[Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for the detection of Japanese encephalitis virus].

OBJECTIVE: To establish a TaqMan based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of Japanese encephalitis virus. METHODS: The gene sequences of Japanese encephalitis virus downloaded from the GenBank was aligned, using the biologic software. Specific primers and probes were designed in the conserved region of the C gene for Japanese encephalitis virus. The real-time RT-PCR reactive condition was optimized and the sensitivity, specificity and the stability of the assay were evaluated. Mosquitoes collected from Zhejiang province were detected by this assay. RESULTS: Mg2+, primer and probe were optimized at 5 mmol/L, 0.2 micromol/L and 0.1 micromol/L respectively. The specificity of the assay was high and there were no cross reactions with dengue virus, rabies virus, seoul virus or hantan virus. The detection limits of the assay was 0.1 TCID50. Results from preliminary application showed that TaqMan RT-PCR for Japanese encephalitis virus was sensitive, easier and faster to perform the process of traditional virus isolation and identification. It took only three hours to extract viral RNA and perform the real-time RT-PCR. CONCLUSION: This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.

[The distributions of mosquito vectors carrying Japanese encephalitis virus in Zhejiang province].

OBJECTIVE: To investigate the natural infection and genotype of Japanese encephalitis virus in mosquitoes and swine in some areas of Zhejiang province. METHODS: Samples 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. RESULTS: Seven 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. CONCLUSION: The 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.

[Complete genome sequence analysis of Japanese encephalitis virus newly isolated in China].

OBJECTIVE: To study the complete genome sequence of Japanese encephalitis virus (JEV) strain XJ69 isolated in ZheJiang province and explore its evolution. METHODS: Overlapping 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. RESULTS: The 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. CONCLUSION: The 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.

[Isolation and identification of Japanese encephalitis virus from mosquitoes in Zhejiang province].

OBJECTIVE: To study the situation of arboviruses carried by mosquitoes in Zhejiang province. METHODS: Mosquitoes were collected from Zhejiang province in 2007. Virus strains were isolated by the inoculation of homogenates of the mosquitoes onto BHK-21 cell line. The isolated strains were identified by serological (IFA) and molecular methods (RT-PCR). RESULTS: Two strains were isolated from mosquitoes causing cytopathogenic effect (CPE) in BHK-21 cells. Results from serological tests showed that both of the two strains were positive for the antibody to Japanese encephalitis virus (JEV). PrM and E gene were then cloned and sequenced. Results from the phylogenetic analysis showed that the isolates belonged to genotype I JEV while through sequence analysis it showed that the homology of nucleotide sequences and amino acid sequences between the two strains were 99.0% and 98.8% respectively. Compared with the JEV vaccine strain SA14-14-2 and two strains, the homology of nucleotide sequences was up to 87.7% and homology of amino acid sequences was up to 96.4%. When comparing with the vaccine strain SA14-14-2, there were 14 common amino acid variations in all the two strains. CONCLUSION: Two strains of JEV were isolated from mosquitoes collected in Zhejiang province which was the first isolation of genotype I JEV in the province in recent years.