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

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

To conduct sequencing of full-length genomes of two Japanese encephalitis virus strains (JEV) newly isolated in 2009 in China and analyze the characteristics of complete nucleotide sequences. The complete genomic sequences were obtained by RT-PCR and sequencing directly. Bioinformatics was used to analyze the nucleic acid data, deduced amino acid sequence and phylogenetic trees. The result of sequence analysis showed that the genomes of YN0911 and YN0967 strains were both 10965nt in length, which coded 3432 amino acid polyprotein. The homology of genome ranged from 83.3% to 98.9% in nt and from 94.8% to 99.7% in aa, respectively, when compared with selected JEV strains in GenBank. There were 13 amino acid divergences which were not the key virulence sites in E protein when compared with vaccine strain SA14-14-2. There were 11nt deletions in the 3' UTR region. Phylogenetic analyses based on C/ PrM, E gene and full-length genome all showed that YN0911 and YN0967 strains belonged to genotype I. The result also showed that two new JEVs had close phylogenetic relationship with the strains from Viet Nam, Sichuan Province, Guizhou Province, Guangxi Province, China. This study indicated that JEV strains newly isolated in 2009 in China were the members of JEV genotype I. The key virulence sites in E protein did not change.
Amino Acid Sequence ; Base Sequence ; China ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Encephalitis, Japanese ; virology ; Genome, Viral ; genetics ; Humans ; Molecular Sequence Data ; 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 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 investigate the natural infection and genotype of Japanese encephalitis virus in mosquitoes and swine in some areas of Zhejiang province.

METHODSSamples of mosquitoes and sera of swine were collected in three counties (Xianju, Longquan and Cixi) of Zhejiang province from May to October in 2007. 10 662 mosquitoes were collected during 2007, of which, C.pipiens pallens and C.quinquefasciatus were the most dominant species and 204 pig serum samples were detected. Japanese encephalitis virus in mosquitoes were detected by virus isolation and real time RT-PCR. The antibody against Japanese encephalitis virus in swine was detected by ELISA. The isolated strain were identified by real time RT-PCR. The PrM gene of the isolated strain was amplified by RT-PCR. Three strains were typed by the gene of PrM.

RESULTSSeven positive mosquitoe samples were identified by real time RT-PCR. Three strains were isolated and identified by real time RT-PCR. The PrM gene was cloned and sequenced. The phylogenetic analysis showed that three isolates belong to genotype I of Japanese encephalitis virus. Of 204 swine serum samples, 121 positive samples were identified positives. Above 50% sera samples from swine were positive in June.

CONCLUSIONThe vector of Japanese encephalitis virus existed and carried the Japanese encephalitis virus in these areas of Zhejiang province. Three strains of Japanese encephalitis virus were isolated from mosquito pools collected in Zhejiang province. It should be the first isolation of genotype I Japanese encephalitis virus in Zhejiang province in recent years.

Animals ; Antibodies, Viral ; blood ; Arboviruses ; classification ; isolation & purification ; China ; Culicidae ; virology ; Disease Vectors ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Genotype ; Swine ; virology

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia and domestic pigs serve as the amplifying hosts. In the present study, the full genomic sequences of two JEV strains (HEN0701 and SH0601) isolated from pigs in China were determined and compared with other 12 JEV strains deposited in GenBank. These two strains had an 88.8% nucleotide sequence similarity and 97.9% deduced amino acid sequence homology. HEN0701 had high nucleotide sequence and high amino acid sequence identity with genotype I (GI) strains, while SH0601 had high nucleotide sequence and high amino acid sequence identity with GIII strains at both the gene and full genome levels. Further phylogenetic analysis showed that HEN0701 belonged to the JEV GI group and SH0601 was classified as a GIII strain. Analysis of codon usage showed there were a few differences between the GI and GIII strains in nucleotide composition and codon usage for the open reading frames.
Animals ; Cell Line ; Cricetinae ; Encephalitis Virus, Japanese/classification/*genetics ; Encephalitis, Japanese/epidemiology/*veterinary/virology ; Gene Expression Regulation, Viral/physiology ; Genome, Viral ; Molecular Epidemiology ; Phylogeny ; Swine ; Swine Diseases/epidemiology/*virology

