses 10 965 nucleotides. An open reading frame from 97 to 10 392 including 10 296 nucleotides is capable of coding for a 3432 amino acid polyprotein. Comparison of strain LN0828 genomic sequence with those of 32 JEV isolates in GenBank showed that nucleotide sequence divergence ranges from 1.6% to 16.4%, which resulted in amino acid sequence divergence from 0.3% to 5.1%. In comparison with live attenuated vaccine stain SA14-14-2 in open reading frame, strain LN0828 has a total of 1186 nucleotide substitutions, 86 amino acid divergences. Based on phylogenetic analysis, the strain LN0828 belongs to the genotype I JEV.

CONCLUSIONThe whole genome of strain LN0828 is close to those of isolates from Liaoning in 2002 and 2007, which were grouped into genotype I JEV.

China ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Encephalitis, Japanese ; virology ; Genome, Viral ; Humans ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics

OBJECTIVETo study the complete genome sequence of Japanese encephalitis virus (JEV) strain XJ69 isolated in ZheJiang province and explore its evolution.

METHODSOverlapping primers were designed according to the full-length genomes from GenBank. RT-PCR was used to amplify the fragments and RT-PCR products were cloned T vector, sequenced and analyzed.

RESULTSThe genome of strain XJ69 and XJP613 were 10 964 nucleotides in length with a single open reading frame encoding 3432 amino acids. Comparison of the complete genome sequences of different JEV isolates showed XJ69 and XJP613 were 83.5%-99.2% and 83.4%-99.4% nucleotide sequence homology among them respectively, which resulted in 94.8%-99.7% amino acid sequence homology. Phylogenetic analysis through PrM/C,E and full-length genome showed that the XJ69 and XJP613 strain belonged to genotype I.

CONCLUSIONThe nucleotitede sequence and deduced amino acid sequence of XJ69 and XJP613 strain were similar to that of those of genotype I of Japanese encephalitis virus. It belonged to genotype I and were close to the isolates SH17M-07.

Animals ; Cell Line ; China ; Cricetinae ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Encephalitis, Japanese ; virology ; Genome, Viral ; Humans ; Molecular Sequence Data ; Phylogeny