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

• Toxicological Research
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• v.13 no.3
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• pp.281-291
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• 1997

The subacute toxicity of the combined vaccine (KGCC-95VI) for the prophylaxis against Japanese encephalitis and Hantaan virus infection, recently developed by Korea Green Cross Corporation, was investigated. KGCC-95VI was subcutaneously administered into the both sexes of New Zealand White rabbits at the dosage of 0, 10. 50 and 250 ml/kg body weight (20, 100 and 500 times the expected clincal dose) once a day for 30 days. There were no deaths and clinical findings during the experiment period. In both sexes. there were no statistically significant differences between the treated and control groups in urinalysis tests, hematological tests, blood chemistry tests and pathological examinations. The KGCC-95VI is considered not to have the subacute toxicity in the rabbits.

• Korean Journal of Veterinary Research
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• v.57 no.1
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• pp.31-36
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• 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
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• v.30 no.2
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• pp.115-123
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• 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.

• Molecules and Cells
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• v.21 no.1
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• pp.112-120
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• 2006

It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese encephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homologous DI virus intimately associated with JEV persistence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a persistent JEV infection in which the DI RNA coreplicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated during its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identification of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules retained their open reading frames despite a large deletion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these observations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection.

• Pediatric Infection and Vaccine
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• v.20 no.3
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• pp.131-138
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• 2013

Purpose: This study aimed to study the antibody response of Japanese encephalitis vaccination in children using different kinds of vaccines (inactivated vaccine, live attenuated vaccine or interchanged) and evaluate the effectiveness of the vaccines to provide the basis of efficient immunization schedule of Japanese encephalitis. Methods: Measurement of the neutralization antibody (NTAb) titers following Japanese encephalitis vaccination using different vaccines for 170 children, 2-6 year of age, who visited six university hospitals and are confirmed by immunization records. Results: Among 170 children who were given primary immunization on Japanese encephalitis, 103 children were given inactivated vaccine, 64 children were given live attenuated vaccine and 3 children were given interchangeably. NTAb titers were more than 1:10 in all children of three groups. The geographic mean antibody titer was 322 in inactivated vaccine group and 266 in live attenuated vaccine group. However, there was no significant difference between two groups. In both groups, the NTAb titer showed the peak at 1-4 months after the third immunization and declined. The NTAb titers of three children who were given two kinds of vaccines alternately were 1:135, 1:632, and 1:2511, respectively. Conclusion: According to the results of this study in children younger than 6 years old, there is no significant difference in effectiveness between inactivated and live attenuated vaccines. However, further studies for the changes of antibody titers for a longer period of time on larger population are required.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.221-229
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• 2002

Japanese encephalitis virus (JEV) persistence was established and maintained in Vero cell culture for over 1 year. Eleven clones of plaque morphology mutant JEV, with large and small plaque sizes, were obtained from the cell culture supernatant. Genomic RNA replication efficiency of the mutants in naive Vero cell appeared to correspond to their different plaque sizes. No significant changes in envelop protein ORF or in non-coding regions at both ends of the RNA genome suggested that there could be an unidentified factor(s) playing role in JEV attenuation. Unlike to the replication of wild-type JEV, the mutants did not induce severe degree of cytopathic effect in Vero cells upon infection. While obvious decrease of Bcl-2 and its mRNA expression and sharp increase of p53 in naive Vero cells infected with either wild-type JEV or the large plaque-forming mutant, those changes were not observed with the small plaque-forming one. Together with these observation, internucleosomal DNA fragmentation and chromosomal DNA profile in the Vero cells infected with the mutants suggest that an overall changes in cytopathic effect in the plaque morphology mutants-infected cells should be primarily due to the reduced genomic RNA replication and the compromised degree of p53-independent apoptosis by the virus infection at least in part.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.217.1-217.1
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• 2002

• Korean Journal of Microbiology
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• v.9 no.4
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• pp.175-178
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• 1971

Because of the cases of Japanese Encephalitis(J.E.) were reported every year in Korea. We, Dong-A Pharmaceutical Co., Ltd., produced J.E. virus vaccine, with lower price, since 1970 in order to prevent ourselves from being infected by the disease. And inoculated the J.E. virus vaccine for the children with a great success. We are going to report several questions which brought about in producing the J.E. virus vaccine by alcohol precipitation, protamine sulfate treatment method. The results obtained were as folows ; 1) In process treated with 40% alcohol, we used to ethanol made in Germany, but it was too expensive to use it. As the result which we had studied about it, we were satisfied with J.E. virus vaccine which produced with alcohol made in Korea, and then, we treated with accurate specific gravity of 40% ethanol for the precipitation of the virus. And also, we knew that it was the best method to be treated it for 3hrs, $13^{\circ}C$. 2) When we treated with protamine sulfate (0.025mg/ml), we acquired the highest potent titer, and suited into purpose for the nitrogen concentration. 3) The filtration of the purified J.E. virus vaccine, in case of millipore filter paper of large pore size was not suitable for the sterility. Therefore the pore size less than 0.8.$\mu$ (AA filter paper) in millipore filter paper was very suitable. But it seemed to be important subhects that the smaller was the pore size, the lower was the potent titer.

• The Journal of the Korean Society for Microbiology
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• v.3 no.1
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• pp.43-49
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• 1968

Japanese encephalitis(JE) shows its explosive epidemicity in the temperate zone of Asia but little is known on the overwintering mechanism. One of the hypotheses on the overwintering mechanism is that the virus overwinters in the hibernating animals. There has been no report on the proliferation of JE virus(JEV) or antibody formation in the snakes. The purpose of this experiment is to explore the mutual relationship between JEV and snake and to clarify whether JEV proliferates and induce antibody formation in snakes. Three species of non-poisonous common snakes were employed. Precipitation test was carried out after injecting calf serum and, HI and neutralization tests were done by injecting JEV into the snakes. The gamma globulin fraction of pre- and post-injection serum were compared by paper chromatography. According to the results, precipitating antibody reaction to calf serum could be observed only at $4^{\circ}C$. It was failed to demonstrate HI antibody formation but neutralizing antibody could be detected in one of the 9 snakes. Although antibody could not be detected in test-tube, tile result of paper chromatography shows the remarkable increase of gamma globulin fraction after the injection. Above results are strongly indicating the antibody formation in the snakes.