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia and domestic pigs serve as the amplifying hosts. In the present study, the full genomic sequences of two JEV strains (HEN0701 and SH0601) isolated from pigs in China were determined and compared with other 12 JEV strains deposited in GenBank. These two strains had an 88.8% nucleotide sequence similarity and 97.9% deduced amino acid sequence homology. HEN0701 had high nucleotide sequence and high amino acid sequence identity with genotype I (GI) strains, while SH0601 had high nucleotide sequence and high amino acid sequence identity with GIII strains at both the gene and full genome levels. Further phylogenetic analysis showed that HEN0701 belonged to the JEV GI group and SH0601 was classified as a GIII strain. Analysis of codon usage showed there were a few differences between the GI and GIII strains in nucleotide composition and codon usage for the open reading frames.
Animals ; Cell Line ; Cricetinae ; Encephalitis Virus, Japanese/classification/*genetics ; Encephalitis, Japanese/epidemiology/*veterinary/virology ; Gene Expression Regulation, Viral/physiology ; Genome, Viral ; Molecular Epidemiology ; Phylogeny ; Swine ; Swine Diseases/epidemiology/*virology

BACKGROUNDTo study arboviruses carried by mosquitoes in Liaoning Province in 2002.

METHODSTotally 4927 mosquitoes were collected from Liaoning Province in July 2002. Virus strains were isolated by inoculating BHK-21, C6/36 and Vero cells. The newly isolated strains were identified by serological (ELISA and IFA) and molecular methods (Real-Time PCR and RT-PCR).

RESULTSTwo strains were isolated from mosquitoes causing cytopathogenic effect (CPE) on cells and were fatal to suckling mice. Serological tests showed that both were positive for the antibody to JEV. The PrM and E gene were cloned and sequenced. The phylogenetic analysis showed that the new isolates belonged to genotype I, JEV. Sequence analysis showed that the homology of nucleotide sequences and amino acid (AA) sequences between the two strains was 100%. Compared with the nucleotide sequences between the two strains and the standard JEV vaccine strain SA14-14-2, the difference was up to 4.11%, and the difference of AA was 0.6%.

CONCLUSIONTwo strains of JEV were isolated and identified in Liaoning province, both belonged to genotype I JEV.

Animals ; Cell Line ; Cercopithecus aethiops ; China ; Culicidae ; virology ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Analysis, DNA ; Vero Cells ; Viral Envelope Proteins ; genetics

OBJECTIVETo sequence and analyze the complete genome of two new Japanese encephalitis virus (JEV) strains isolated from mosquitoes collected in Hubei province in 2008, and to understand the molecular biological characteristics of JEV in this area.

METHODSRT-PCR was used to amplify the fragments of HBZG08-09 strain and HBZG08-55 strain with 16 pairs overlapping primers after they had been recovered and identified, then the full-length genome was obtained by sequencing and splicing. Biological sequence alignment, nucleotide and amino acid sequence analysis, phylogenetic analysis and analysis of amino acid differences were performed by the software of Clustal X (1.83), MegAlign, Mega (4.0) and Genedoc (3.2).

RESULTSThe genome of two new strains were both 10 965 nucleotides in length with a single open reading frame from 96 to 10 392 coding for a 3432 amino acid poly-protein, the homology of nucleotide and amino acid sequence between two isolates were 98.2% and 99.7% respectively. Further study showed that the new strains were both belonging to genotype I. Two new strains were most closely related to isolates obtained from Henan and Zhejiang province in recent years. Compared with the live attenuated vaccine strain SA-14-14-2 in China, HBZG08-09 strain had 82 amino acid divergence; HBZG08-55 had 84 amino acid divergences. But the amino acid difference occurred in sites were not the key ones affecting the toxicity or antigenic of JEV.

CONCLUSIONTwo new JEV isolates were both belonging to genotype I, and the key sites of amino acid were not changed.

Animals ; China ; Culicidae ; virology ; Encephalitis Virus, Japanese ; classification ; genetics ; isolation & purification ; Genome, Viral ; Genotype ; Molecular Sequence Data ; Phylogeny ; RNA, Viral ; Sequence Alignment