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

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 evaluate the susceptibility of Aedes albopictus and Culex pipiens quinquefasciatus to oral infection with bat Japanese encephalitis virus isolates (GD1 and HN2 strains).

METHODSAedes albopictus and Culex pipiens quinquefasciatus were infected orally by GD1 and HN2 strains of bat Japanese encephalitis virus. TaqMan real-time PCR was used to detect the virus and monitor the changes in the viral loads in Aedes albopictus and Culex pipiens quinquefasciatus at a 2-day interval, starting from 4 days till 20 days after the infection.

RESULTSThe infected Aedes albopictus and Culex pipiens quinquefasciatus were found positive for the Japanese encephalitis virus from day 4 to day 20. Both Aedes albopictus and Culex pipiens quinquefasciatus were susceptible to infection by GD1 and HN2 strains, but the latter showed a greater susceptibility. The HN2 strain virus appeared to have a greater virulence than the GD1 strain.

CONCLUSIONAedes albopictus and Culex pipiens quinquefasciatus can carry GD1 and HN2 strains of bat Japanese encephalitis virus isolates.

Aedes ; virology ; Animals ; Chiroptera ; virology ; Culex ; virology ; Disease Susceptibility ; Encephalitis Virus, Japanese ; isolation & purification

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

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

In order to reveal the phenotypic characteristics of 17 JE virus strains isolated from different years, plaque sizes, mice neurovirulence and mice neuroinvasiveness of the isolates were studied and compared. BHK21 cell monolayers were used for testing the plaque sizes. The virus neurovirulence was tested in 9-11g mice inoculated intracerebrally and the virus neuroinvasiveness was tested in 9-11g and 14-16g by subcutaneous inoculation. Results showed that all the viruses produced clear plaques on the BHK21 cell monolayers with different sizes and all the virus strains appeared high neurovirulence in the mice with higher than lg8. 0/0.03 mL virus titers, while no apparent difference among them. The neuroinvasiveness (subcutaneous virulence) tested in the 9-11g mice had shown a little difference, but when tested in the 12-14 g mice,the difference was apparent. The results demonstrated that JEV in nature were highly neurovirulent with no apparent difference. However the neuroinvasiveness of the JEV in nature was greatly different, which didn't relate to the years of isolation and genotypes, but most of the viruses isolated from patients showed higher neuroinvasiveness.
Animals ; Cell Line ; China ; Culicidae ; virology ; Encephalitis Virus, Japanese ; genetics ; isolation & purification ; pathogenicity ; Encephalitis, Japanese ; virology ; Genotype ; Humans ; Mice ; Phenotype ; Viral Plaque Assay ; Virulence

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

Objective: To investigate the distribution patterns of mosquitoes, midges and related arboviruses in Sichuan province. Methods: Blood-sucking insects were collected from houses and pens, using the ultraviolet lights. Mosquito samples were classified according to morphologic characteristics and then stored at liquid nitrogen. All samples were incubated with BHK-21 and C6/36 cells for virus isolation and then detected for their viral genes. Sequences of the virus were identified and analyzed by molecular biological software, such as BioEdit 7.0.5.3, MEGA 6.0. Results: In total, 17 019 mosquitoes from 3 genera and 4 species and 12 700 midges were collected from the southeast regions of Sichuan province in 2016 and 2017. Among them, 79.4% (13 519/17 019) belonged to Culex tritaeniorhynchus with 11.1% (1 897/17 019) as Armigeres subalbatus, 5.5% (930/17 019) were Anopheles sinensis and 4.0% (673/17 019) were Anopheles sinensis 3 virus strains that isolated from Culex tritaeniorhynchus were identified as typeⅠ Japanese encephalitis virus. Seven pools of mosquitoes isolated from Hejiang county were identified Japanese encephalitis virus gene positive through PCR amplification. With 4 pool midges were detected positive for Akabane virus through PCR gene amplification while midges samples didn't have virus isolates. Conclusions: Culex tritaeniorhynchus appeared the predominant species in the southeast regions of Sichuan. Japanese encephalitis virus transmitted by mosquitoes and Akabane virus by midges were prevalent in southeast Sichuan province.
Animals ; Arboviruses ; Culicidae ; Encephalitis Virus, Japanese/isolation & purification* ; Encephalitis, Japanese/diagnosis* ; Genes, Viral ; Nucleic Acid Amplification Techniques ; Phylogeny ; Polymerase Chain Reaction

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