OBJECTIVETo detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed.

METHODSBy aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.

RESULTSWith the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%.

CONCLUSIONA TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.

Animals ; Culicidae ; virology ; Encephalitis Virus, Japanese ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity

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

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 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 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

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

OBJECTIVETo learn the characteristics of pathogen spectrum of Encephalitis /Meningitis in northwestern area of China.

METHODSBetween January 1st 2009 and March 31st 2011, a total of 569 patients with clinical symptoms of Encephalitis/Meningitis were selected from the hospitals in Gansu, Qinghai,Inner Mongolia and Xinjiang province. 1514 samples of specimen were collected from the 515 patients, to detect the IgM of Japanese encephalitis virus (JEV), enterovirus (EV, including Coxsackie virus, ECHO virus and enterovirus 71), Mumps virus, Herpes simplex virus (HSV) in blood and cerebrospinal fluid. Meanwhile, Neisseria meningitis (Nm), Haemophilus influenzae Type B (Hib), Staphylococcus, Streptococcus pneumonia, Streptococcus Suis, E. Coli and Cryptococci were also identified. The detection results were analyzed by different region, time and age range.

RESULTSPathogenic bacteria were identified in the specimen from 16 patients, with the rate at 3.65%, of which the dominant ones were Streptococcus pneumonia (7 patients, 43.75%). Virus were identified in the specimen from 132 patients, with the rate at 27.05%, of which the dominant types were EV and HSV, accounting for 33.33% (44 cases) and 31.82% (42 cases) respectively. The detection rate of virus showed a significant seasonal trend, with the peak appearing between June and November each year. The peak of EV detection was between July and September, with 24 cases detected out; the peak of HSV was between June and August (11 cases detected out); mumps virus was mainly found between July and December (25 cases). There was no significant time-distribution found in the detection of bacteria. The EV and HSV were mainly distributed in Gansu and Qinghai province (70 cases) ;most of mumps virus were found in Gansu province (24 cases);and JEV were only found in Gansu province (20 cases). The viral pathogen spectrum was identified in all ages, and the EV and mumps virus were mainly found in children aged 0-14 years old (42 and 17 cases respectively) ; JEV were identified in people over 15 years old, with 13 detected out of the 20 patients.

CONCLUSIONThe main pathogen of acute encephalitis and meningitis in northwestern area of China was virus, and the main pathogens of encephalitis and meningitis in children under 15 years were Herpes simplex virus and Mumps virus.

Adolescent ; Adult ; Antibodies, Viral ; blood ; cerebrospinal fluid ; Child ; Child, Preschool ; China ; epidemiology ; Encephalitis ; epidemiology ; microbiology ; virology ; Encephalitis Virus, Japanese ; isolation & purification ; Enterovirus ; isolation & purification ; Female ; Humans ; Immunoglobulin M ; blood ; cerebrospinal fluid ; Infant ; Infant, Newborn ; Male ; Meningitis ; epidemiology ; microbiology ; virology ; Middle Aged ; Mumps virus ; isolation & purification ; Simplexvirus ; isolation & purification ; Young Adult

OBJECTIVETo investigate the abundance and seasonal dynamics of mosquitoes, and to detect Japanese encephalitis virus (JEV) in these mosquitoes at the nesting colony of ardeid birds.

METHODSMosquitoes were collected bimonthly from July 2009 to May 2010 by Centers for Disease Control. Light traps and dry ice, as a source of CO2, were employed to attract mosquitoes. Mosquitoes were first identified, pooled into groups of upto 50 mosquitoes by species, and tested for JEV infection by viral isolation and reverse transcriptase polymerase chain reaction.

RESULTSA total of 20 370 mosquitoes comprising 14 species in five genera were collected. The five most abundant mosquito species collected were Culex tritaeniorhynchus (95.46%), Culex vishnui (2.68%), Culex gelidus (0.72%), Anopheles peditaeniatus (0.58%) and Culex quinquefasciatus (0.22%). Mosquito peak densities were observed in July. All of 416 mosquito pools were negative for JEV.

CONCLUSIONSThis study provides new information about mosquito species and status of JEV infection in mosquitoes in Thailand. Further study should be done to continue a close survey for the presence of this virus in the ardeid birds.

Animals ; Bird Diseases ; epidemiology ; virology ; Birds ; Culicidae ; physiology ; virology ; Encephalitis Virus, Japanese ; isolation & purification ; Encephalitis, Japanese ; epidemiology ; veterinary ; virology ; Population Dynamics ; Reverse Transcriptase Polymerase Chain Reaction ; veterinary ; Seasons ; Thailand ; epidemiology ; Virus Cultivation ; veterinary

Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) is currently available in virus detection and defined as the simultaneous amplification of two or more DNA/RNA targets in a single reaction vessel. In this study, we attempted to modify the conventional mRT-PCR technique on a basis of GenomeLab Genetic Analysis System (GeXP). Initially, we optimized the analytical validation of the GeXP analyzer and its design of workflow and simultaneously detected eight arboviruses that related to epidemic encephalitis by verifying the specificity of mRT-PCR with Japanese encephalitis virus(JEV) cell cultures and positive strains identified previously and determining the sensitivity with in vitro-transcribed RNA of serial dilutions. The GeXP system after optimization could amplify the specific fragments related to the viruses and exposed specifically a total of 13 target genes out of eight types of arboviruses at the level of 10(2) copies/microL, and the findings suggest that the novel protocol we developed can be high-throughput and highly specific and sensitive as well as quickness in screening of the encephalitis viruses, and is promising in detection of encephalitis-associated viruses for molecular epidemiological studies.
Arboviruses ; genetics ; isolation & purification ; Encephalitis Virus, Japanese ; genetics ; Encephalitis Viruses ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

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