• Korean Journal of Microbiology
• /
• v.38 no.3
• /
• pp.213-220
• /
• 2002

Inducible expression system for the three structural proteins, capsid (C), precursor membrane (prM/M), and envelop (E) of Japanese encephalitis virus (JEV) was established in BHK-21 cells. Doxycycline, a tetracycline analog, was utilized as an inducer. Transfectants BHK-21/IV (vector only), BHK-21/IC (for C), BHK-21/IP3 (for prM), and BHK-21/IE1 (for E) were selected and cloned in the presence of G4l8 or hygromycin. Transcribed mRNAs for the corresponding genes were observed after doxycycline induction. Effects by the JEV structural gene expression on the transfectants were monitored via cell growth, chromatin condensation, internucleosomal DNA fragmentation, and DNA contents analyses. Clear cell growth retardation and chromatin condensation were observed in all three transfectants while only BHK-2/IC corresponded to the induction status in the DNA fragmentation and DNA content analyses. Combined results, therefore, suggested that JEV capsid protein should be one of the direct and independent factors in apoptotic cell death induced by IEV infection.

• Korean Journal of Microbiology
• /
• v.26 no.3
• /
• pp.149-154
• /
• 1988

Japanese Encephalitis Virus(JEV) can be detected conveniently by the use of biotinylated cDNA probe. To prepare biotinylated probe aminoallyl dUTP was first synthesized chemically to reverse transcribe the virial RNA. The allylamine-labeled cDNA was then converted to the biotin-cDNA by the reaction with an activated biotin ester, NHS-ACA-biotin. The JEV genomic RNA was hybridized to the biotinylated cDNA probe on nitrocellulose filter and visualized colorimetrically by streptavidin complexes with alkaline phosphatase polymer. Sensitivity of the detection system was determined by estimating the amount of the JEV genomic RNA through comparison with signals generated from the biotinylated and $^{32/P}$ -labeled probes. It was found that the biotin probe was as sensitive as $^{32/P}$ -cDNA probe which can detect 50pgs of the target RNA.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.11
• /
• pp.1580-1587
• /
• 2012

Japanese encephalitis virus (JEV) envelope (E) protein holds great promise for use in the development of a recombinant vaccine. Purified recombinant E (rE) protein may be useful for numerous clinical applications; however, there are limitations in using the Escherichia coli expression system for producing high-quality rE protein. Therefore, in this study, the yeast expression system was used to generate the rE protein. For protein production using the yeast system, the full-length JEV E gene was cloned into Pichia pastoris. SDS-PAGE and immunoblotting analysis demonstrated that the rE protein had a molecular mass of 58 kDa and was glycosylated. The predicted size of the mature unmodified E protein is 53 kDa, suggesting that post-translational modifications resulted in the higher molecular mass. The rE protein was purified to greater than 95% purity using combined ammonium sulfate precipitation and a SP-Sepharose Fast Flow column. This purified rE protein was evaluated for immunogenicity and protective efficacy in mice. The survival rates of mice immunized with the rE protein were significantly increased over that of Hyphantria cunea nuclear polyhedrosis virus E protein (HcE). Our results indicate that the rE protein expressed in the P. pastoris expression system holds great promise for use in the development of a subunit vaccine against JEV.

• The Journal of Korean Society of Virology
• /
• v.27 no.2
• /
• pp.197-207
• /
• 1997

To investigate the NS4 region of JEV, NS4 cDNA of K94P05 (JEV strain isolated from Korea in 1994) was amplified by RT-PCR and analyzed by sequencing PCR product. Genomic size of NS4 was 1212bp and nucleotide sequence was compared with that of other JEV strains. Nucleotide homology between JaOAr582 and K94P05 was 91.1% and that between Beijing and K94P05 was 89.8%, respectively. But the nucleotide sequence of E region of JaOAr582 and K94P05 showed 97.0% homology and that of Beijing and K94P05 did 95.8% homology. NS4 protein was expressed as a form of fusion protein by a prokaryotic expression system. The induced fusion product showed a lower molecular weight than predicted size and remained insoluble. The NS4 protein might be cleavaged by E. coli protease. Concluding above results, high hydrophobicity of the NS4 protein supported the fact that this protein played a role as a membrane component and the poor nucleotide sequence conservativity among JEV strains suggested that this region might be important to adapt each viral growth environment.

• Korean Journal of Veterinary Research
• /
• v.57 no.1
• /
• pp.31-36
• /
• 2017

Japanese encephalitis (JE) is an important zoonosis caused by the mosquito-transmitted JE virus (JEV), which is a causative agent of reproductive failure in pregnant sows. Detection of JEV antibodies in swine is performed by hemagglutination inhibition (HI), virus neutralization (VN), and the plaque reduction neutralization test (PRNT). The most stringent PRNT is the 90% endpoint PRNT ($PRNT_{90}$). These conventional assays are difficult to carry out in diagnostic laboratories with insufficient instruments or cell culture systems. An alternative assay that is easily conducted and time efficient is required. In this study, we improved the indirect enzyme-linked immunosorbent assay (I-ELISA) with clarified antigen for the detection of JEV antibodies. The I-ELISA results obtained from 175 swine serum samples were compared with HI, VN, and $PRNT_{90}$ results. The sensitivity of I-ELISA was 91.8%, 95.0%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. The specificity of I-ELISA was 92.2%, 94.7%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. Moreover, the I-ELISA results were significantly correlated with the HI (r = 0.93), VN (r = 0.95), and $PRNT_{90}$ (r = 0.92) results. These results suggest that the improved I-ELISA is useful for serosurveillance of JEV in swine.

• Korean Journal of Microbiology
• /
• v.30 no.2
• /
• pp.115-123
• /
• 1992

A total of453 wilci hats inhabiting in Korea were captured ancl .he IgG antibodies againstJapanese Encephalitis Virus(J1IV) were detected by the heniagglut:nation inhibition te5t. 35501' the 453 blood sera showecl positive reaction to JEV with titers of I0 up to 40. Positiverates of male and kniale hats were 70.0'%1 anel 78.1'k. rcspectivclv. Positive ratci accordingto area were 74.7%) in Chungnan~. 72.h'\ulcorner6 in Kangwon and 74.3'"; in C'hungbuk. the resultbof which indicated no dil'krencc in areii. Whereas positive ratus according to hats specie5were 87.5(% f i ~ rC i..cpc~rtilios upernns. fi~llowedb y 83 3'%, k)r Mpoii.\ i ~ ~ : t r u ~ i tci.i~lis~. ~75l.0. '\4 1 forRhitrolol~h.\ '||'&'||',rurn~uic,r~unal ncl 59.6'!41 for Minioprc,ru.s schrc~ibersii.I t was Ibund by incli rrctini~nunofluorosce~icae nd clectron microscope techniques that the virus particles 01. JEVcould infect the brain of a Korean wilcl bats and proliferatn ill the brain cells.he brain cells.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.1
• /
• pp.121-127
• /
• 2004

Amphotericin B (AmB), an amphipathic polyene macrolide, is an antifungal drug produced by Streptomyces nodosus. Recently, AmB has been shown to exert antiviral activity against rubella virus and human immunodeficiency virus by different mechanisms. In this study, we evaluated the antiviral effect of AmB against Japanese encephalitis virus (JEV) and investigated which step of the viral life cycle was inhibited by AmB to understand the mechanism of antiviral action of AmB. AmB reduced both plaque size and number in the infected cells in a dose-dependent manner. In addition, a 200-fold reduction of infectious virus titer was observed by treatment of infected cells with $5\mug/ml$ of AmB. AmB acted at the post virus-infection step, but not during adsorption of virus to host cells. Western blot analysis revealed that the accumulated level of JEV envelope protein dramatically decreased in the infected cells by treatment with $5-10\mug/ml$ of AmB. Our results indicate that AmB inhibits the replication of JEV at the postinfection step by interfering with viral replication and/or by inhibiting the synthesis of viral proteins.

• Korean Journal of Veterinary Service
• /
• v.26 no.1
• /
• pp.11-18
• /
• 2003

A total of 1,024 sera were collected from cattle(227), pigs(465), chickens(257) and dogs(75) raised or slaughtered in Daejeon metropolitan city from April to September 2002. Japanses encephalitis virus(JEV) antibodies in sera were detected by the haemagglutination inhibition test. The prevalence rates of JEV antibodies were 99.1 %, 54.0 %, 63.0 % and 98.7 % in cattle, pigs, chickens and dogs, respectively. In case of cattle and dogs, the monthly antibody-positive rates were as high as 85.7∼100.0 % and there were no differences among six months. In case of pigs, the monthly antibody-positive rate showed the lowest in April(6.4 %) and the highest in July(100.0 %) and it remained above 50 % during the summer-time. In case of chickens, the monthly antibody-positive rate was 100.0 % in July & August, 80.5 % in June, 40.0 % in May, 7.5 % in September and 5.0 % in April in order and there were distinct differences in seasons.

• The Journal of the Korean Society for Microbiology
• /
• v.3 no.1
• /
• pp.51-65
• /
• 1968

The site of the synthesis of Japanese encephalitis virus(JEV) in the actinomycin-treated and infecter PS Y15 cells(a porcine kidney stable cell line) was observed by the immunofluorescent antibody technique, acridine orange staining, and the autoradiographic analysis. In the parallel studies by immunofluorescent technique and acridine orange staining it the infected cells, Viral protein(as an antigen) and viral RNA were detected at the same site of cytoplasm. In the autoradiographic analysis, the cytoplasmic labeling of $^3H$-uridine was due to the synthesis of JEV-RNA, while the nucleolus and nucleus were not involved. In the autoradiographic studies on the secton of infected cells, the $^3H$-uridine was frequently incorporated around the cytoplasmic vacuoles. This localization of labeling agreed with the site of acridine orange positive granules. The results suggest that the syntheses of the viral RNA and viral protein occurred in the similar site of cytoplasm of the infected cells, and also the virus particles seem to be assembled in the sites of the viral RNA and protein syntheses.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.8
• /
• pp.955-959
• /
• 2022

Japanese encephalitis (JE) is a vaccine-preventable mosquito-borne disease caused by infection with the Japanese encephalitis virus (JEV). JEV has five genotypes, including genotype V (GV), which is considered ancestral to the other genotypes. The first GV strain, GV Muar, was isolated from a Malayan patient in 1952 and GV did not reappear for 57 years until GV XZ0934 was isolated from a mosquito sample in China. Since 2010, 21 GV strains have been identified in Republic of Korea (ROK). Both GV Muar and GV XZ0934 are more pathogenic than other GI/GIII strains and are serologically distinct. However, because the ROK's GV strains have not been experimentally tested, their characteristics are not known. Characterization of the ROK's isolates is needed to enable development of effective GV strain-based vaccines to protect against GV infections.